Arousal of cancer-associated stroma: overexpression of palladin activates fibroblasts to promote tumor invasion.

PubMed

Brentnall, Teresa A; Lai, Lisa A; Coleman, Joshua; Bronner, Mary P; Pan, Sheng; Chen, Ru

2012-01-01

fibroblasts can be triggered by k-ras expression in adjacent epithelial cells. This data supports a model whereby palladin-activated fibroblasts facilitate stromal-dependent metastasis and outgrowth of tumorigenic epithelium.

Mesenchymal stromal cells reverse hypoxia-mediated suppression of α-smooth muscle actin expression in human dermal fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Faulknor, Renea A.; Olekson, Melissa A.; Nativ, Nir I.

During wound healing, fibroblasts deposit extracellular matrix that guides angiogenesis and supports the migration and proliferation of cells that eventually form the scar. They also promote wound closure via differentiation into α-smooth muscle actin (SMA)-expressing myofibroblasts, which cause wound contraction. Low oxygen tension typical of chronic nonhealing wounds inhibits fibroblast collagen production and differentiation. It has been suggested that hypoxic mesenchymal stromal cells (MSCs) secrete factors that promote wound healing in animal models; however, it is unclear whether these factors are equally effective on the target cells in a hypoxic wound environment. Here we investigated the impact of MSC-derived solublemore » factors on the function of fibroblasts cultured in hypoxic fibroblast-populated collagen lattices (FPCLs). Hypoxia alone significantly decreased FPCL contraction and α-SMA expression. MSC-conditioned medium restored hypoxic FPCL contraction and α-SMA expression to levels similar to normoxic FPCLs. (SB431542), an inhibitor of transforming growth factor-β{sub 1} (TGF-β{sub 1})-mediated signaling, blocked most of the MSC effect on FPCL contraction, while exogenous TGF-β{sub 1} at levels similar to that secreted by MSCs reproduced the MSC effect. These results suggest that TGF-β{sub 1} is a major paracrine signal secreted by MSCs that can restore fibroblast functions relevant to the wound healing process and that are impaired in hypoxia. - Highlights: • Fibroblasts were cultured in collagen lattices (FPCLs) as model contracting wounds. • Hypoxia decreased FPCL contraction and fibroblast α-smooth muscle actin expression. • Mesenchymal stromal cells (MSCs) restored function of hypoxic fibroblasts. • MSCs regulate fibroblast function mainly via secreted transforming growth factor-β{sub 1}.« less

Syndecan-2 is upregulated in colorectal cancer cells through interactions with extracellular matrix produced by stromal fibroblasts.

PubMed

Vicente, Carolina Meloni; Ricci, Ritchelli; Nader, Helena Bonciani; Toma, Leny

2013-05-25

The extracellular matrix (ECM) influences the structure, viability and functions of cells and tissues. Recent evidence indicates that tumor cells and stromal cells interact through direct cell-cell contact, the production of ECM components and the secretion of growth factors. Syndecans are a family of transmembrane heparan sulfate proteoglycans that are involved in cell adhesion, motility, proliferation and differentiation. Syndecan-2 has been found to be highly expressed in colorectal cancer cell lines and appears to be critical for cancer cell behavior. We have examined the effect of stromal fibroblast-produced ECM on the production of proteoglycans by colorectal cancer cell lines. Our results showed that in a highly metastatic colorectal cancer cell line, HCT-116, syndecan-2 expression is enhanced by fibroblast ECM, while the expression of other syndecans decreased. Of the various components of the stromal ECM, fibronectin was the most important in stimulating the increase in syndecan-2 expression. The co-localization of syndecan-2 and fibronectin suggests that these two molecules are involved in the adhesion of HCT-116 cells to the ECM. Additionally, we demonstrated an increase in the expression of integrins alpha-2 and beta-1, in addition to an increase in the expression of phospho-FAK in the presence of fibroblast ECM. Furthermore, blocking syndecan-2 with a specific antibody resulted in a decrease in cell adhesion, migration, and organization of actin filaments. Overall, these results show that interactions between cancer cells and stromal ECM proteins induce significant changes in the behavior of cancer cells. In particular, a shift from the expression of anti-tumorigenic syndecans to the tumorigenic syndecan-2 may have implications in the migratory behavior of highly metastatic tumor cells.

Kallikrein-related peptidase 4 induces cancer-associated fibroblast features in prostate-derived stromal cells.

PubMed

Kryza, Thomas; Silva, Lakmali M; Bock, Nathalie; Fuhrman-Luck, Ruth A; Stephens, Carson R; Gao, Jin; Samaratunga, Hema; Lawrence, Mitchell G; Hooper, John D; Dong, Ying; Risbridger, Gail P; Clements, Judith A

2017-10-01

The reciprocal communication between cancer cells and their microenvironment is critical in cancer progression. Although involvement of cancer-associated fibroblasts (CAF) in cancer progression is long established, the molecular mechanisms leading to differentiation of CAFs from normal fibroblasts are poorly understood. Here, we report that kallikrein-related peptidase-4 (KLK4) promotes CAF differentiation. KLK4 is highly expressed in prostate epithelial cells of premalignant (prostatic intraepithelial neoplasia) and malignant lesions compared to normal prostate epithelia, especially at the peristromal interface. KLK4 induced CAF-like features in the prostate-derived WPMY1 normal stromal cell line, including increased expression of alpha-smooth muscle actin, ESR1 and SFRP1. KLK4 activated protease-activated receptor-1 in WPMY1 cells increasing expression of several factors (FGF1, TAGLN, LOX, IL8, VEGFA) involved in prostate cancer progression. In addition, KLK4 induced WPMY1 cell proliferation and secretome changes, which in turn stimulated HUVEC cell proliferation that could be blocked by a VEGFA antibody. Importantly, the genes dysregulated by KLK4 treatment of WPMY1 cells were also differentially expressed between patient-derived CAFs compared to matched nonmalignant fibroblasts and were further increased by KLK4 treatment. Taken together, we propose that epithelial-derived KLK4 promotes tumour progression by actively promoting CAF differentiation in the prostate stromal microenvironment. © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

Stromal fibroblasts are associated with collagen IV in scar tissues of alkali-burned and lacerated corneas.

PubMed

Ishizaki, M; Shimoda, M; Wakamatsu, K; Ogro, T; Yamanaka, N; Kao, C W; Kao, W W

1997-04-01

Corneal wound healing frequently leads to the formation of opaque scar tissue. We examined whether stromal fibroblastic cells of injured corneas express collagen IV and contributes to the formation of a basal lamina-like structure. Rabbits were anesthetized, and central corneal alkali burn (8 mm in diameter; 1 M NaOH, 1 min) or laceration (8 mm long) were produced. The injured corneas, which had healed for 1, 7, 21 and 45 days, were subjected to histological and immunohistochemical studies with goat anti-collagen IV antibodies, using light and electron microscopy, and in situ hybridization with an antisense digoxigenin-labeled riboprobe of collagen alpha 1(IV) mRNA. For comparison, twenty-day-old fetal corneas were subjected to immunohistochemical study and transmission electron microscopy (TEM). TEM examinations revealed that the stromal collagenous matrix was organized in orthogonal lamellae during corneal development, whereas that of alkali-burned cornea, which had healed for 3 weeks, was disorganized. The stroma of twenty-day-old fetal cornea was not labeled by the anti-collagen IV antibodies. In contrast, one week after injury, specific collagen IV immunostaining was detected in the injured stroma. As the healing proceeded (21-45 days), the antibodies reacted with fibroblastic cells and the extracellular matrix of scar tissues located in the anterior portion of alkali-burned corneas, as well as the posterior portion of lacerated corneas. The middle portion of the stromal tissues was weakly labeled by the anti-collagen IV antibodies with the exception of the blood vessel wall. Immuno-electron microscopic study showed that collagen IV and fibronectin were closely associated with the fibroblastic cells. In situ hybridization demonstrated that epithelial and endothelial cells and fibroblastic cells in the wounded corneal stroma and retro-corneal membrane expressed alpha 1(IV) mRNA, whereas in normal corneas the expression of alpha 1(IV) mRNA was limited to

Adipose-Derived Stromal Vascular Fraction Differentially Expands Breast Progenitors in Tissue Adjacent to Tumors Compared to Healthy Breast Tissue

PubMed Central

Chatterjee, Sumanta; Laliberte, Mike; Blelloch, Sarah; Ratanshi, Imran; Safneck, Janice; Buchel, Ed

2015-01-01

Background: Autologous fat grafts supplemented with adipose-derived stromal vascular fraction are used in reconstructive and cosmetic breast procedures. Stromal vascular fraction contains adipose-derived stem cells that are thought to encourage wound healing, tissue regeneration, and graft retention. Although use of stromal vascular fraction has provided exciting perspectives for aesthetic procedures, no studies have yet been conducted to determine whether its cells contribute to breast tissue regeneration. The authors examined the effect of these cells on the expansion of human breast epithelial progenitors. Methods: From patients undergoing reconstructive breast surgery following mastectomies, abdominal fat, matching tissue adjacent to breast tumors, and the contralateral non–tumor-containing breast tissue were obtained. Ex vivo co-cultures using breast epithelial cells and the stromal vascular fraction cells were used to study the expansion potential of breast progenitors. Breast reduction samples were collected as a source of healthy breast cells. Results: The authors observed that progenitors present in healthy breast tissue or contralateral non–tumor-containing breast tissue showed significant and robust expansion in the presence of stromal vascular fraction (5.2- and 4.8-fold, respectively). Whereas the healthy progenitors expanded up to 3-fold without the stromal vascular fraction cells, the expansion of tissue adjacent to breast tumor progenitors required the presence of stromal vascular fraction cells, leading to a 7-fold expansion, which was significantly higher than the expansion of healthy progenitors with stromal vascular fraction. Conclusions: The use of stromal vascular fraction might be more beneficial to reconstructive operations following mastectomies compared with cosmetic corrections of the healthy breast. Future studies are required to examine the potential risk factors associated with its use. CLINICAL QUESTION/LEVEL OF EVIDENCE

Exosomes released by chronic lymphocytic leukemia cells induce the transition of stromal cells into cancer-associated fibroblasts

PubMed Central

Paggetti, Jerome; Haderk, Franziska; Seiffert, Martina; Janji, Bassam; Distler, Ute; Ammerlaan, Wim; Kim, Yeoun Jin; Adam, Julien; Lichter, Peter; Solary, Eric; Berchem, Guy

2015-01-01

Exosomes derived from solid tumor cells are involved in immune suppression, angiogenesis, and metastasis, but the role of leukemia-derived exosomes has been less investigated. The pathogenesis of chronic lymphocytic leukemia (CLL) is stringently associated with a tumor-supportive microenvironment and a dysfunctional immune system. Here, we explore the role of CLL-derived exosomes in the cellular and molecular mechanisms by which malignant cells create this favorable surrounding. We show that CLL-derived exosomes are actively incorporated by endothelial and mesenchymal stem cells ex vivo and in vivo and that the transfer of exosomal protein and microRNA induces an inflammatory phenotype in the target cells, which resembles the phenotype of cancer-associated fibroblasts (CAFs). As a result, stromal cells show enhanced proliferation, migration, and secretion of inflammatory cytokines, contributing to a tumor-supportive microenvironment. Exosome uptake by endothelial cells increased angiogenesis ex vivo and in vivo, and coinjection of CLL-derived exosomes and CLL cells promoted tumor growth in immunodeficient mice. Finally, we detected α-smooth actin–positive stromal cells in lymph nodes of CLL patients. These findings demonstrate that CLL-derived exosomes actively promote disease progression by modulating several functions of surrounding stromal cells that acquire features of cancer-associated fibroblasts. PMID:26100252

Let-7b inhibits cancer-promoting effects of breast cancer-associated fibroblasts through IL-8 repression

PubMed Central

Al-Harbi, Bothina; Hendrayani, Siti-Fauziah; Silva, Gabriela; Aboussekhra, Abdelilah

2018-01-01

Cancer-associated fibroblasts (CAFs) are major players in the development and spread of breast carcinomas through non-cell-autonomous signaling. These paracrine effects are under the control of several genes and microRNAs. We present here clear evidence that let-7b, a tumor suppressor microRNA, plays key roles in the persistent activation of breast stromal fibroblasts and their functional interplay with cancer cells. We have first shown that let-7b is down-regulated in CAFs as compared to their corresponding normal adjacent fibroblasts, and transient specific let-7b inhibition permanently activated breast fibroblasts through induction of the IL-6-related positive feedback loop. More importantly, let-7b-deficient cells promoted the epithelial-to-mesenchymal transition process in breast cancer cells in an IL-8-dependent manner, and also enhanced orthotopic tumor growth in vivo. On the other hand, overexpression of let-7b by mimic permanently suppressed breast myofibroblasts through blocking the positive feedback loop, which inhibited their paracrine pro-carcinogenic effects. Furthermore, we have shown that let-7b negatively controls IL-8, which showed higher expression in the majority of CAF cells as compared to their adjacent normal counterparts, indicating that IL-8 plays a major role in the carcinoma/stroma cross-talk. These findings support targeting active stromal fibroblasts through restoration of let-7b/IL-8 expression as a therapeutic option for breast carcinomas. PMID:29707149

Reproducible isolation of lymph node stromal cells reveals site-dependent differences in fibroblastic reticular cells.

PubMed

Fletcher, Anne L; Malhotra, Deepali; Acton, Sophie E; Lukacs-Kornek, Veronika; Bellemare-Pelletier, Angelique; Curry, Mark; Armant, Myriam; Turley, Shannon J

2011-01-01

Within lymph nodes, non-hematopoietic stromal cells organize and interact with leukocytes in an immunologically important manner. In addition to organizing T and B cell segregation and expressing lymphocyte survival factors, several recent studies have shown that lymph node stromal cells shape the naïve T cell repertoire, expressing self-antigens which delete self-reactive T cells in a unique and non-redundant fashion. A fundamental role in peripheral tolerance, in addition to an otherwise extensive functional portfolio, necessitates closer study of lymph node stromal cell subsets using modern immunological techniques; however this has not routinely been possible in the field, due to difficulties reproducibly isolating these rare subsets. Techniques were therefore developed for successful ex vivo and in vitro manipulation and characterization of lymph node stroma. Here we discuss and validate these techniques in mice and humans, and apply them to address several unanswered questions regarding lymph node composition. We explored the steady-state stromal composition of lymph nodes isolated from mice and humans, and found that marginal reticular cells and lymphatic endothelial cells required lymphocytes for their normal maturation in mice. We also report alterations in the proportion and number of fibroblastic reticular cells (FRCs) between skin-draining and mesenteric lymph nodes. Similarly, transcriptional profiling of FRCs revealed changes in cytokine production from these sites. Together, these methods permit highly reproducible stromal cell isolation, sorting, and culture.

Modeling triple-negative breast cancer heterogeneity: effects of stromal macrophages, fibroblasts and tumor vasculature.

PubMed

Norton, Kerri-Ann; Jin, Kideok; Popel, Aleksander S

2018-05-08

A hallmark of breast tumors is its spatial heterogeneity that includes its distribution of cancer stem cells and progenitor cells, but also heterogeneity in the tumor microenvironment. In this study we focus on the contributions of stromal cells, specifically macrophages, fibroblasts, and endothelial cells on tumor progression. We develop a computational model of triple-negative breast cancer based on our previous work and expand it to include macrophage infiltration, fibroblasts, and angiogenesis. In vitro studies have shown that the secretomes of tumor-educated macrophages and fibroblasts increase both the migration and proliferation rates of triple-negative breast cancer cells. In vivo studies also demonstrated that blocking signaling of selected secreted factors inhibits tumor growth and metastasis in mouse xenograft models. We investigate the influences of increased migration and proliferation rates on tumor growth, the effect of the presence on fibroblasts or macrophages on growth and morphology, and the contributions of macrophage infiltration on tumor growth. We find that while the presence of macrophages increases overall tumor growth, the increase in macrophage infiltration does not substantially increase tumor growth and can even stifle tumor growth at excessive rates. Copyright © 2018. Published by Elsevier Ltd.

Combined CSL and p53 downregulation promotes cancer-associated fibroblast activation

PubMed Central

Procopio, Maria-Giuseppina; Laszlo, Csaba; Labban, Dania Al; Kim, Dong Eun; Bordignon, Pino; Jo, Seunghee; Goruppi, Sandro; Menietti, Elena; Ostano, Paola; Ala, Ugo; Provero, Paolo; Hoetzenecker, Wolfram; Neel, Victor; Kilarski, Witek; Swartz, Melody A.; Brisken, Cathrin; Lefort, Karine; Dotto, G. Paolo

2015-01-01

Stromal fibroblast senescence has been linked to aging-associated cancer risk. However, density and proliferation of cancer-associated fibroblasts (CAF) are frequently increased. Loss or down-modulation of the Notch effector CSL/RBP-Jκ in dermal fibroblasts is sufficient for CAF activation and ensuing keratinocyte-derived tumors. We report that CSL silencing induces senescence of primary fibroblasts from dermis, oral mucosa, breast and lung. CSL functions in these cells as direct repressor of multiple senescence- and CAF-effector genes. It also physically interacts with p53, repressing its activity. CSL is down-modulated in stromal fibroblasts of premalignant skin actinic keratosis lesions and squamous cell carcinomas (SCC), while p53 expression and function is down-modulated only in the latter, with paracrine FGF signaling as likely culprit. Concomitant loss of CSL and p53 overcomes fibroblast senescence, enhances expression of CAF effectors and promotes stromal and cancer cell expansion. The findings support a CAF activation/stromal co-evolution model under convergent CSL/p53 control. PMID:26302407

Periostin expression in intra-tumoral stromal cells is prognostic and predictive for colorectal carcinoma via creating a cancer-supportive niche

PubMed Central

Tan, Xiaojie; Ding, Yibo; Luo, Yanxin; Cai, Hui; Liu, Yan; Gao, Xianhua; Liu, Qizhi; Yu, Yongwei; Du, Yan; Wang, Hao; Ma, Liye; Wang, Jianping; Chen, Kun; Ding, Yanqing; Fu, Chuangang; Cao, Guangwen

2016-01-01

Periostin (POSTN) expression in cancer cells and circulation has been related to poor prognosis of colorectal carcinoma (CRC). However, the role of POSTN expressed in intra-tumoral stroma on CRC progression remains largely unknown. This study enrolled 1098 CRC patients who received surgical treatment in Shanghai and Guangzhou, Mainland China. In Shanghai cohort, immunohistochemistry score of stromal POSTN expression increased consecutively from adjacent mucosa, primary CRC tissues, to metastatic CRC tissues (P < 0.001), while medium- and high-stromal POSTN expression, rather than epithelial POSTN expression, independently predicted unfavorable prognoses of CRC, adjusted for covariates including TNM stage and postoperative chemotherapy in multivariate Cox models. The results in Shanghai cohort were faithfully replicated in Guangzhou cohort. Stromal POSTN expression dose-dependently predicted an unfavorable prognosis of stage III CRC patients with postoperative chemotherapy in both cohorts. POSTN derived from colonic fibroblasts or recombinant POSTN significantly promoted proliferation, anchorage independent growth, invasion, and chemo-resistance of CRC cells; whereas these effects were counteracted via targeting to PI3K/Akt or Wnt/β-catenin signaling pathway. CRC cell RKO-derived factor(s) significantly induced POSTN production in colonic fibroblasts and autocrine POSTN promoted proliferation, migration, and anchorage independent growth of fibroblasts. Conclusively, stromal POSTN is prognostic and predictive for CRC via creating a niche to facilitate cancer progression. Targeting POSTN-induced signaling pathways may be therapeutic options for metastatic or chemoresistant CRC. PMID:26556874

The Initiator Methionine tRNA Drives Secretion of Type II Collagen from Stromal Fibroblasts to Promote Tumor Growth and Angiogenesis

PubMed Central

Clarke, Cassie J.; Berg, Tracy J.; Birch, Joanna; Ennis, Darren; Mitchell, Louise; Cloix, Catherine; Campbell, Andrew; Sumpton, David; Nixon, Colin; Campbell, Kirsteen; Bridgeman, Victoria L.; Vermeulen, Peter B.; Foo, Shane; Kostaras, Eleftherios; Jones, J. Louise; Haywood, Linda; Pulleine, Ellie; Yin, Huabing; Strathdee, Douglas; Sansom, Owen; Blyth, Karen; McNeish, Iain; Zanivan, Sara; Reynolds, Andrew R.; Norman, Jim C.

2016-01-01

Summary Expression of the initiator methionine tRNA (tRNAiMet) is deregulated in cancer. Despite this fact, it is not currently known how tRNAiMet expression levels influence tumor progression. We have found that tRNAiMet expression is increased in carcinoma-associated fibroblasts, implicating deregulated expression of tRNAiMet in the tumor stroma as a possible contributor to tumor progression. To investigate how elevated stromal tRNAiMet contributes to tumor progression, we generated a mouse expressing additional copies of the tRNAiMet gene (2+tRNAiMet mouse). Growth and vascularization of subcutaneous tumor allografts was enhanced in 2+tRNAiMet mice compared with wild-type littermate controls. Extracellular matrix (ECM) deposited by fibroblasts from 2+tRNAiMet mice supported enhanced endothelial cell and fibroblast migration. SILAC mass spectrometry indicated that elevated expression of tRNAiMet significantly increased synthesis and secretion of certain types of collagen, in particular type II collagen. Suppression of type II collagen opposed the ability of tRNAiMet-overexpressing fibroblasts to deposit pro-migratory ECM. We used the prolyl hydroxylase inhibitor ethyl-3,4-dihydroxybenzoate (DHB) to determine whether collagen synthesis contributes to the tRNAiMet-driven pro-tumorigenic stroma in vivo. DHB had no effect on the growth of syngeneic allografts in wild-type mice but opposed the ability of 2+tRNAiMet mice to support increased angiogenesis and tumor growth. Finally, collagen II expression predicts poor prognosis in high-grade serous ovarian carcinoma. Taken together, these data indicate that increased tRNAiMet levels contribute to tumor progression by enhancing the ability of stromal fibroblasts to synthesize and secrete a type II collagen-rich ECM that supports endothelial cell migration and angiogenesis. PMID:26948875

The Initiator Methionine tRNA Drives Secretion of Type II Collagen from Stromal Fibroblasts to Promote Tumor Growth and Angiogenesis.

PubMed

Clarke, Cassie J; Berg, Tracy J; Birch, Joanna; Ennis, Darren; Mitchell, Louise; Cloix, Catherine; Campbell, Andrew; Sumpton, David; Nixon, Colin; Campbell, Kirsteen; Bridgeman, Victoria L; Vermeulen, Peter B; Foo, Shane; Kostaras, Eleftherios; Jones, J Louise; Haywood, Linda; Pulleine, Ellie; Yin, Huabing; Strathdee, Douglas; Sansom, Owen; Blyth, Karen; McNeish, Iain; Zanivan, Sara; Reynolds, Andrew R; Norman, Jim C

2016-03-21

Expression of the initiator methionine tRNA (tRNAi(Met)) is deregulated in cancer. Despite this fact, it is not currently known how tRNAi(Met) expression levels influence tumor progression. We have found that tRNAi(Met) expression is increased in carcinoma-associated fibroblasts, implicating deregulated expression of tRNAi(Met) in the tumor stroma as a possible contributor to tumor progression. To investigate how elevated stromal tRNAi(Met) contributes to tumor progression, we generated a mouse expressing additional copies of the tRNAi(Met) gene (2+tRNAi(Met) mouse). Growth and vascularization of subcutaneous tumor allografts was enhanced in 2+tRNAi(Met) mice compared with wild-type littermate controls. Extracellular matrix (ECM) deposited by fibroblasts from 2+tRNAi(Met) mice supported enhanced endothelial cell and fibroblast migration. SILAC mass spectrometry indicated that elevated expression of tRNAi(Met) significantly increased synthesis and secretion of certain types of collagen, in particular type II collagen. Suppression of type II collagen opposed the ability of tRNAi(Met)-overexpressing fibroblasts to deposit pro-migratory ECM. We used the prolyl hydroxylase inhibitor ethyl-3,4-dihydroxybenzoate (DHB) to determine whether collagen synthesis contributes to the tRNAi(Met)-driven pro-tumorigenic stroma in vivo. DHB had no effect on the growth of syngeneic allografts in wild-type mice but opposed the ability of 2+tRNAi(Met) mice to support increased angiogenesis and tumor growth. Finally, collagen II expression predicts poor prognosis in high-grade serous ovarian carcinoma. Taken together, these data indicate that increased tRNAi(Met) levels contribute to tumor progression by enhancing the ability of stromal fibroblasts to synthesize and secrete a type II collagen-rich ECM that supports endothelial cell migration and angiogenesis. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Anti-proliferative activities of finasteride in benign prostate epithelial cells require stromal fibroblasts and c-Jun gene.

PubMed

Wang, Kai; Jin, Song; Fan, Dongdong; Wang, Mingshuai; Xing, Nianzeng; Niu, Yinong

2017-01-01

This study aimed to identify the role of mouse fibroblast-mediated c-Jun and IGF-1 signaling in the therapeutic effect of finasteride on benign prostatic epithelial cells. BPH-1 cells, alone or with fibroblasts (c-Jun+/+ or c-Jun-/-), were implanted subcutaneously in male nude mice who were then treated with finasteride. The degrees of cell proliferation, apoptosis, and sizes of the xenografts were determined. BPH-1 cells were grown alone or co-cultured with mouse fibroblasts in the presence of finasteride and the level of IGF-1 secreted into the medium by the fibroblasts was determined. The proliferation-associated signaling pathway in BPH-1 cells was also evaluated. Fibroblasts and c-Jun promoted xenograft growth, stimulated Ki-67 expression, and inhibited BPH-1 apoptosis. Finasteride did not induce the shrinkage of xenografts in the combined-grafted groups despite repressing Ki-67 expression and inducing cell apoptosis. The addition of c-Jun-/- fibroblasts did not promote xenograft growth. In the absence of c-Jun and fibroblasts, finasteride did not alter xenograft growth, Ki-67 expression, or cell apoptosis. The in vitro results demonstrated that when BPH-1 cells were grown in monoculture, treatment with finasteride did not induce cell death and stimulated the expression of pro-proliferative signaling molecules, while in the presence of fibroblasts containing c-Jun, finasteride treatment repressed epithelial cell proliferation, the level of IGF-1 in the medium, and the activation of downstream pro-proliferative signaling pathways. Taken together, our results suggest that fibroblasts, c-Jun, and IGF-1 play key roles in mediating stromal-epithelial interactions that are required for the therapeutic effects of finasteride in benign prostate epithelial cells.

Anti-proliferative activities of finasteride in benign prostate epithelial cells require stromal fibroblasts and c-Jun gene

PubMed Central

Fan, Dongdong; Wang, Mingshuai; Xing, Nianzeng; Niu, Yinong

2017-01-01

This study aimed to identify the role of mouse fibroblast-mediated c-Jun and IGF-1 signaling in the therapeutic effect of finasteride on benign prostatic epithelial cells. BPH-1 cells, alone or with fibroblasts (c-Jun+/+ or c-Jun-/-), were implanted subcutaneously in male nude mice who were then treated with finasteride. The degrees of cell proliferation, apoptosis, and sizes of the xenografts were determined. BPH-1 cells were grown alone or co-cultured with mouse fibroblasts in the presence of finasteride and the level of IGF-1 secreted into the medium by the fibroblasts was determined. The proliferation-associated signaling pathway in BPH-1 cells was also evaluated. Fibroblasts and c-Jun promoted xenograft growth, stimulated Ki-67 expression, and inhibited BPH-1 apoptosis. Finasteride did not induce the shrinkage of xenografts in the combined-grafted groups despite repressing Ki-67 expression and inducing cell apoptosis. The addition of c-Jun-/- fibroblasts did not promote xenograft growth. In the absence of c-Jun and fibroblasts, finasteride did not alter xenograft growth, Ki-67 expression, or cell apoptosis. The in vitro results demonstrated that when BPH-1 cells were grown in monoculture, treatment with finasteride did not induce cell death and stimulated the expression of pro-proliferative signaling molecules, while in the presence of fibroblasts containing c-Jun, finasteride treatment repressed epithelial cell proliferation, the level of IGF-1 in the medium, and the activation of downstream pro-proliferative signaling pathways. Taken together, our results suggest that fibroblasts, c-Jun, and IGF-1 play key roles in mediating stromal-epithelial interactions that are required for the therapeutic effects of finasteride in benign prostate epithelial cells. PMID:28196103

Oncogenic KRAS Regulates Tumor Cell Signaling via Stromal Reciprocation

PubMed Central

Tape, Christopher J.; Ling, Stephanie; Dimitriadi, Maria; McMahon, Kelly M.; Worboys, Jonathan D.; Leong, Hui Sun; Norrie, Ida C.; Miller, Crispin J.; Poulogiannis, George; Lauffenburger, Douglas A.; Jørgensen, Claus

2016-01-01

Summary Oncogenic mutations regulate signaling within both tumor cells and adjacent stromal cells. Here, we show that oncogenic KRAS (KRASG12D) also regulates tumor cell signaling via stromal cells. By combining cell-specific proteome labeling with multivariate phosphoproteomics, we analyzed heterocellular KRASG12D signaling in pancreatic ductal adenocarcinoma (PDA) cells. Tumor cell KRASG12D engages heterotypic fibroblasts, which subsequently instigate reciprocal signaling in the tumor cells. Reciprocal signaling employs additional kinases and doubles the number of regulated signaling nodes from cell-autonomous KRASG12D. Consequently, reciprocal KRASG12D produces a tumor cell phosphoproteome and total proteome that is distinct from cell-autonomous KRASG12D alone. Reciprocal signaling regulates tumor cell proliferation and apoptosis and increases mitochondrial capacity via an IGF1R/AXL-AKT axis. These results demonstrate that oncogene signaling should be viewed as a heterocellular process and that our existing cell-autonomous perspective underrepresents the extent of oncogene signaling in cancer. Video Abstract PMID:27087446

Stromal loss of TGFβ drives cancer growth in the epithelium via inflammation | Center for Cancer Research

Cancer.gov

Interactions between epithelial and stromal cells play an important role in cancer development and progression. Epithelial cancers develop when changes occur to tumor suppressor genes in stromal fibroblast cells. For example, loss of tumor suppressor, p53, in stromal fibroblasts leads to p53 inactivation in the epithelium in a prostate cancer model, and disruption of the transforming growth factor-b receptor II (TGF-βRII) in stromal fibroblasts results in intraepithelial dysplasia in prostate cancer and invasive squamous cell carcinoma (SCC) in mouse forestomach.

Mesenchymal stromal cells reverse hypoxia-mediated suppression of α-smooth muscle actin expression in human dermal fibroblasts.

PubMed

Faulknor, Renea A; Olekson, Melissa A; Nativ, Nir I; Ghodbane, Mehdi; Gray, Andrea J; Berthiaume, François

2015-02-27

During wound healing, fibroblasts deposit extracellular matrix that guides angiogenesis and supports the migration and proliferation of cells that eventually form the scar. They also promote wound closure via differentiation into α-smooth muscle actin (SMA)-expressing myofibroblasts, which cause wound contraction. Low oxygen tension typical of chronic nonhealing wounds inhibits fibroblast collagen production and differentiation. It has been suggested that hypoxic mesenchymal stromal cells (MSCs) secrete factors that promote wound healing in animal models; however, it is unclear whether these factors are equally effective on the target cells in a hypoxic wound environment. Here we investigated the impact of MSC-derived soluble factors on the function of fibroblasts cultured in hypoxic fibroblast-populated collagen lattices (FPCLs). Hypoxia alone significantly decreased FPCL contraction and α-SMA expression. MSC-conditioned medium restored hypoxic FPCL contraction and α-SMA expression to levels similar to normoxic FPCLs. SB431542, an inhibitor of transforming growth factor-β1 (TGF-β1)-mediated signaling, blocked most of the MSC effect on FPCL contraction, while exogenous TGF-β1 at levels similar to that secreted by MSCs reproduced the MSC effect. These results suggest that TGF-β1 is a major paracrine signal secreted by MSCs that can restore fibroblast functions relevant to the wound healing process and that are impaired in hypoxia. Copyright © 2015. Published by Elsevier Inc.

Role of Stromal Paracrine Signals in Proliferative Diseases of the Aging Human Prostate

PubMed Central

Takahashi, Sanai; Sugimura, Yoshiki

2018-01-01

Androgens are essential for the development, differentiation, growth, and function of the prostate through epithelial–stromal interactions. However, androgen concentrations in the hypertrophic human prostate decrease significantly with age, suggesting an inverse correlation between androgen levels and proliferative diseases of the aging prostate. In elderly males, age- and/or androgen-related stromal remodeling is spontaneously induced, i.e., increased fibroblast and myofibroblast numbers, but decreased smooth muscle cell numbers in the prostatic stroma. These fibroblasts produce not only growth factors, cytokines, and extracellular matrix proteins, but also microRNAs as stromal paracrine signals that stimulate prostate epithelial cell proliferation. Surgical or chemical castration is the standard systemic therapy for patients with advanced prostate cancer. Androgen deprivation therapy induces temporary remission, but the majority of patients eventually progress to castration-resistant prostate cancer, which is associated with a high mortality rate. Androgen deprivation therapy-induced stromal remodeling may be involved in the development and progression of castration-resistant prostate cancer. In the tumor microenvironment, activated fibroblasts stimulating prostate cancer cell proliferation are called carcinoma-associated fibroblasts. In this review, we summarize the role of stromal paracrine signals in proliferative diseases of the aging human prostate and discuss the potential clinical applications of carcinoma-associated fibroblast-derived exosomal microRNAs as promising biomarkers. PMID:29614830

Stromagenesis: the changing face of fibroblastic microenvironments during tumor progression.

PubMed

Beacham, Dorothy A; Cukierman, Edna

2005-10-01

During tumorigenesis, reciprocal changes in stromal fibroblasts and tumor cells induce changes to the neoplastic microenvironmental landscape. In stromagenesis, both the complex network of bi-directional stromal fibroblastic signaling pathways and the stromal extracellular matrix are modified. The presence of a 'primed' stroma during the early, reversible stage of tumorigenesis is optimal for stromal-directed therapeutic intervention. Three-dimensional (3D) cell culture systems have been developed that mimic the in vivo microenvironment. These systems provide unique experimental tools to identify early alterations in stromagenesis that are supportive of tumor progression with the ultimate goal of blocking neoplastic permissiveness and restoring normal phenotypes.

Stromal loss of TGFβ drives cancer growth in the epithelium via inflammation | Center for Cancer Research

Cancer.gov

Interactions between epithelial and stromal cells play an important role in cancer development and progression. Epithelial cancers develop when changes occur to tumor suppressor genes in stromal fibroblast cells. For example, loss of tumor suppressor, p53, in stromal fibroblasts leads to p53 inactivation in the epithelium in a prostate cancer model, and disruption of the

Tumor cells induce the cancer associated fibroblast phenotype via caveolin-1 degradation: implications for breast cancer and DCIS therapy with autophagy inhibitors.

PubMed

Martinez-Outschoorn, Ubaldo E; Pavlides, Stephanos; Whitaker-Menezes, Diana; Daumer, Kristin M; Milliman, Janet N; Chiavarina, Barbara; Migneco, Gemma; Witkiewicz, Agnieszka K; Martinez-Cantarin, Maria P; Flomenberg, Neal; Howell, Anthony; Pestell, Richard G; Lisanti, Michael P; Sotgia, Federica

2010-06-15

Loss of stromal caveolin 1 (Cav-1) is a novel biomarker for cancer-associated fibroblasts that predicts poor clinical outcome in breast cancer and DCIS patients. We hypothesized that epithelial cancer cells may have the ability to drive Cav-1 downregulation in adjacent normal fibroblasts, thereby promoting the cancer associated fibroblast phenotype. To test this hypothesis directly, here we developed a novel co-culture model employing (i) human breast cancer cells (MCF7), and (ii) immortalized fibroblasts (hTERT-BJ1), which are grown under defined experimental conditions. Importantly, we show that co-culture of immortalized human fibroblasts with MCF7 breast cancer cells leads to Cav-1 downregulation in fibroblasts. These results were also validated using primary cultures of normal human mammary fibroblasts co-cultured with MCF7 cells. In this system, we show that Cav-1 downregulation is mediated by autophagic/lysosomal degradation, as pre-treatment with lysosome-specific inhibitors rescues Cav-1 expression. Functionally, we demonstrate that fibroblasts co-cultured with MCF7 breast cancer cells acquire a cancer associated fibroblast phenotype, characterized by Cav-1 downregulation, increased expression of myofibroblast markers and extracellular matrix proteins, and constitutive activation of TGFβ/Smad2 signaling. siRNA-mediated Cav-1 downregulation mimics several key changes that occur in co-cultured fibroblasts, clearly indicating that a loss of Cav-1 is a critical initiating factor, driving stromal fibroblast activation during tumorigenesis. As such, this co-culture system can now be used as an experimental model for generating "synthetic" cancer associated fibroblasts (CAFs). More specifically, these "synthetic" CAFs could be used for drug screening to identify novel therapeutics that selectively target the Cav-1-negative tumor micro-environment. Our findings also suggest that chloroquine, or other autophagy/lysosome inhibitors, may be useful as anti

Effects of human umbilical cord blood-derived mesenchymal stromal cells and dermal fibroblasts on diabetic wound healing.

PubMed

Moon, Kyung-Chul; Lee, Jong-Seok; Han, Seung-Kyu; Lee, Hyup-Woo; Dhong, Eun-Sang

2017-07-01

A previous study demonstrated that human umbilical cord blood-derived mesenchymal stromal cells (hUCB-MSCs) have superior wound-healing activity compared with fibroblasts in vitro. However, wound healing in vivo is a complex process that involves multiple factors. The purpose of this study was to compare the effects of hUCB-MSCs and fibroblasts on diabetic wound healing in vivo. This study especially focused on collagen synthesis and angiogenesis, which are considered to be the important factors affecting diabetic wound healing. Porous polyethylene discs were loaded with either fibroblasts or hUCB-MSCs, and a third group, which served as a control, was not loaded with cells. The discs were then implanted in the back of diabetic mice. During the first and the second week after implantation, the discs were harvested, and collagen level and microvascular density were compared. In terms of collagen synthesis, the hUCB-MSC group showed the highest collagen level (117.7 ± 8.9 ng/mL), followed by the fibroblast group (83.2 ± 5.2 ng/mL) and the no-cell group (60.0 ± 4.7 ng/mL) in the second week after implantation. In terms of angiogenesis, the microvascular density in the hUCB-MSC group was 56.8 ± 16.4, which was much higher than that in the fibroblast group (14.3 ± 4.0) and the no-cell group (5.7 ± 2.1) in the second week after implantation. These results demonstrate that hUCB-MSCs are superior to fibroblasts in terms of their effect on diabetic wound healing in vivo. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Oncogenes and inflammation rewire host energy metabolism in the tumor microenvironment: RAS and NFκB target stromal MCT4.

PubMed

Martinez-Outschoorn, Ubaldo E; Curry, Joseph M; Ko, Ying-Hui; Lin, Zhao; Tuluc, Madalina; Cognetti, David; Birbe, Ruth C; Pribitkin, Edmund; Bombonati, Alessandro; Pestell, Richard G; Howell, Anthony; Sotgia, Federica; Lisanti, Michael P

2013-08-15

Here, we developed a model system to evaluate the metabolic effects of oncogene(s) on the host microenvironment. A matched set of "normal" and oncogenically transformed epithelial cell lines were co-cultured with human fibroblasts, to determine the "bystander" effects of oncogenes on stromal cells. ROS production and glucose uptake were measured by FACS analysis. In addition, expression of a panel of metabolic protein biomarkers (Caveolin-1, MCT1, and MCT4) was analyzed in parallel. Interestingly, oncogene activation in cancer cells was sufficient to induce the metabolic reprogramming of cancer-associated fibroblasts toward glycolysis, via oxidative stress. Evidence for "metabolic symbiosis" between oxidative cancer cells and glycolytic fibroblasts was provided by MCT1/4 immunostaining. As such, oncogenes drive the establishment of a stromal-epithelial "lactate-shuttle", to fuel the anabolic growth of cancer cells. Similar results were obtained with two divergent oncogenes (RAS and NFκB), indicating that ROS production and inflammation metabolically converge on the tumor stroma, driving glycolysis and upregulation of MCT4. These findings make stromal MCT4 an attractive target for new drug discovery, as MCT4 is a shared endpoint for the metabolic effects of many oncogenic stimuli. Thus, diverse oncogenes stimulate a common metabolic response in the tumor stroma. Conversely, we also show that fibroblasts protect cancer cells against oncogenic stress and senescence by reducing ROS production in tumor cells. Ras-transformed cells were also able to metabolically reprogram normal adjacent epithelia, indicating that cancer cells can use either fibroblasts or epithelial cells as "partners" for metabolic symbiosis. The antioxidant N-acetyl-cysteine (NAC) selectively halted mitochondrial biogenesis in Ras-transformed cells, but not in normal epithelia. NAC also blocked stromal induction of MCT4, indicating that NAC effectively functions as an "MCT4 inhibitor". Taken

The role of stromal cells in the persistence of chronic inflammation

PubMed Central

Naylor, A J; Filer, A; Buckley, C D

2013-01-01

Inflammation is an unstable state; it either resolves or persists. Inflammatory reactions often have a propensity for specific anatomical sites. Why inflammation persists with specific tissue tropism remains obscure. Increasing evidence suggests that stromal cells which define tissue architecture are the key cells involved, and therefore make attractive therapeutic targets. Research on stromal cells in general and fibroblasts in particular has so far been hampered by a lack of fibroblast-specific cell markers. This review highlights our increasing understanding of the role of fibroblasts in inflammation, and suggests that these cells provide the cellular basis for site specific chronic inflammation. PMID:23199320

IFATS Collection: Adipose-Derived Stromal Cells Improve the Foreign Body Response

PubMed Central

Prichard, Heather L.; Reichert, William; Klitzman, Bruce

2015-01-01

Many implanted devices fail due to the formation of an avascular capsule surrounding the device. Additionally, fat has long been known to promote healing and vascularization. The goals of this study were to identify potential mechanisms of the provascular actions of adipose-derived stromal cells (ASCs) and to improve implant biocompatibility. First, adult ASCs and fibroblasts from rats were attached to polyurethane and polystyrene in vitro and their cytokine secretion profile was analyzed. Secretion of vascular endothelial growth factor (VEGF) from ASCs was 10 –70 times higher than fibroblasts after 3 and 6 days. Next, polyurethane, bare and with cellular coatings, was implanted subcutaneously in rats. The fibrous capsule surrounding bare polyurethane implants was 17%–32% thicker and the amount of collagen was 27% greater than the capsule surrounding ASC-coated implants. Finally, the microvessel density adjacent to ASC-coated polyurethane was approximately 50%–80% higher than bare polyurethane. In summary, ASCs attached to polyurethane have a dramatically increased VEGF production compared with fibroblasts in vitro, and these cells also produce an increased microvessel density in the surrounding tissue when implanted subcutaneously in rats. PMID:18436858

Stromal signatures in endometrioid endometrial carcinomas.

PubMed

Espinosa, Iñigo; Catasus, Lluis; D' Angelo, Emanuela; Mozos, Ana; Pedrola, Nuria; Bértolo, Cristina; Ferrer, Irene; Zannoni, Gian Franco; West, Robert B; van de Rijn, Matt; Matias-Guiu, Xavier; Prat, Jaime

2014-04-01

The pattern of myometrial invasion in endometrioid endometrial carcinomas varies considerably; ie, from widely scattered glands and cell nests, often associated with a fibromyxoid stromal reaction (desmoplasia) and/or a lymphocytic infiltrate, to invasive glands with little or no stromal response. Recently, two distinct stromal signatures derived from a macrophage response (colony-stimulating factor 1, CSF1) and a fibroblastic response (desmoid-type fibromatosis, DTF) were identified in breast carcinomas and correlated with clinicopathologic features including outcome. In this study, we explored whether these stromal signatures also apply to endometrioid carcinomas and how their expression patterns correlated with morphologic changes. We studied the stromal signatures both by immunohistochemistry and in situ hybridization in 98 primary endometrioid carcinomas with (87 cases) and without (11 cases) myometrial invasion as well as in the corresponding regional lymph nodes metatases of 9 myoinvasive tumors. Desmoplasia correlated positively with the DTF expression signature. Likewise, mononuclear infiltrates were found in the stroma of tumors expressing CSF1. Twenty-four out of eighty-seven (27%) myoinvasive endometrioid carcinomas were positive for the macrophage signature and thirteen out of eighty-seven (15%) expressed the fibroblast signature. Eleven additional cases were positive for both DTF and CSF1 signatures (11/87; 13%). However, over half of the cases (39/87; 45%) and the majority of the non-myoinvasive tumors (8/11; 73%) failed to express any of the two stromal signatures. The macrophage response (CSF1) was associated with higher tumor grade, lymphovascular invasion, and PIK3CA mutations (P<0.05). There was a concordance in the expression of the CSF1 signature in the primary tumors and their corresponding lymph node metastases. This study is the first characterization of stromal signatures in endometrioid carcinomas. Our findings shed new light on the

Effect of Fibroblast-Like Cells of Mesenchymal Origin of Cytotoxic Activity of Lymphocytes against NK-Sensitive Target Cells.

PubMed

Lupatov, A Yu; Kim, Ya S; Bystrykh, O A; Vakhrushev, I V; Pavlovich, S V; Yarygin, K N; Sukhikh, G T

2017-02-01

We studied immunosuppressive properties of skin fibroblasts and mesenchymal stromal cells against NK cells. In vitro experiments showed that mesenchymal stromal cells isolated from human umbilical cord and human skin fibroblasts can considerably attenuate cytotoxic activity of NK cells against Jurkat cells sensitive to NK-mediated lysis. NK cells cultured in lymphocyte population exhibited higher cytotoxic activity than isolated NK cells. Mesenchymal stromal cells or fibroblasts added 1:1 to lymphocyte culture almost completely suppressed NK cell cytotoxicity. This suggests that fibroblast-like cells can suppress not only isolated NK cells, but also NK cells in natural cell microenvironment.

Burn Eschar Stimulates Fibroblast and Adipose Mesenchymal Stromal Cell Proliferation and Migration but Inhibits Endothelial Cell Sprouting

PubMed Central

Monsuur, Hanneke N.; van den Broek, Lenie J.; Jhingoerie, Renushka L.; Vloemans, Adrianus F. P. M.

2017-01-01

The majority of full-thickness burn wounds heal with hypertrophic scar formation. Burn eschar most probably influences early burn wound healing, since granulation tissue only forms after escharotomy. In order to investigate the effect of burn eschar on delayed granulation tissue formation, burn wound extract (BWE) was isolated from the interface between non-viable eschar and viable tissue. The influence of BWE on the activity of endothelial cells derived from dermis and adipose tissue, dermal fibroblasts and adipose tissue-derived mesenchymal stromal cells (ASC) was determined. It was found that BWE stimulated endothelial cell inflammatory cytokine (CXCL8, IL-6 and CCL2) secretion and migration. However, BWE had no effect on endothelial cell proliferation or angiogenic sprouting. Indeed, BWE inhibited basic Fibroblast Growth Factor (bFGF) induced endothelial cell proliferation and sprouting. In contrast, BWE stimulated fibroblast and ASC proliferation and migration. No difference was observed between cells isolated from dermis or adipose tissue. The inhibitory effect of BWE on bFGF-induced endothelial proliferation and sprouting would explain why excessive granulation tissue formation is prevented in full-thickness burn wounds as long as the eschar is still present. Identifying the eschar factors responsible for this might give indications for therapeutic targets aimed at reducing hypertrophic scar formation which is initiated by excessive granulation tissue formation once eschar is removed. PMID:28820426

Curcumin suppresses crosstalk between colon cancer stem cells and stromal fibroblasts in the tumor microenvironment: potential role of EMT.

PubMed

Buhrmann, Constanze; Kraehe, Patricia; Lueders, Cora; Shayan, Parviz; Goel, Ajay; Shakibaei, Mehdi

2014-01-01

Interaction of stromal and tumor cells plays a dynamic role in initiating and enhancing carcinogenesis. In this study, we investigated the crosstalk between colorectal cancer (CRC) cells with stromal fibroblasts and the anti-cancer effects of curcumin and 5-Fluorouracil (5-FU), especially on cancer stem cell (CSC) survival in a 3D-co-culture model that mimics in vivo tumor microenvironment. Colon carcinoma cells HCT116 and MRC-5 fibroblasts were co-cultured in a monolayer or high density tumor microenvironment model in vitro with/without curcumin and/or 5-FU. Monolayer tumor microenvironment co-cultures supported intensive crosstalk between cancer cells and fibroblasts and enhanced up-regulation of metastatic active adhesion molecules (β1-integrin, ICAM-1), transforming growth factor-β signaling molecules (TGF-β3, p-Smad2), proliferation associated proteins (cyclin D1, Ki-67) and epithelial-to-mesenchymal transition (EMT) factor (vimentin) in HCT116 compared with tumor mono-cultures. High density tumor microenvironment co-cultures synergistically increased tumor-promoting factors (NF-κB, MMP-13), TGF-β3, favored CSC survival (characterized by up-regulation of CD133, CD44, ALDH1) and EMT-factors (increased vimentin and Slug, decreased E-cadherin) in HCT116 compared with high density HCT116 mono-cultures. Interestingly, this synergistic crosstalk was even more pronounced in the presence of 5-FU, but dramatically decreased in the presence of curcumin, inducing biochemical changes to mesenchymal-epithelial transition (MET), thereby sensitizing CSCs to 5-FU treatment. Enrichment of CSCs, remarkable activation of tumor-promoting factors and EMT in high density co-culture highlights that the crosstalk in the tumor microenvironment plays an essential role in tumor development and progression, and this interaction appears to be mediated at least in part by TGF-β and EMT. Modulation of this synergistic crosstalk by curcumin might be a potential therapy for CRC and

Curcumin Suppresses Crosstalk between Colon Cancer Stem Cells and Stromal Fibroblasts in the Tumor Microenvironment: Potential Role of EMT

PubMed Central

Buhrmann, Constanze; Kraehe, Patricia; Lueders, Cora; Shayan, Parviz; Goel, Ajay; Shakibaei, Mehdi

2014-01-01

Objective Interaction of stromal and tumor cells plays a dynamic role in initiating and enhancing carcinogenesis. In this study, we investigated the crosstalk between colorectal cancer (CRC) cells with stromal fibroblasts and the anti-cancer effects of curcumin and 5-Fluorouracil (5-FU), especially on cancer stem cell (CSC) survival in a 3D-co-culture model that mimics in vivo tumor microenvironment. Methods Colon carcinoma cells HCT116 and MRC-5 fibroblasts were co-cultured in a monolayer or high density tumor microenvironment model in vitro with/without curcumin and/or 5-FU. Results Monolayer tumor microenvironment co-cultures supported intensive crosstalk between cancer cells and fibroblasts and enhanced up-regulation of metastatic active adhesion molecules (β1-integrin, ICAM-1), transforming growth factor-β signaling molecules (TGF-β3, p-Smad2), proliferation associated proteins (cyclin D1, Ki-67) and epithelial-to-mesenchymal transition (EMT) factor (vimentin) in HCT116 compared with tumor mono-cultures. High density tumor microenvironment co-cultures synergistically increased tumor-promoting factors (NF-κB, MMP-13), TGF-β3, favored CSC survival (characterized by up-regulation of CD133, CD44, ALDH1) and EMT-factors (increased vimentin and Slug, decreased E-cadherin) in HCT116 compared with high density HCT116 mono-cultures. Interestingly, this synergistic crosstalk was even more pronounced in the presence of 5-FU, but dramatically decreased in the presence of curcumin, inducing biochemical changes to mesenchymal-epithelial transition (MET), thereby sensitizing CSCs to 5-FU treatment. Conclusion Enrichment of CSCs, remarkable activation of tumor-promoting factors and EMT in high density co-culture highlights that the crosstalk in the tumor microenvironment plays an essential role in tumor development and progression, and this interaction appears to be mediated at least in part by TGF-β and EMT. Modulation of this synergistic crosstalk by curcumin might be

Quantum dots-based immunofluorescent imaging of stromal fibroblasts Caveolin-1 and light chain 3B expression and identification of their clinical significance in human gastric cancer.

PubMed

He, Yuyu; Zhao, Xianda; Gao, Jun; Fan, Lifang; Yang, Guifang; Cho, William Chi-Shing; Chen, Honglei

2012-10-24

Caveolin-1 (Cav-1) expression deficiency and autophagy in tumor stromal fibroblasts (hereafter fibroblasts) are involved in tumor proliferation and progression, particularly in breast and prostate cancer. The aim of this study was to detect the expression of fibroblastic Cav-1 and LC3B, markers of autophagy, in gastric cancer (GC) and to analyze their clinical significances. Furthermore, because Epstein-Barr virus (EBV)-associated GC (EBVaGC) is a unique subtype of GC; we compared the differential expression of fibroblastic Cav-1 and LC3B in EBVaGC and non-EBVaGC. Quantum dots (QDs)-based immunofluorescence histochemistry was used to examine the expression of fibroblastic Cav-1 and LC3B in 118 cases of GC with adequate stroma. QDs-based double immunofluorescence labeling was performed to detect the coexpression of Cav-1 and LC3B proteins. EBV-encoded small RNA was detected by QDs-based fluorescence in situ hybridization to identify EBVaGC. Multivariate analysis indicated that low fibroblastic Cav-1 level was an independent prognosticator (p = 0.029) that predicted poorer survival of GC patients. Positive fibroblastic LC3B was correlated with lower invasion (p = 0.032) and was positively associated with Cav-1 expression (r = 0.432, p < 0.001). EBV infection did not affect fibroblastic Cav-1 and LC3B expression. In conclusion, positive fibroblastic LC3B correlates with lower invasion, and low expression of fibroblastic Cav-1 is a novel predictor of poor GC prognosis.

Carcinoma-associated fibroblasts: orchestrating the composition of malignancy

PubMed Central

Gascard, Philippe; Tlsty, Thea D.

2016-01-01

The tumor stroma is no longer seen solely as physical support for mutated epithelial cells but as an important modulator and even a driver of tumorigenicity. Within the tumor stromal milieu, heterogeneous populations of fibroblast-like cells, collectively termed carcinoma-associated fibroblasts (CAFs), are key players in the multicellular, stromal-dependent alterations that contribute to malignant initiation and progression. This review focuses on novel insights into the contributions of CAFs to disease progression, emergent events leading to the generation of CAFs, identification of CAF-specific biomarkers predictive of disease outcome, and recent therapeutic approaches aimed at blunting or reverting detrimental protumorigenic phenotypes associated with CAFs. PMID:27151975

Regulation of HGF and SDF-1 expression by oral fibroblasts--implications for invasion of oral cancer.

PubMed

Daly, Aisling J; McIlreavey, Leanne; Irwin, Chris R

2008-07-01

Invasion and metastasis of oral squamous cell carcinoma (OSCC) is dependent on signals received from stromal fibroblasts present in the surrounding connective tissue. The aim of this study was to investigate the regulation of expression of two important signaling molecules--HGF and SDF-1--by both stromal fibroblasts and their 'activated' form, myofibroblasts, and to determine the role of these two factors in stimulating OSCC cell invasion in vitro. Fibroblasts and myofibroblasts produced similar levels of HGF and SDF-1. IL-1alpha and OSCC cell conditioned medium both stimulated HGF and SDF-1 expression, while TGF-beta(1) inhibited production of each factor. Myofibroblast-derived conditioned medium stimulated OSCC cell invasion through matrigel. Blocking antibodies to both HGF and SDF-1 reduced the level of invasion. In fibroblast-free organotypic raft cultures, addition of HGF and SDF-1 stimulated OSCC cell invasion into the underlying collagen gel, although the pattern of invasion differed from that induced by fibroblasts. Fibroblast-derived HGF and SDF-1 appear to play central roles in the reciprocal interactions between OSCC cells and underlying stromal fibroblasts leading to the local invasion of oral cancer.

Genome-wide analysis of AR binding and comparison with transcript expression in primary human fetal prostate fibroblasts and cancer associated fibroblasts.

PubMed

Nash, Claire; Boufaied, Nadia; Mills, Ian G; Franco, Omar E; Hayward, Simon W; Thomson, Axel A

2017-05-05

The androgen receptor (AR) is a transcription factor, and key regulator of prostate development and cancer, which has discrete functions in stromal versus epithelial cells. AR expressed in mesenchyme is necessary and sufficient for prostate development while loss of stromal AR is predictive of prostate cancer progression. Many studies have characterized genome-wide binding of AR in prostate tumour cells but none have used primary mesenchyme or stroma. We applied ChIPseq to identify genomic AR binding sites in primary human fetal prostate fibroblasts and patient derived cancer associated fibroblasts, as well as the WPMY1 cell line overexpressing AR. We identified AR binding sites that were specific to fetal prostate fibroblasts (7534), cancer fibroblasts (629), WPMY1-AR (2561) as well as those common among all (783). Primary fibroblasts had a distinct AR binding profile versus prostate cancer cell lines and tissue, and showed a localisation to gene promoter binding sites 1 kb upstream of the transcriptional start site, as well as non-classical AR binding sequence motifs. We used RNAseq to define transcribed genes associated with AR binding sites and derived cistromes for embryonic and cancer fibroblasts as well as a cistrome common to both. These were compared to several in vivo ChIPseq and transcript expression datasets; which identified subsets of AR targets that were expressed in vivo and regulated by androgens. This analysis enabled us to deconvolute stromal AR targets active in stroma within tumour samples. Taken together, our data suggest that the AR shows significantly different genomic binding site locations in primary prostate fibroblasts compared to that observed in tumour cells. Validation of our AR binding site data with transcript expression in vitro and in vivo suggests that the AR target genes we have identified in primary fibroblasts may contribute to clinically significant and biologically important AR-regulated changes in prostate tissue

Collaborative and Defensive Fibroblasts in Tumor Progression and Therapy Resistance.

PubMed

Chiavarina, Barbara; Turtoi, Andrei

2017-01-01

Tumor microenvironment is a complex network of epithelial cancer cells and non-transformed stromal cells. Of the many stromal cell types, fibroblasts are the most numerous ones and are traditionally viewed as supportive elements of cancer progression. Many studies show that cancer cells engage in active crosstalk with associated fibroblasts in order to obtain key resources, such as growth factors and nutrients. The facets of fibroblast "complicity to murder" in cancer are multiple. However, recent therapeutic attempts aiming at depleting fibroblasts from tumors, perturbed rather simplistic picture. Contrary to the expectations, tumors devoid of fibroblasts accelerated their progression while patients faced poorer outcomes. These studies remind us of the physiologic roles fibroblasts have in maintaining tissue homeostasis even in the presence of cancer. It is becoming increasingly clear that our research focus on advanced tumors has biased our understanding of fibroblast role in tumor biology. The numerous events where the fibroblasts protect the tissue from malignant transformation remain largely unacknowledged, as the tumors are invisible. The present review has the ambition to offer a more balanced view of fibroblasts functions in cancer progression and therapy resistance. We will address the question whether it is possible to synergize the efforts with fibroblasts as the therapeutic concept against tumor progression and therapy resistance. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Very late antigen-5 facilitates stromal progenitor cell differentiation into myofibroblast.

PubMed

Sen, Namita; Weingarten, Mark; Peter, Yakov

2014-11-01

Fibrotic disease is associated with abrogated stromal cell proliferation and activity. The precise identity of the cells that drive fibrosis remains obscure, in part because of a lack of information on their lineage development. To investigate the role of an early stromal progenitor cell (SPC) on the fibrotic process, we selected for, and monitored the stages of, fibroblast development from a previously reported free-floating anchorage-independent cell (AIC) progenitor population. Our findings demonstrate that organotypic pulmonary, cardiac, and renal fibroblast commitment follows a two-step process of attachment and remodeling in culture. Cell differentiation was confirmed by the inability of SPCs to revert to the free-floating state and functional mesenchymal stem/stromal cell (MSC) differentiation into osteoblast, adipocyte, chondrocyte, and fibroblastic lineages. The myofibroblastic phenotype was reflected by actin stress-fiber formation, α-smooth muscle production, and a greater than threefold increase in proliferative activity compared with that of the progenitors. SPC-derived pulmonary myofibroblasts demonstrated a more than 300-fold increase in fibronectin-1 (Fn1), collagen, type 1, α1, integrin α-5 (Itga5), and integrin β-1 (Itgb1) transcript levels. Very late antigen-5 (ITGA5/ITGB1) protein cluster formations were also prevalent on the differentiated cells. Normalized SPC-derived myofibroblast expression patterns reflected those of primary cultured lung myofibroblasts. Intratracheal implantation of pulmonary AICs into recipient mouse lungs resulted in donor cell FN1 production and evidence of epithelial derivation. SPC derivation into stromal tissue in vitro and in vivo and the observation that MSC and fibroblast lineages share a common ancestor could potentially lead to personalized antifibrotic therapies. ©AlphaMed Press.

Influence of Ionizing Radiation on Stromal-Epithelial Intercellular Communication in Esophageal Carcinogenesis

NASA Technical Reports Server (NTRS)

Patel, Zarana S.; Kalabis, Jiri; Rustgi, Anil K.; Cucinotta, Francis A.; Huff, Janice L.

2010-01-01

Esophageal cancer is the 6th leading cause of cancer death worldwide. Its development is associated with a variety of risk factors including tobacco use, heavy alcohol consumption, human papilloma virus infection, and certain dietary factors such as trace mineral and vitamin deficiencies. An association with ionizing radiation exposure is revealed by the high excess relative risk for squamous cell carcinoma of the esophagus observed in the survivors of the atomic bomb detonations in Japan. It is also seen as a secondary malignancy in patients who received radiotherapy for breast and thoracic cancers; additionally, patients with head/neck and oral squamous cell cancers are at increased risk for metachronous esophageal squamous cell cancers. This malignancy is rapidly fatal, mainly because it remains asymptomatic until late, advanced stages when the disease is rarely curable. The stromal microenvironment plays an essential role in the maintenance and modulation of normal epithelial cell growth and differentiation and cross talk between the epithelial and stromal compartments can influence many aspects of malignant progression, including tumor cell proliferation, migration, invasion and recruitment of new blood vessels. To test the hypothesis that radiation exposure plays a role in esophageal carcinogenesis via non-targeted mechanisms involving stromal-epithelial cell communication, we are studying radiation effects on hTERT-immortalized human esophageal epithelial cells and genetic variants grown in co-culture with human esophageal stromal fibroblasts (Okawa et al., Genes & Dev. 2007. 21: 2788-2803). We examined how radiation treatment of stromal fibroblasts affected epithelial migration and invasion, behaviors associated with cancer promotion and progression. Chemotactic and haptotactic migration of epithelial cells stimulated by conditioned media from irradiated fibroblasts was measured using assays conducted in Transwell cell culture chambers. Our results using

Tissue engineering of ligaments: a comparison of bone marrow stromal cells, anterior cruciate ligament, and skin fibroblasts as cell source.

PubMed

Van Eijk, F; Saris, D B F; Riesle, J; Willems, W J; Van Blitterswijk, C A; Verbout, A J; Dhert, W J A

2004-01-01

Anterior cruciate ligament (ACL) reconstruction surgery still has important problems to overcome, such as "donor site morbidity" and the limited choice of grafts in revision surgery. Tissue engineering of ligaments may provide a solution for these problems. Little is known about the optimal cell source for tissue engineering of ligaments. The aim of this study is to determine the optimal cell source for tissue engineering of the anterior cruciate ligament. Bone marrow stromal cells (BMSCs), ACL, and skin fibroblasts were seeded onto a resorbable suture material [poly(L-lactide/glycolide) multifilaments] at five different seeding densities, and cultured for up to 12 days. All cell types tested attached to the suture material, proliferated, and synthesized extracellular matrix rich in collagen type I. On day 12 the scaffolds seeded with BMSCs showed the highest DNA content (p < 0.01) and the highest collagen production (p < 0.05 for the two highest seeding densities). Scaffolds seeded with ACL fibroblasts showed the lowest DNA content and collagen production. Accordingly, BMSCs appear to be the most suitable cells for further study and development of tissue-engineered ligament.

COX-2 and Prostaglandin EP3/EP4 Signaling Regulate the Tumor Stromal Proangiogenic Microenvironment via CXCL12-CXCR4 Chemokine Systems

PubMed Central

Katoh, Hiroshi; Hosono, Kanako; Ito, Yoshiya; Suzuki, Tatsunori; Ogawa, Yasufumi; Kubo, Hidefumi; Kamata, Hiroki; Mishima, Toshiaki; Tamaki, Hideaki; Sakagami, Hiroyuki; Sugimoto, Yukihiko; Narumiya, Shuh; Watanabe, Masahiko; Majima, Masataka

2010-01-01

Bone marrow (BM)–derived hematopoietic cells, which are major components of tumor stroma, determine the tumor microenvironment and regulate tumor phenotypes. Cyclooxygenase (COX)−2 and endogenous prostaglandins are important determinants for tumor growth and tumor-associated angiogenesis; however, their contributions to stromal formation and angiogenesis remain unclear. In this study, we observed that Lewis lung carcinoma cells implanted in wild-type mice formed a tumor mass with extensive stromal formation that was markedly suppressed by COX-2 inhibition, which reduced the recruitment of BM cells. Notably, COX-2 inhibition attenuated CXCL12/CXCR4 expression as well as expression of several other chemokines. Indeed, in a Matrigel model, prostaglandin (PG) E2 enhanced stromal formation and CXCL12/CXCR4 expression. In addition, a COX-2 inhibitor suppressed stromal formation and reduced expression of CXCL12/CXCR4 and a fibroblast marker (S100A4) in a micropore chamber model. Moreover, stromal formation after tumor implantation was suppressed in EP3−/− mice and EP4−/− mice, in which stromal expression of CXCL12/CXCR4 and S100A4 was reduced. The EP3 or EP4 knockout suppressed S100A4+ fibroblasts, CXCL12+, and/or CXCR4+ stromal cells as well. Immunofluorescent analyses revealed that CXCL12+CXCR4+S100A4+ fibroblasts mainly comprised stromal cells and most of these were recruited from the BM. Additionally, either EP3- or EP4-specific agonists stimulated CXCL12 expression by fibroblasts in vitro. The present results address the novel activities of COX-2/PGE2-EP3/EP4 signaling that modulate tumor biology and show that CXCL12/CXCR4 axis may play a crucial role in tumor stromal formation and angiogenesis under the control of prostaglandins. PMID:20110411

Influence of Ionizing Radiation on Stromal-Epithelial Communication in Esophageal Carcinogenesis

NASA Astrophysics Data System (ADS)

Huff, Janice; Patel, Zarana; Grugan, Katharine; Rustgi, Anil; Cucinotta, Francis A.

Esophageal cancer is the 6th leading cause of cancer death worldwide and is associated with a variety of risk factors including tobacco use, heavy alcohol consumption, human papilloma virus infection, and certain dietary factors such as trace mineral and vitamin deficiencies. A connection with ionizing radiation exposure is revealed by the high excess relative risk for esophageal squamous cell carcinoma observed in the survivors of the atomic bomb detonations in Japan. Esophageal carcinomas are also seen as secondary malignancies in patients who received radiotherapy for breast and thoracic cancers; additionally, patients with head/neck and oral squamous cell cancers are at increased risk for metachronous esophageal squamous cell cancers. This malignancy is rapidly fatal, mainly because it remains asymptomatic until late, advanced stages when the disease is rarely responsive to treatment. In normal epithelium, the stromal microenvironment is essential for the maintenance and modulation of cell growth and differentiation. Cross talk between the epithelial and stromal compartments can influence many aspects of malignant progression, including tumor cell proliferation, migration, invasion and recruitment of new blood vessels. To test the hypothesis that radiation exposure plays a role in esophageal carcinogenesis via non-targeted mechanisms involving stromal-epithelial cell communication, we are studying radiation effects on hTERT-immortalized human esophageal epithelial cells and genetic variants grown in co-culture with human esophageal stromal fibrob-lasts (Okawa et al., Genes Dev. 2007. 21: 2788-2803). We examined how irradiation of stromal fibroblasts affected epithelial migration and invasion, behaviors associated with cancer promotion and progression. These assays were conducted in modified Boyden chambers using conditioned media from irradiated fibroblasts. Our results using low LET gamma radiation showed a dose-dependent increase in migration of epithelial

HIF-1 maintains a functional relationship between pancreatic cancer cells and stromal fibroblasts by upregulating expression and secretion of Sonic hedgehog

PubMed Central

Katagiri, Tomohiro; Kobayashi, Minoru; Yoshimura, Michio; Morinibu, Akiyo; Itasaka, Satoshi; Hiraoka, Masahiro; Harada, Hiroshi

2018-01-01

Hypoxic and stroma-rich microenvironments, characteristic features of pancreatic cancers, are strongly associated with a poor prognosis. However, whether and how hypoxia increases stromal compartments remain largely unknown. Here, we investigated the potential importance of a master regulator of the cellular adaptive response to hypoxia, hypoxia-inducible factor-1 (HIF-1), in the formation of stroma-rich microenvironments of pancreatic tumors. We found that pancreatic cancer cells secreted more Sonic hedgehog protein (SHH) under hypoxia by upregulating its expression and efficiency of secretion in a HIF-1-dependent manner. Recombinant SHH, which was confirmed to activate the hedgehog signaling pathway, accelerated the growth of fibroblasts in a dose-dependent manner. The SHH protein secreted from pancreatic cancer cells under hypoxic conditions promoted the growth of fibroblasts by stimulating their Sonic hedgehog signaling pathway. These results suggest that the increased secretion of SHH by HIF-1 is potentially responsible for the formation of detrimental and stroma-rich microenvironments in pancreatic cancers, therefore providing a rational basis to target it in cancer therapy. PMID:29535824

Distinct function of estrogen receptor α in smooth muscle and fibroblast cells in prostate development.

PubMed

Vitkus, Spencer; Yeh, Chiuan-Ren; Lin, Hsiu-Hsia; Hsu, Iawen; Yu, Jiangzhou; Chen, Ming; Yeh, Shuyuan

2013-01-01

Estrogen signaling, through estrogen receptor (ER)α, has been shown to cause hypertrophy in the prostate. Our recent report has shown that epithelial ERα knockout (KO) will not affect the normal prostate development or homeostasis. However, it remains unclear whether ERα in different types of stromal cells has distinct roles in prostate development. This study proposed to elucidate how KO of ERα in the stromal smooth muscle or fibroblast cells may interrupt cross talk between prostate stromal and epithelial cells. Smooth muscle ERαKO (smERαKO) mice showed decreased glandular infolding with the proximal area exhibiting a significant decrease. Fibroblast ERαKO mouse prostates did not exhibit this phenotype but showed a decrease in the number of ductal tips. Additionally, the amount of collagen observed in the basement membrane was reduced in smERαKO prostates. Interestingly, these phenotypes were found to be mutually exclusive among smERαKO or fibroblast ERαKO mice. Compound KO of ERα in both fibroblast and smooth muscle showed combined phenotypes from each of the single KO. Further mechanistic studies showed that IGF-I and epidermal growth factor were down-regulated in prostate smooth muscle PS-1 cells lacking ERα. Together, our results indicate the distinct functions of fibroblast vs. smERα in prostate development.

Regulating Cancer-Associated Fibroblast Biology in Prostate Cancer

DTIC Science & Technology

2017-10-01

AWARD NUMBER: W81XWH-15-1-0512 TITLE: Regulating Cancer-Associated Fibroblast Biology in Prostate Cancer PRINCIPAL INVESTIGATOR: Andrew...SUBTITLE 5a. CONTRACT NUMBER Regulating Cancer-Associated Fibroblast Biology in Prostate Cancer 5b. GRANT NUMBER W81XWH-15-1-0512 5c. PROGRAM...blocked by the addition of Pim inhibitors. These results suggest that the Pim protein kinase can regulate stromal cell biology to modulate epithelial

TSPAN12 is a critical factor for cancer–fibroblast cell contact-mediated cancer invasion

PubMed Central

Otomo, Ryo; Otsubo, Chihiro; Matsushima-Hibiya, Yuko; Miyazaki, Makoto; Tashiro, Fumio; Ichikawa, Hitoshi; Kohno, Takashi; Ochiya, Takahiro; Yokota, Jun; Nakagama, Hitoshi; Taya, Yoichi; Enari, Masato

2014-01-01

Communication between cancer cells and their microenvironment controls cancer progression. Although the tumor suppressor p53 functions in a cell-autonomous manner, it has also recently been shown to function in a non–cell-autonomous fashion. Although functional defects have been reported in p53 in stromal cells surrounding cancer, including mutations in the p53 gene and decreased p53 expression, the role of p53 in stromal cells during cancer progression remains unclear. We herein show that the expression of α-smooth muscle actin (α-SMA), a marker of cancer-associated fibroblasts (CAFs), was increased by the ablation of p53 in lung fibroblasts. CAFs enhanced the invasion and proliferation of lung cancer cells when cocultured with p53-depleted fibroblasts and required contact between cancer and stromal cells. A comprehensive analysis using a DNA chip revealed that tetraspanin 12 (TSPAN12), which belongs to the tetraspanin protein family, was derepressed by p53 knockdown. TSPAN12 knockdown in p53-depleted fibroblasts inhibited cancer cell proliferation and invasion elicited by coculturing with p53-depleted fibroblasts in vitro, and inhibited tumor growth in vivo. It also decreased CXC chemokine ligand 6 (CXCL6) secretion through the β-catenin signaling pathway, suggesting that cancer cell contact with TSPAN12 in fibroblasts transduced β-catenin signaling into fibroblasts, leading to the secretion of CXCL6 to efficiently promote invasion. These results suggest that stroma-derived p53 plays a pivotal role in epithelial cancer progression and that TSPAN12 and CXCL6 are potential targets for lung cancer therapy. PMID:25512506

Downstream effects of ROCK signaling in cultured human corneal stromal cells: microarray analysis of gene expression.

PubMed

Harvey, Stephen A K; Anderson, Susan C; SundarRaj, Nirmala

2004-07-01

Rho-associated coiled-coil-containing protein kinase (ROCK) is a downstream target of Rho GTPase signaling and regulates the assembly of stress fibers. Previous reports indicate that Rho/ROCK signaling is involved in the regulation of several cellular processes, some of which may be cell-type specific and are probably critical to corneal stromal cell activation. The present study identified ROCK-regulated gene expression in corneal stromal cells. Corneal stromal cells derived from eyes of three different donors were cultured to yield the following designated phenotypes: baseline fibroblasts (DMEM with 10% serum), activated fibroblasts (10% serum+bFGF+heparin), and myofibroblasts (1% serum+TGF-beta 1). Cells were exposed to the ROCK inhibitor Y-27632 or vehicle for 12 hours, and transcript levels altered by ROCK inhibition were identified with oligonucleotide microarrays (GeneChips; Affymetrix, Santa Clara, CA). In these phenotypes, Y-27632 caused marked (twofold or more) increases or decreases in 14/4, 12/3, and 15/10 transcripts. In both fibroblast groups Y-27632-treatment increased expression of endothelin receptors and of parathyroid hormone-like hormone. The upregulation of alpha-smooth muscle actin in myofibroblasts was attenuated by Y-27632. Combining data from all groups identified ROCK-supported (Y-27632 inhibitable) expression of 10 transcripts, including ribonucleotide reductase M2, the cyclin B1-CDC2-CKS2 system, and four mitotic spindle-associated proteins. ROCK inhibition causes broad inhibition of DNA synthesis and mitosis and causes changes that are different between (bFGF-activated) fibroblasts and (TGF-beta 1-induced) myofibroblasts. Thus, Rho/ROCK signaling regulates both common and distinct downstream events in corneal stromal cells activated (differentiated) to fibroblast or myofibroblast phenotype.

Lactate is a mediator of metabolic cooperation between stromal carcinoma associated fibroblasts and glycolytic tumor cells in the tumor microenvironment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rattigan, Yanique I.; Patel, Brijesh B.; Department of Pharmacology, The Cancer Institute of New Jersey, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08901

2012-02-15

Human mesenchymal stem cells (hMSCs) are bone marrow-derived stromal cells, which play a role in tumor progression. We have shown earlier that breast cancer cells secrete higher levels of interleukin-6 (IL-6) under hypoxia, leading to the recruitment of hMSCs towards hypoxic tumor cells. We found that (i) MDA-MB-231 cells secrete significantly higher levels of lactate (3-fold more) under hypoxia (1% O{sub 2}) than under 20% O{sub 2} and (ii) lactate recruits hMSCs towards tumor cells by activating signaling pathways to enhance migration. The mRNA and protein expression of functional MCT1 in hMSCs is increased in response to lactate exposure. Thus,more » we hypothesized that hMSCs and stromal carcinoma associated fibroblasts (CAFs) in the tumor microenvironment have the capacity to take up lactate expelled from tumor cells and use it as a source of energy. Our {sup 13}C NMR spectroscopic measurements indicate that {sup 13}C-lactate is converted to {sup 13}C-alpha ketoglutarate in hMSCs and CAFs supporting this hypothesis. To our knowledge this is the first in vitro model system demonstrating that hMSCs and CAFs can utilize lactate produced by tumor cells.« less

Depletion of stromal cells expressing fibroblast activation protein-α from skeletal muscle and bone marrow results in cachexia and anemia

PubMed Central

Roberts, Edward W.; Deonarine, Andrew; Jones, James O.; Denton, Alice E.; Feig, Christine; Lyons, Scott K.; Espeli, Marion; Kraman, Matthew; McKenna, Brendan; Wells, Richard J.B.; Zhao, Qi; Caballero, Otavia L.; Larder, Rachel; Coll, Anthony P.; O’Rahilly, Stephen; Brindle, Kevin M.; Teichmann, Sarah A.; Tuveson, David A.

2013-01-01

Fibroblast activation protein-α (FAP) identifies stromal cells of mesenchymal origin in human cancers and chronic inflammatory lesions. In mouse models of cancer, they have been shown to be immune suppressive, but studies of their occurrence and function in normal tissues have been limited. With a transgenic mouse line permitting the bioluminescent imaging of FAP+ cells, we find that they reside in most tissues of the adult mouse. FAP+ cells from three sites, skeletal muscle, adipose tissue, and pancreas, have highly similar transcriptomes, suggesting a shared lineage. FAP+ cells of skeletal muscle are the major local source of follistatin, and in bone marrow they express Cxcl12 and KitL. Experimental ablation of these cells causes loss of muscle mass and a reduction of B-lymphopoiesis and erythropoiesis, revealing their essential functions in maintaining normal muscle mass and hematopoiesis, respectively. Remarkably, these cells are altered at these sites in transplantable and spontaneous mouse models of cancer-induced cachexia and anemia. Thus, the FAP+ stromal cell may have roles in two adverse consequences of cancer: their acquisition by tumors may cause failure of immunosurveillance, and their alteration in normal tissues contributes to the paraneoplastic syndromes of cachexia and anemia. PMID:23712428

Targeting Stromal Androgen Receptor Suppresses Prolactin-Driven Benign Prostatic Hyperplasia (BPH)

PubMed Central

Lai, Kuo-Pao; Huang, Chiung-Kuei; Fang, Lei-Ya; Izumi, Kouji; Lo, Chi-Wen; Wood, Ronald; Kindblom, Jon; Yeh, Shuyuan

2013-01-01

Stromal-epithelial interaction plays a pivotal role to mediate the normal prostate growth, the pathogenesis of benign prostatic hyperplasia (BPH), and prostate cancer development. Until now, the stromal androgen receptor (AR) functions in the BPH development, and the underlying mechanisms remain largely unknown. Here we used a genetic knockout approach to ablate stromal fibromuscular (fibroblasts and smooth muscle cells) AR in a probasin promoter-driven prolactin transgenic mouse model (Pb-PRL tg mice) that could spontaneously develop prostate hyperplasia to partially mimic human BPH development. We found Pb-PRL tg mice lacking stromal fibromuscular AR developed smaller prostates, with more marked changes in the dorsolateral prostate lobes with less proliferation index. Mechanistically, prolactin mediated hyperplastic prostate growth involved epithelial-stromal interaction through epithelial prolactin/prolactin receptor signals to regulate granulocyte macrophage-colony stimulating factor expression to facilitate stromal cell growth via sustaining signal transducer and activator of transcription-3 activity. Importantly, the stromal fibromuscular AR could modulate such epithelial-stromal interacting signals. Targeting stromal fibromuscular AR with the AR degradation enhancer, ASC-J9®, led to the reduction of prostate size, which could be used in future therapy. PMID:23893956

Prostate stromal cells express the progesterone receptor to control cancer cell mobility.

PubMed

Yu, Yue; Lee, Jennifer Suehyun; Xie, Ning; Li, Estelle; Hurtado-Coll, Antonio; Fazli, Ladan; Cox, Michael; Plymate, Stephen; Gleave, Martin; Dong, Xuesen

2014-01-01

Reciprocal interactions between epithelium and stroma play vital roles for prostate cancer development and progression. Enhanced secretions of cytokines and growth factors by cancer associated fibroblasts in prostate tumors create a favorable microenvironment for cancer cells to grow and metastasize. Our previous work showed that the progesterone receptor (PR) was expressed specifically in prostate stromal fibroblasts and smooth muscle cells. However, the expression levels of PR and its impact to tumor microenvironment in prostate tumors are poorly understood. Immunohistochemistry assays are applied to human prostate tissue biopsies. Cell migration, invasion and proliferation assays are performed using human prostate cells. Real-time PCR and ELISA are applied to measure gene expression at molecular levels. Immunohistochemistry assays showed that PR protein levels were decreased in cancer associated stroma when compared with paired normal prostate stroma. Using in vitro prostate stromal cell models, we showed that conditioned media collected from PR positive stromal cells inhibited prostate cancer cell migration and invasion, but had minor suppressive impacts on cancer cell proliferation. PR suppressed the secretion of stromal derived factor-1 (SDF-1) and interlukin-6 (IL-6) by stromal cells independent to PR ligands. Blocking PR expression by siRNA or supplementation of exogenous SDF-1 or IL-6 to conditioned media from PR positive stromal cells counteracted the inhibitory effects of PR to cancer cell migration and invasion. Decreased expression of the PR in cancer associated stroma may contribute to the elevated SDF-1 and IL-6 levels in prostate tumors and enhance prostate tumor progression.

Distinct Transcriptional Changes and Epithelial-stromal Interactions are Altered in Early Stage Colon Cancer Development

PubMed Central

Mo, Allen; Jackson, Stephen; Varma, Kamini; Carpino, Alan; Giardina, Charles; Devers, Thomas J.; Rosenberg, Daniel W.

2016-01-01

While the progression of mutated colonic cells is dependent upon interactions between the initiated epithelium and surrounding stroma, the nature of these interactions is poorly understood. Here the development of an ultra-sensitive laser-capture microdissection (LCM)/RNA-seq approach for studying the epithelial and stromal compartments of aberrant crypt foci (ACF) is described. ACF are the earliest identifiable pre-neoplastic lesion found within the human colon and are detected using high-definition endoscopy with contrast dye-spray. The current analysis focused on the epithelium of ACF with somatic mutations to either KRAS, BRAF, or APC, with expression patterns compared to normal mucosa from each patient. By comparing gene expression patterns between groups, an increase in a number of pro-inflammatory NF-κB target genes were identified that were specific to ACF epithelium, including TIMP1, RELA and RELB. Distinct transcriptional changes associated with each somatic mutation were observed and a subset display a BRAFV600E-mediated senescence-associated transcriptome characterized by increased expression of CDKN2A. Finally, LCM-captured ACF-associated stroma was found to be transcriptionally distinct from normal stroma, with an up-regulation of genes related to immune cell infiltration and fibroblast activation. Immunofluorescence confirmed increased CD3+ T cells within the stromal microenvironment of ACF and an abundance of activated fibroblasts. Collectively, these results provide new insight into the cellular interplay that occurs at the earliest stages of colonic neoplasia, highlighting the important role of NF-kB, activated stromal fibroblasts and lymphocyte infiltration. Implications Fibroblasts and immune cells in the stromal microenvironment play an important role during the earliest stages of colon carcinogenesis. PMID:27353028

Paracrine interactions of cancer-associated fibroblasts, macrophages and endothelial cells: tumor allies and foes.

PubMed

Ronca, Roberto; Van Ginderachter, Jo A; Turtoi, Andrei

2018-01-01

Tumor stroma is composed of many cellular subtypes, of which the most abundant are fibroblasts, macrophages and endothelial cells. During the process of tissue injury, these three cellular subtypes must coordinate their activity to efficiently contribute to tissue regeneration. In tumor, this mechanism is hijacked by cancer cells, which rewire the interaction of stromal cells to benefit tumor development. The present review aims at summarizing most relevant information concerning both pro-tumorigenic and anti-tumorigenic actions implicating the three stromal cell subtypes as well as their mutual interactions. Although stromal cells are generally regarded as tumor-supportive and at will manipulated by cancer cells, several novel studies point at many defaults in cancer cell-mediated stromal reprograming. Indeed, parts of initial tissue-protective and homeostatic functions of the stromal cells remain in place even after tumor development. Both tumor-supportive and tumor-suppressive functions have been well described for macrophages, whereas similar results are emerging for fibroblasts and endothelial cells. Recent success of immunotherapies have finally brought the long awaited proof that stroma is key for efficient tumor targeting. However, a better understanding of paracrine stromal interactions is needed in order to encourage drug development not only aiming at disruption of tumor-supportive communication but also re-enforcing, existing, tumor-suppressive mechanisms.

Co-culture of 3D tumor spheroids with fibroblasts as a model for epithelial–mesenchymal transition in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Sun-Ah, E-mail: j.sarah.k@gmail.com; Lee, Eun Kyung, E-mail: leeek@catholic.ac.kr; Cancer Evolution Research Center, College of Medicine, The Catholic University of Korea, Seoul 137-701

Epithelial–mesenchymal transition (EMT) acts as a facilitator of metastatic dissemination in the invasive margin of malignant tumors where active tumor–stromal crosstalks take place. Co-cultures of cancer cells with cancer-associated fibroblasts (CAFs) are often used as in vitro models of EMT. We established a tumor–fibroblast proximity co-culture using HT-29 tumor spheroids (TSs) with CCD-18co fibroblasts. When co-cultured with TSs, CCD-18co appeared activated, and proliferative activity as well as cell migration increased. Expression of fibronectin increased whereas laminin and type I collagen decreased in TSs co-cultured with fibroblasts compared to TSs alone, closely resembling the margin of in vivo xenograft tissue. Activemore » TGFβ1 in culture media significantly increased in TS co-cultures but not in 2D co-cultures of cancer cells–fibroblasts, indicating that 3D context-associated factors from TSs may be crucial to crosstalks between cancer cells and fibroblasts. We also observed in TSs co-cultured with fibroblasts increased expression of α-SMA, EGFR and CTGF; reduced expression of membranous β-catenin and E-cadherin, together suggesting an EMT-like changes similar to a marginal region of xenograft tissue in vivo. Overall, our in vitro TS–fibroblast proximity co-culture mimics the EMT-state of the invasive margin of in vivo tumors in early metastasis. - Highlights: • An adjacent co-culture of tumor spheroids and fibroblasts is presented as EMT model. • Activation of fibroblasts and increased cell migration were shown in co-culture. • Expression of EMT-related factors in co-culture was similar to that in tumor tissue. • Crosstalk between spheroids and fibroblasts was demonstrated by secretome analysis.« less

Evidence of two distinct functionally specialized fibroblast lineages in breast stroma.

PubMed

Morsing, Mikkel; Klitgaard, Marie Christine; Jafari, Abbas; Villadsen, René; Kassem, Moustapha; Petersen, Ole William; Rønnov-Jessen, Lone

2016-11-03

The terminal duct lobular unit (TDLU) is the most dynamic structure in the human breast and the putative site of origin of human breast cancer. Although stromal cells contribute to a specialized microenvironment in many organs, this component remains largely understudied in the human breast. We here demonstrate the impact on epithelium of two lineages of breast stromal fibroblasts, one of which accumulates in the TDLU while the other resides outside the TDLU in the interlobular stroma. The two lineages are prospectively isolated by fluorescence activated cell sorting (FACS) based on different expression levels of CD105 and CD26. The characteristics of the two fibroblast lineages are assessed by immunocytochemical staining and gene expression analysis. The differentiation capacity of the two fibroblast populations is determined by exposure to specific differentiating conditions followed by analysis of adipogenic and osteogenic differentiation. To test whether the two fibroblast lineages are functionally imprinted by their site of origin, single cell sorted CD271 low /MUC1 high normal breast luminal epithelial cells are plated on fibroblast feeders for the observation of morphological development. Epithelial structure formation and polarization is shown by immunofluorescence and digitalized quantification of immunoperoxidase-stained cultures. Lobular fibroblasts are CD105 high /CD26 low while interlobular fibroblasts are CD105 low /CD26 high . Once isolated the two lineages remain phenotypically stable and functionally distinct in culture. Lobular fibroblasts have properties in common with bone marrow derived mesenchymal stem cells and they specifically convey growth and branching morphogenesis of epithelial progenitors. Two distinct functionally specialized fibroblast lineages exist in the normal human breast, of which the lobular fibroblasts have properties in common with mesenchymal stem cells and support epithelial growth and morphogenesis. We propose that

Fibroblast spheroids as a model to study sustained fibroblast quiescence and their crosstalk with tumor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Salmenperä, Pertteli, E-mail: pertteli.salmenpera@helsinki.fi; Karhemo, Piia-Riitta; Räsänen, Kati

Stromal fibroblasts have an important role in regulating tumor progression. Normal and quiescent fibroblasts have been shown to restrict and control cancer cell growth, while cancer-associated, i. e. activated fibroblasts have been shown to enhance proliferation and metastasis of cancer cells. In this study we describe generation of quiescent fibroblasts in multicellular spheroids and their effects on squamous cell carcinoma (SCC) growth in soft-agarose and xenograft models. Quiescent phenotype of fibroblasts was determined by global down-regulation of expression of genes related to cell cycle and increased expression of p27. Interestingly, microarray analysis showed that fibroblast quiescence was associated with similarmore » secretory phenotype as seen in senescence and they expressed senescence-associated-β-galactosidase. Quiescent fibroblasts spheroids also restricted the growth of RT3 SCC cells both in soft-agarose and xenograft models unlike proliferating fibroblasts. Restricted tumor growth was associated with marginally increased tumor cell senescence and cellular differentiation, showed with senescence-associated-β-galactosidase and cytokeratin 7 staining. Our results show that the fibroblasts spheroids can be used as a model to study cellular quiescence and their effects on cancer cell progression. - Highlights: • Fibroblasts acquire a sustained quiescence when grown as multicellular spheroids. • This quiescence is associated with drastic change in gene expression. • Fibroblasts spheroids secrete various inflammation-linked cytokines and chemokines. • Fibroblasts spheroids reduced growth of RT3 SCC cells in xenograft model.« less

Epithelial-Stromal Interaction 1 (EPSTI1) Substitutes for Peritumoral Fibroblasts in the Tumor Microenvironment

PubMed Central

de Neergaard, Michala; Kim, Jiyoung; Villadsen, René; Fridriksdottir, Agla J.; Rank, Fritz; Timmermans-Wielenga, Vera; Langerød, Anita; Børresen-Dale, Anne-Lise; Petersen, Ole W.; Rønnov-Jessen, Lone

2010-01-01

Tumor cells can activate stroma, yet the implication of this activation in terms of reciprocal induction of gene expression in tumor cells is poorly understood. Epithelial Stromal Interaction 1 (EPSTI1) is an interferon response gene originally isolated from heterotypic recombinant cultures of human breast cancer cells and activated breast myofibroblasts. Here we describe the first immunolocalization of EPSTI1 in normal and cancerous breast tissue, and we provide evidence for a role of this molecule in the regulation of tumor cell properties and epithelial-mesenchymal transition. In general, no EPSTI1 staining was observed in normal breast epithelial cells from reduction mammoplasties (n = 25). However, in carcinomas, staining was positive in 22 of 40 biopsies and inversely correlated with the level of differentiation. To address the function of EPSTI1, we expressed EPSTI1 ectopically in one cell line and silenced endogenous EPSTI1 by RNA interference in another. Irrespective of the experimental approach, EPSTI1 expression led to an increase in tumorsphere formation—a property associated with breast stem/progenitor cells. Most remarkably, we show that EPSTI1, by conveying spread of tumor cells, can replace peritumoral activated fibroblasts in a tumor environment assay. These observations implicate EPSTI1 as a hitherto unappreciated regulator of tumor cell properties. PMID:20133812

Science to Practice: imaging cancer-associated fibroblasts--no innocent bystanders.

PubMed

Choyke, Peter L

2013-09-01

The era of stromal-based therapies is coming, and methods to image the stroma are likely to become vital to improved understanding of the intricate interrelationships of these cells. Because fibroblasts are so important for the initiation of cancer, stromal-based therapies may serve as preventive regimens in patients who are at high risk for recurrent disease. The method described by Vandsburger et al uses a reporter-gene magnetic resonance (MR) imaging–agent paradigm that withstands dilution from cell division while allowing imaging without ionizing radiation. The requirement for gene transfection makes near-term clinical translation unlikely, but the opportunities for studying cancer-associated fibroblast activity in tumor models and observing and modulating their migratory behavior is an exciting prospect, one that is hoped to bring tangible benefits to patients with cancer.

Gastrointestinal Fibroblasts Have Specialized, Diverse Transcriptional Phenotypes: A Comprehensive Gene Expression Analysis of Human Fibroblasts

PubMed Central

Ishii, Genichiro; Aoyagi, Kazuhiko; Sasaki, Hiroki; Ochiai, Atsushi

2015-01-01

Background Fibroblasts are the principal stromal cells that exist in whole organs and play vital roles in many biological processes. Although the functional diversity of fibroblasts has been estimated, a comprehensive analysis of fibroblasts from the whole body has not been performed and their transcriptional diversity has not been sufficiently explored. The aim of this study was to elucidate the transcriptional diversity of human fibroblasts within the whole body. Methods Global gene expression analysis was performed on 63 human primary fibroblasts from 13 organs. Of these, 32 fibroblasts from gastrointestinal organs (gastrointestinal fibroblasts: GIFs) were obtained from a pair of 2 anatomical sites: the submucosal layer (submucosal fibroblasts: SMFs) and the subperitoneal layer (subperitoneal fibroblasts: SPFs). Using hierarchical clustering analysis, we elucidated identifiable subgroups of fibroblasts and analyzed the transcriptional character of each subgroup. Results In unsupervised clustering, 2 major clusters that separate GIFs and non-GIFs were observed. Organ- and anatomical site-dependent clusters within GIFs were also observed. The signature genes that discriminated GIFs from non-GIFs, SMFs from SPFs, and the fibroblasts of one organ from another organ consisted of genes associated with transcriptional regulation, signaling ligands, and extracellular matrix remodeling. Conclusions GIFs are characteristic fibroblasts with specific gene expressions from transcriptional regulation, signaling ligands, and extracellular matrix remodeling related genes. In addition, the anatomical site- and organ-dependent diversity of GIFs was also discovered. These features of GIFs contribute to their specific physiological function and homeostatic maintenance, and create a functional diversity of the gastrointestinal tract. PMID:26046848

Dissecting the Functions of Autophagy in Breast Cancer Associated Fibroblasts

DTIC Science & Technology

2014-10-01

compound transgenic mouse model of mammary cancer (MMTV-PyMT) harboring genetic deletion of Atg12 in stromal fibroblasts using the fibroblast specific...Cre;MMTV-PyMT mice (months 2-18). Using the breeding strategy outlined in Figure 1, we have successfully generated these quadruple transgenic mice...could then use for generating lysate and interrogation by Western blot (Fig. 7). However, our data suggest that the autophagy incompetent MMFs (from

Fibroblast-induced switching to the mesenchymal-like phenotype and PI3K/mTOR signaling protects melanoma cells from BRAF inhibitors

PubMed Central

Seip, Kotryna; Nygaard, Vigdis; Haugen, Mads H.; Engesæter, Birgit Ø.; Mælandsmo, Gunhild M.; Prasmickaite, Lina

2016-01-01

The knowledge on how tumor-associated stroma influences efficacy of anti-cancer therapy just started to emerge. Here we show that lung fibroblasts reduce melanoma sensitivity to the BRAF inhibitor (BRAFi) vemurafenib only if the two cell types are in close proximity. In the presence of fibroblasts, the adjacent melanoma cells acquire de-differentiated mesenchymal-like phenotype. Upon treatment with BRAFi, such melanoma cells maintain high levels of phospho ribosomal protein S6 (pS6), i.e. active mTOR signaling, which is suppressed in the BRAFi sensitive cells without stromal contacts. Inhibitors of PI3K/mTOR in combination with BRAFi eradicate pS6high cell subpopulations and potentiate anti-cancer effects in melanoma protected by the fibroblasts. mTOR and BRAF co-inhibition also delayed the development of early-stage lung metastases in vivo. In conclusion, we demonstrate that upon influence from fibroblasts, melanoma cells undergo a phenotype switch to the mesenchymal state, which can support PI3K/mTOR signaling. The lost sensitivity to BRAFi in such cells can be overcome by co-targeting PI3K/mTOR. This knowledge could be explored for designing BRAFi combination therapies aiming to eliminate both stroma-protected and non-protected counterparts of metastases. PMID:26918352

Stromal cell markers are differentially expressed in the synovial tissue of patients with early arthritis.

PubMed

Choi, Ivy Y; Karpus, Olga N; Turner, Jason D; Hardie, Debbie; Marshall, Jennifer L; de Hair, Maria J H; Maijer, Karen I; Tak, Paul P; Raza, Karim; Hamann, Jörg; Buckley, Christopher D; Gerlag, Danielle M; Filer, Andrew

2017-01-01

Previous studies have shown increased expression of stromal markers in synovial tissue (ST) of patients with established rheumatoid arthritis (RA). Here, ST expression of stromal markers in early arthritis in relationship to diagnosis and prognostic outcome was studied. ST from 56 patients included in two different early arthritis cohorts and 7 non-inflammatory controls was analysed using immunofluorescence to detect stromal markers CD55, CD248, fibroblast activation protein (FAP) and podoplanin. Diagnostic classification (gout, psoriatic arthritis, unclassified arthritis (UA), parvovirus associated arthritis, reactive arthritis and RA), disease outcome (resolving vs persistent) and clinical variables were determined at baseline and after follow-up, and related to the expression of stromal markers. We observed expression of all stromal markers in ST of early arthritis patients, independent of diagnosis or prognostic outcome. Synovial expression of FAP was significantly higher in patients developing early RA compared to other diagnostic groups and non-inflammatory controls. In RA FAP protein was expressed in both lining and sublining layers. Podoplanin expression was higher in all early inflammatory arthritis patients than controls, but did not differentiate diagnostic outcomes. Stromal marker expression was not associated with prognostic outcomes of disease persistence or resolution. There was no association with clinical or sonographic variables. Stromal cell markers CD55, CD248, FAP and podoplanin are expressed in ST in the earliest stage of arthritis. Baseline expression of FAP is higher in early synovitis patients who fulfil classification criteria for RA over time. These results suggest that significant fibroblast activation occurs in RA in the early window of disease.

Role of fibroblast growth factor receptors (FGFR) and FGFR like-1 (FGFRL1) in mesenchymal stromal cell differentiation to osteoblasts and adipocytes.

PubMed

Kähkönen, T E; Ivaska, K K; Jiang, M; Büki, K G; Väänänen, H K; Härkönen, P L

2018-02-05

Fibroblast growth factors (FGF) and their receptors (FGFRs) regulate many developmental processes including differentiation of mesenchymal stromal cells (MSC). We developed two MSC lines capable of differentiating to osteoblasts and adipocytes and studied the role of FGFRs in this process. We identified FGFR2 and fibroblast growth factor receptor like-1 (FGFRL1) as possible actors in MSC differentiation with gene microarray and qRT-PCR. FGFR2 and FGFRL1 mRNA expression strongly increased during MSC differentiation to osteoblasts. FGF2 treatment, resulting in downregulation of FGFR2, or silencing FGFR2 expression with siRNAs inhibited osteoblast differentiation. During adipocyte differentiation expression of FGFR1 and FGFRL1 increased and was down-regulated by FGF2. FGFR1 knockdown inhibited adipocyte differentiation. Silencing FGFR2 and FGFR1 in MSCs was associated with decreased FGFRL1 expression in osteoblasts and adipocytes, respectively. Our results suggest that FGFR1 and FGFR2 regulate FGFRL1 expression. FGFRL1 may mediate or modulate FGFR regulation of MSC differentiation together with FGFR2 in osteoblastic and FGFR1 in adipocytic lineage. Copyright © 2017 Elsevier B.V. All rights reserved.

Possibility of Aggravation of Tissue Sclerosis after Injection of Multipotent Mesenchymal Stromal Cells Near the Forming Cicatrix in the Experiment.

PubMed

Maiborodin, I V; Morozov, V V; Anikeev, A A; Figurenko, N F; Maslov, R V; Matveeva, V A; Chastikina, G A; Maiborodina, V I

2017-08-01

The peculiarities of tissue sclerosis after injection of autologous bone marrow multipotent mesenchymal stromal cells transfected with GFP gene and stained with Vybrant CM-Dil cell membrane dye were studied by light microscopy with luminescence. The surgical intervention consisting in ligation of the great vein was followed by tissue sclerotic transformation caused by direct damage and chronic inflammation caused by the presence of slowly resorbed ligature. Injection of stromal cells after this intervention led to formation of more extensive scar. This can attest to the possibility of stromal cells differentiation into connective tissue cells, fibroblasts, and stimulation of proliferation and collagen synthesis by host fibroblasts. A decrease in the volume of dense fibrous connective tissue due to scar reorganization at latter terms cannot not excluded.

Adipose-derived stromal cells for the reconstruction of a human vesical equivalent.

PubMed

Rousseau, Alexandre; Fradette, Julie; Bernard, Geneviève; Gauvin, Robert; Laterreur, Véronique; Bolduc, Stéphane

2015-11-01

Despite a wide panel of tissue-engineering models available for vesical reconstruction, the lack of a differentiated urothelium remains their main common limitation. For the first time to our knowledge, an entirely human vesical equivalent, free of exogenous matrix, has been reconstructed using the self-assembly method. Moreover, we tested the contribution of adipose-derived stromal cells, an easily available source of mesenchymal cells featuring many potential advantages, by reconstructing three types of equivalent, named fibroblast vesical equivalent, adipose-derived stromal cell vesical equivalent and hybrid vesical equivalent--the latter containing both adipose-derived stromal cells and fibroblasts. The new substitutes have been compared and characterized for matrix composition and organization, functionality and mechanical behaviour. Although all three vesical equivalents displayed adequate collagen type I and III expression, only two of them, fibroblast vesical equivalent and hybrid vesical equivalent, sustained the development of a differentiated and functional urothelium. The presence of uroplakins Ib, II and III and the tight junction marker ZO-1 was detected and correlated with impermeability. The mechanical resistance of these tissues was sufficient for use by surgeons. We present here in vitro tissue-engineered vesical equivalents, built without the use of any exogenous matrix, able to sustain mechanical stress and to support the formation of a functional urothelium, i.e. able to display a barrier function similar to that of native tissue. Copyright © 2013 John Wiley & Sons, Ltd.

TGF-β induction of FGF-2 expression in stromal cells requires integrated smad3 and MAPK pathways.

PubMed

Strand, Douglas W; Liang, Yao-Yun; Yang, Feng; Barron, David A; Ressler, Steven J; Schauer, Isaiah G; Feng, Xin-Hua; Rowley, David R

2014-01-01

Transforming Growth Factor-β (TGF-β) regulates the reactive stroma microenvironment associated with most carcinomas and mediates expression of many stromal derived factors important for tumor progression, including FGF-2 and CTGF. TGF-β is over-expressed in most carcinomas, and FGF-2 action is important in tumor-induced angiogenesis. The signaling mechanisms of how TGF-β regulates FGF-2 expression in the reactive stroma microenvironment are not understood. Accordingly, we have assessed key signaling pathways that mediate TGF-β1-induced FGF-2 expression in prostate stromal fibroblasts and mouse embryo fibroblasts (MEFs) null for Smad2 and Smad3. TGF-β1 induced phosphorylation of Smad2, Smad3, p38 and ERK1/2 proteins in both control MEFs and prostate fibroblasts. Of these, Smad3, but not Smad2 was found to be required for TGF-β1 induction of FGF-2 expression in stromal cells. ChIP analysis revealed a Smad3/Smad4 complex was associated with the -1.9 to -2.3 kb upstream proximal promoter of the FGF-2 gene, further suggesting a Smad3-specific regulation. In addition, chemical inhibition of p38 or ERK1/2 MAPK activity also blocked TGF-β1-induced FGF-2 expression in a Smad3-independent manner. Conversely, inhibition of JNK signaling enhanced FGF-2 expression. Together, these data indicate that expression of FGF-2 in fibroblasts in the tumor stromal cell microenvironment is coordinately dependent on both intact Smad3 and MAP kinase signaling pathways. These pathways and key downstream mediators of TGF-β action in the tumor reactive stroma microenvironment, may evolve as putative targets for therapeutic intervention.

Fibroblast-mediated in vivo and in vitro growth promotion of tumorigenic rat thyroid carcinoma cells but not normal Fisher rat thyroid follicular cells.

PubMed

Saitoh, Ohki; Mitsutake, Norisato; Nakayama, Toshiyuki; Nagayama, Yuji

2009-07-01

It is known that genetic abnormalities in oncogenes and/or tumor suppressor genes promote carcinogenesis. Numerous recent articles, however, have demonstrated that epithelial-stromal interaction also plays a critical role for initiation and progression of carcinoma cells. Furthermore, ionizing radiation induces alterations in the tissue microenvironments that promote carcinogenesis. There is little or no information on epithelial-stromal interaction in thyroid carcinoma cells. The objective of this study was to determine if epithelial-stromal interaction influenced the growth of thyroid carcinoma cells in vivo and in vitro and to determine if radiation had added or interacting effects. Normal Fisher rat thyroid follicular cells (FRTL5 cells) and tumorigenic rat thyroid carcinoma cells (FRTL-Tc cells) derived from FRTL5 cells were employed. The cells were injected into thyroids or subcutaneously into left flanks of rats alone or in combination with skin-derived fibroblasts. In groups of rats, fibroblasts were irradiated with 0.1 or 4 Gy x-ray 3 days before inoculation. In vitro growth of FRTL-Tc and FRTL-5 cells were evaluated using the fibroblast-conditioned medium and in a co-culture system with fibroblasts. The in vivo experiments demonstrated that FRTL-Tc cells injected intrathyroidally grew faster than those injected subcutaneously, and that admixed fibroblasts enhanced growth of subcutaneous FRTL-Tc tumors, indicating that the intrathyroidal milieu, particularly in the presence of fibroblasts, confer growth-promoting advantage to thyroid carcinoma cells. This in vivo growth-promoting effect of fibroblasts on FRTL-Tc cells was duplicated in the in vitro experiments using the fibroblast-conditioned medium. Thus, our data demonstrate that this effect is mediated by soluble factor(s), is reversible, and is comparable to that of 10% fetal bovine serum. However, normal FRTL5 cells did not respond to the fibroblast-conditioned medium. Furthermore, high- and low

Marginal reticular cells: a stromal subset directly descended from the lymphoid tissue organizer

PubMed Central

Katakai, Tomoya

2012-01-01

The architecture of secondary lymphoid organs (SLOs) is supported by several non-hematopoietic stromal cells. Currently it is established that two distinct stromal subsets, follicular dendritic cells and fibroblastic reticular cells, play crucial roles in the formation of tissue compartments within SLOs, i.e., the follicle and T zone, respectively. Although stromal cells in the anlagen are essential for SLO development, the relationship between these primordial cells and the subsets in adulthood remains poorly understood. In addition, the roles of stromal cells in the entry of antigens into the compartments through some tissue structures peculiar to SLOs remain unclear. A recently identified stromal subset, marginal reticular cells (MRCs), covers the margin of SLOs that are primarily located in the outer edge of follicles and construct a unique reticulum. MRCs are closely associated with specialized endothelial or epithelial structures for antigen transport. The similarities in marker expression profiles and successive localization during development suggest that MRCs directly descend from organizer stromal cells in the anlagen. Therefore, MRCs are thought to be a crucial stromal component for the organization and function of SLOs. PMID:22807928

Understanding tumor-stroma interplays for targeted therapies by armed mesenchymal stromal progenitors: the Mesenkillers

PubMed Central

Grisendi, Giulia; Bussolari, Rita; Veronesi, Elena; Piccinno, Serena; Burns, Jorge S; De Santis, Giorgio; Loschi, Pietro; Pignatti, Marco; Di Benedetto, Fabrizio; Ballarin, Roberto; Di Gregorio, Carmela; Guarneri, Valentina; Piccinini, Lino; Horwitz, Edwin M; Paolucci, Paolo; Conte, PierFranco; Dominici, Massimo

2011-01-01

A tumor represents a complex structure containing malignant cells strictly coupled with a large variety of surrounding cells constituting the tumor stroma (TS). In recent years, the importance of TS for cancer initiation, development, local invasion and metastases has become increasingly clear allowing the identification of TS as one of the possible ways to indirectly target tumors. Inside the heterogeneous stromal cell population, tumor associated fibroblasts (TAF) play a crucial role providing both functional and supportive environments. During both tumor and stroma development, several findings suggest that TAF could be recruited from different sources such as locally derived host fibroblasts, via epithelial/endothelial mesenchymal transitions or from circulating pools of fibroblasts deriving form mesenchymal progenitors, namely mesenchymal stem/stromal cells (MSC). These insights prompted scientists to identify multimodal approaches to target TS by biomolecules, monoclonal antibodies, and more recently via cell based strategies. These latter strategies appear extremely promising, although still associated with debated and unclear findings. This review discusses crosstalk between cancers and their stroma, dissecting specific tumor types, such as sarcoma, pancreatic and breast carcinoma, where stroma plays distinct paradigmatic roles. The recognition of these distinct stromal functions may help in planning effective and safer approaches aimed either to eradicate or to substitute TS by novel compounds and/or MSC having specific killing activities. PMID:22016827

LRRC15 is a novel mesenchymal protein and stromal target for antibody-drug conjugates.

PubMed

Purcell, James W; Tanlimco, Sonia G; Hickson, Jonathan A; Fox, Melvin; Sho, Mien; Durkin, Lisa; Uziel, Tamar; Powers, Rick; Foster-Duke, Kelly D; McGonigal, Thomas; Kumar, Subashri; Samayoa, Josue; Zhang, Dong; Palma, Joann P; Mishra, Sasmita; Hollenbaugh, Diane; Gish, Kurt; Morgan-Lappe, Susan E; Hsi, Eric D; Chao, Debra T

2018-05-15

Progress in understanding tumor stromal biology has been constrained in part because cancer-associated fibroblasts (CAF) are a heterogeneous population with limited cell type-specific protein markers. Using RNA expression profiling, we identified the membrane protein leucine rich repeat containing 15 (LRRC15) as highly expressed in multiple solid tumor indications with limited normal tissue expression. LRRC15 was expressed on stromal fibroblasts in many solid tumors (e.g., breast, head and neck, lung, pancreatic) as well as directly on a subset of cancer cells of mesenchymal origin (e.g., sarcoma, melanoma, glioblastoma). LRRC15 expression was induced by TGFβ on activated fibroblasts (αSMA+) as well as on mesenchymal stem cells (MSC). These collective findings suggested LRRC15 as a novel CAF and mesenchymal marker with utility as a therapeutic target for the treatment of cancers with LRRC15-positive stromal desmoplasia or cancers of mesenchymal origin. ABBV-085 is a monomethyl auristatin E (MMAE)-containing antibody-drug conjugate (ADC) directed against LRRC15 and it demonstrated robust preclinical efficacy against LRRC15 stromal-positive/cancer-negative, and LRRC15 cancer-positive models as a monotherapy, or in combination with standard of care therapies. ABBV-085's unique mechanism of action relied upon the cell-permeable properties of MMAE to preferentially kill cancer cells over LRRC15-positive CAF while also increasing immune infiltrate (e.g., F4/80+ macrophages) in the tumor microenvironment. In summary, these findings validate LRRC15 as a novel therapeutic target in multiple solid tumor indications and support the ongoing clinical development of the LRRC15-targeted ADC ABBV-085. Copyright ©2018, American Association for Cancer Research.

Carbon Monoxide Improves Efficacy of Mesenchymal Stromal Cells During Sepsis by Production of Specialized Proresolving Lipid Mediators.

PubMed

Tsoyi, Konstantin; Hall, Sean R R; Dalli, Jesmond; Colas, Romain A; Ghanta, Sailaja; Ith, Bonna; Coronata, Anna; Fredenburgh, Laura E; Baron, Rebecca M; Choi, Augustine M K; Serhan, Charles N; Liu, Xiaoli; Perrella, Mark A

2016-12-01

Mesenchymal stromal cells are being investigated as a cell-based therapy for a number of disease processes, with promising results in animal models of systemic inflammation and sepsis. Studies are ongoing to determine ways to further improve the therapeutic potential of mesenchymal stromal cells. A gas molecule that improves outcome in experimental sepsis is carbon monoxide. We hypothesized that preconditioning of mesenchymal stromal cells with carbon monoxide ex vivo would promote further therapeutic benefit when cells are administered in vivo after the onset of polymicrobial sepsis in mice. Animal study and primary cell culture. Laboratory investigation. BALB/c mice. Polymicrobial sepsis was induced by cecal ligation and puncture. Mesenchymal stromal cells, mesenchymal stromal cells-conditioned with carbon monoxide, fibroblasts, or fibroblasts-conditioned with carbon monoxide were delivered by tail vein injections to septic mice. The mice were assessed for survival, bacterial clearance, and the inflammatory response during sepsis in each of the groups. Mesenchymal stromal cells were also assessed for their ability to promote bacterial phagocytosis by neutrophils, the production of specialized proresolving lipid mediators, and their importance for mesenchymal stromal cells function using gene silencing. Ex vivo preconditioning with carbon monoxide allowed mesenchymal stromal cells to be administered later after the onset of sepsis (6 hr), and yet maintain their therapeutic effect with increased survival. Carbon monoxide preconditioned mesenchymal stromal cells were also able to alleviate organ injury, improve bacterial clearance, and promote the resolution of inflammation. Mesenchymal stromal cells exposed to carbon monoxide, with docosahexaenoic acid substrate, produced specialized proresolving lipid mediators, particularly D-series resolvins, which promoted survival. Silencing of lipoxygenase pathways (5-lipoxygenase and 12/15-lipoxygenase), which are

Know thy neighbor: stromal cells can contribute oncogenic signals

NASA Technical Reports Server (NTRS)

Tlsty, T. D.; Hein, P. W.

2001-01-01

Although the stroma within carcinogenic lesions is known to be supportive and responsive to tumors, new data increasingly show that the stroma also has a more active, oncogenic role in tumorigenesis. Stromal cells and their products can transform adjacent tissues in the absence of pre-existing tumor cells by inciting phenotypic and genomic changes in the epithelial cells. The oncogenic action of distinctive stromal components has been demonstrated through a variety of approaches, which provide clues about the cellular pathways involved.

Epithelial-stromal interface in normal and neoplastic human bladder epithelium.

PubMed

Alroy, J; Gould, V E

1980-01-01

The ultrastructure of the epithelial-stromal interface of the human urinary bladder was studied in biopsy specimens that included 7 normal controls, 1 inverted papilloma, 18 noninvasive papillary carcinomas, and 19 invasive transitional cell carcinomas. In the invasive foci of the transitional cell carcinomas, the underlying basal lamina was attenuated or absent and the number of hemidesmosomes was decreased. These neoplastic cells displayed notably increased numbers of lysosomes, some of which appeared to be in the process of exocytosis. Increased numbers of cytoplasmic filaments adjacent to the plasma membranes at the invading pole of these cells were also observed. Tight junctions and junctional complexes were noticed adjacent to the tumor-stromal interface. None of the aforementioned features was observed in normal transitional epithelium, in inverted papilloma, in noninvasive papillary carcinomas, or in the noninvasive portions of invasive transitional cell carcinomas. Alterations of the epithelial-stromal interface deserve additional studies for they may constitute important parameters in the evaluation of actual or potential invasiveness in the various types of carcinoma of the bladder.

Carbon nanotubes functionalized with fibroblast growth factor accelerate proliferation of bone marrow-derived stromal cells and bone formation

NASA Astrophysics Data System (ADS)

Hirata, Eri; Ménard-Moyon, Cécilia; Venturelli, Enrica; Takita, Hiroko; Watari, Fumio; Bianco, Alberto; Yokoyama, Atsuro

2013-11-01

Multi-walled carbon nanotubes (MWCNTs) were functionalized with fibroblast growth factor (FGF) and the advantages of their use as scaffolds for bone augmentation were evaluated in vitro and in vivo. The activity of FGF was assessed by measuring the effect on the proliferation of rat bone marrow stromal cells (RBMSCs). The presence of FGF enhanced the proliferation of RBMSCs and the FGF covalently conjugated to the nanotubes (FGF-CNT) showed the same effect as FGF alone. In addition, FGF-CNT coated sponges were implanted between the parietal bone and the periosteum of rats and the formation of new bone was investigated. At day 14 after implantation, a larger amount of newly formed bone was clearly observed in most pores of FGF-CNT coated sponges. These findings indicated that MWCNTs accelerated new bone formation in response to FGF, as well as the integration of particles into new bone during its formation. Scaffolds coated with FGF-CNT could be considered as promising novel substituting materials for bone regeneration in future tissue engineering applications.

Inhibition of Breast Cancer Progression by Blocking Heterocellular Contact Between Epithelial Cells and Fibroblasts

DTIC Science & Technology

2013-04-01

by employing a microfluidic -based compartmentalized 3D co-culture platform enabling both contact-free and contact-associated co-cultures. 15...SUBJECT TERMS Heterocellular contact between cancer cells and stromal fibroblasts, Microfluidics , 3D 16. SECURITY CLASSIFICATION OF: 17. LIMITATION...and human mammary fibroblasts (HMFs) in breast cancer progression by employing a microfluidic - based compartmentalized 3D co-culture platform

Wound-healing potential of human umbilical cord blood-derived mesenchymal stromal cells in vitro--a pilot study.

PubMed

You, Hi-Jin; Namgoong, Sik; Han, Seung-Kyu; Jeong, Seong-Ho; Dhong, Eun-Sang; Kim, Woo-Kyung

2015-11-01

Our previous studies demonstrated that human bone marrow-derived mesenchymal stromal cells have great potential for wound healing. However, it is difficult to clinically utilize cultured stem cells. Recently, human umbilical cord blood-derived mesenchymal stromal cells (hUCB-MSCs) have been commercialized for cartilage repair as a first cell therapy product that uses allogeneic stem cells. Should hUCB-MSCs have a superior effect on wound healing as compared with fibroblasts, which are the main cell source in current cell therapy products for wound healing, they may possibly replace fibroblasts. The purpose of this in vitro study was to compare the wound-healing activity of hUCB-MSCs with that of fibroblasts. This study was particularly designed to compare the effect of hUCB-MSCs on diabetic wound healing with those of allogeneic and autologous fibroblasts. Healthy (n = 5) and diabetic (n = 5) fibroblasts were used as the representatives of allogeneic and autologous fibroblasts for diabetic patients in the control group. Human UCB-MSCs (n = 5) were used in the experimental group. Cell proliferation, collagen synthesis and growth factor (basic fibroblast growth factor, vascular endothelial growth factor and transforming growth factor-β) production were compared among the three cell groups. Human UCB-MSCs produced significantly higher amounts of vascular endothelial growth factor and basic fibroblast growth factor when compared with both fibroblast groups. Human UCB-MSCs were superior to diabetic fibroblasts but not to healthy fibroblasts in collagen synthesis. There were no significant differences in cell proliferation and transforming growth factor-β production. Human UCB-MSCs may have greater capacity for diabetic wound healing than allogeneic or autologous fibroblasts, especially in angiogenesis. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

The corneal fibrosis response to epithelial-stromal injury

PubMed Central

Torricelli, Andre A. M.; Santhanam, Abirami; Wu, Jiahui; Singh, Vivek; Wilson, Steven E.

2014-01-01

The corneal wound healing response, including the development of stromal opacity in some eyes, is a process that often leads to scarring that occurs after injury, surgery or infection to the cornea. Immediately after epithelial and stromal injury, a complex sequence of processes contributes to wound repair and regeneration of normal corneal structure and function. In some corneas, however, often depending on the type and extent of injury, the response may also lead to the development of mature vimentin+ α-smooth muscle actin+ desmin+ myofibroblasts. Myofibroblasts are specialized fibroblastic cells generated in the cornea from keratocyte-derived or bone marrow-derived precursor cells. The disorganized extracellular matrix components secreted by myofibroblasts, in addition to decreased expression of corneal crystallins in these cells, are central biological processes that result in corneal stromal fibrosis associated with opacity or “haze”. Several factors are associated with myofibroblast generation and haze development after PRK surgery in rabbits, a reproducible model of scarring, including the amount of tissue ablated, which may relate to the extent of keratocyte apoptosis in the early response to injury, irregularity of stromal surface after surgery, and changes on corneal stromal proteoglycans, but normal regeneration of the epithelial basement membrane (EBM) appears to be a critical factor determining whether a cornea heals with relative transparency or vision-limiting stromal opacity. Structural and functional abnormalities of the regenerated EBM facilitate prolonged entry of epithelium-derived growth factors such as transforming growth factor β (TGF-β) and platelet-derived growth factor (PDGF) into the stroma that both drive development of mature myofibroblasts from precursor cells and lead to persistence of the cells in the anterior stroma. A major discovery that has contributed to our understanding of haze development is that keratocytes and

SDF-1 in Mammary Fibroblasts of Bovine with Mastitis Induces EMT and Inflammatory Response of Epithelial Cells.

PubMed

He, Guiliang; Ma, Mengru; Yang, Wei; Wang, Hao; Zhang, Yong; Gao, Ming-Qing

2017-01-01

Fibroblasts constitute the majority of the stromal cells within bovine mammary gland, yet the functional contributions of these cells to mastitis and fibrosis and the mechanism are poorly understood. In this study, we demonstrate that inflammation-associated fibroblasts (INFs) extracted from bovine mammary glands with clinical mastitis had different expression pattern regarding to several extracellular matrix (ECM) proteins, chemokines and cytokines compared to normal fibroblasts (NFs) from dairy cows during lactation. The INFs induced epithelial-mesenchymal transition (EMT) and inflammatory responses of mammary epithelial cells in a vitro co-culture model. These functional contributions of INFs to normal epithelial cells were mediated through their ability to secrete stromal cell-derived factor 1 (SDF-1). SDF-1 was highly secreted/expressed by INFs, lipopolysaccharide (LPS) -treated NFs, lipoteichoic acid (LTA) -treated NFs, as well as mastitic tissue compared to their counterparts. Exogenous SDF-1 promoted EMT on epithelial cells through activating NF-κB pathway, induced inflammation response and inhibited proliferation of epithelial cells. In addition, SDF-1 was able to induce mastitis and slight fibrosis of mouse mammary gland, which was attenuated by a specific inhibitor of the receptor of SDF-1. Our findings indicate that stromal fibroblasts within mammary glands with mastitis contribute to EMT and inflammatory responses of epithelial cells through the secretion of SDF-1, which could result in the inflammation spread and fibrosis within mammary gland.

Loss of Stromal Caveolin-1 Expression: A Novel Tumor Microenvironment Biomarker That Can Predict Poor Clinical Outcomes for Pancreatic Cancer

PubMed Central

Shan, Tao; Lu, Hongwei; Ji, Hong; Li, Yiming; Guo, Jian; Chen, Xi; Wu, Tao

2014-01-01

Aims Cancer development and progression is not only associated with the tumor cell proliferation but also depends on the interaction between tumor cells and the stromal microenvironment. A new understanding of the role of the tumor microenvironment suggests that the loss of stromal caveolin-1 (Cav-1) as a key regulator may become a potential therapy target. This study aims to elucidate whether stromal Cav-1 expression in pancreatic cancer can be a strong prognosis biomarker. Methods Tissue samples from 45 pancreatic cancer patients were studied. Parenchyma and stroma were separated and purified using laser capture microdissection. Stromal Cav-1 expression was measured from pancreatic cancer, paraneoplastic, and normal tissue using immunohistochemistry. We analyzed the correlation of stromal Cav-1 expression with clinicopathologic features and prognostic indicators, such as tumor marker HER-2/neu gene. Results Specimens from six patients (13.3%) showed high levels of stromal Cav-1 staining, those from eight patients (17.8%) showed a lower, intermediate level of staining, whereas those from 31 patients (68.9%) showed an absence of staining. Cav-1 expression in cancer-associated fibroblasts was lower than that in paracancer-associated and in normal fibroblasts. Stromal Cav-1 loss was associated with TNM stage (P = 0.018), lymph node metastasis (P = 0.014), distant metastasis (P = 0.027), and HER-2/neu amplification (P = 0.007). The relationships of age, sex, histological grade, and tumor size with stromal Cav-1 expression were not significant (P>0.05). A negative correlation was found between circulating tumor cells and stromal Cav-1 expression (P<0.05). Conclusion The loss of stromal Cav-1 in pancreatic cancer was an independent prognostic indicator, thus suggesting that stromal Cav-1 may be an effective therapeutic target for patients with pancreatic cancer. PMID:24949874

Stromal Androgen Receptor in Prostate Cancer Development and Progression

PubMed Central

Leach, Damien A.; Buchanan, Grant

2017-01-01

Prostate cancer development and progression is the result of complex interactions between epithelia cells and fibroblasts/myofibroblasts, in a series of dynamic process amenable to regulation by hormones. Whilst androgen action through the androgen receptor (AR) is a well-established component of prostate cancer biology, it has been becoming increasingly apparent that changes in AR signalling in the surrounding stroma can dramatically influence tumour cell behavior. This is reflected in the consistent finding of a strong association between stromal AR expression and patient outcomes. In this review, we explore the relationship between AR signalling in fibroblasts/myofibroblasts and prostate cancer cells in the primary site, and detail the known functions, actions, and mechanisms of fibroblast AR signaling. We conclude with an evidence-based summary of how androgen action in stroma dramatically influences disease progression. PMID:28117763

Stromal Fibroblasts Induce CCL20 through IL6/C/EBPβ to Support the Recruitment of Th17 Cells during Cervical Cancer Progression.

PubMed

Walch-Rückheim, Barbara; Mavrova, Russalina; Henning, Melanie; Vicinus, Benjamin; Kim, Yoo-Jin; Bohle, Rainer Maria; Juhasz-Böss, Ingolf; Solomayer, Erich-Franz; Smola, Sigrun

2015-12-15

Cervical cancer is a consequence of persistent infection with human papillomaviruses (HPV). Progression to malignancy is linked to an inflammatory microenvironment comprising T-helper-17 (Th17) cells, a T-cell subset with protumorigenic properties. Neoplastic cells express only low endogenous levels of the Th17 chemoattractant CCL20, and therefore, it is unclear how Th17 cells are recruited to the cervical cancer tissue. In this study, we demonstrate that CCL20 was predominantly expressed in the stroma of cervical squamous cell carcinomas in situ. This correlated with stromal infiltration of CD4(+)/IL17(+) cells and with advancing International Federation of Gynecology and Obstetrics (FIGO) stage. Furthermore, we show that cervical cancer cells instructed primary cervical fibroblasts to produce high levels of CCL20 and to attract CD4/IL17/CCR6-positive cells, generated in vitro, in a CCL20/CCR6-dependent manner. Further mechanistic investigations identified cervical cancer cell-derived IL6 as an important mediator of paracrine CCL20 induction at the promoter, mRNA, and protein level in fibroblasts. CCL20 was upregulated through the recently described CCAAT/enhancer-binding protein β (C/EBPβ) pathway as shown with a dominant-negative version of C/EBPβ and through siRNA-mediated knockdown. In summary, our study defines a novel molecular mechanism by which cervical neoplastic cells shape their local microenvironment by instructing fibroblasts to support Th17 cell infiltration in a paracrine IL6/C/EBPβ-dependent manner. Th17 cells may in turn maintain chronic inflammation within high-grade cervical lesions to further promote cancer progression. ©2015 American Association for Cancer Research.

Comparison of human umbilical cord blood-derived mesenchymal stem cells with healthy fibroblasts on wound-healing activity of diabetic fibroblasts.

PubMed

Jung, Jae-A; Yoon, Young-Don; Lee, Hyup-Woo; Kang, So-Ra; Han, Seung-Kyu

2018-02-01

Various types of skin substitutes composed of fibroblasts and/or keratinocytes have been used for the treatment of diabetic ulcers. However, the effects have generally not been very dramatic. Recently, human umbilical cord blood-derived mesenchymal stromal cells (hUCB-MSCs) have been commercialised for cartilage repair as a first cell therapy product using allogeneic stem cells. In a previous pilot study, we reported that hUCB-MSCs have a superior wound-healing capability compared with fibroblasts. The present study was designed to compare the treatment effect of hUCB-MSCs with that of fibroblasts on the diabetic wound healing in vitro. Diabetic fibroblasts were cocultured with healthy fibroblasts or hUCB-MSCs. Five groups were evaluated: group I, diabetic fibroblasts without coculture; groups II and III, diabetic fibroblasts cocultured with healthy fibroblasts or hUCB-MSCs; and groups IV and V, no cell cocultured with healthy fibroblasts or hUCB-MSCs. After a 3-day incubation, cell proliferation, collagen synthesis levels and glycosaminoglycan levels, which are the major contributing factors in wound healing, were measured. As a result, a hUCB-MSC-treated group showed higher cell proliferation, collagen synthesis and glycosaminoglycan level than a fibroblast-treated group. In particular, there were significant statistical differences in collagen synthesis and glycosaminoglycan levels (P = 0·029 and P = 0·019, respectively). In conclusion, these results demonstrate that hUCB-MSCs may have a superior effect to fibroblasts in stimulating diabetic wound healing. © 2017 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

Fibroblast growth factor receptor mediates fibroblast-dependent growth in EMMPRIN-depleted head and neck cancer tumor cells.

PubMed

Liu, Zhiyong; Hartman, Yolanda E; Warram, Jason M; Knowles, Joseph A; Sweeny, Larissa; Zhou, Tong; Rosenthal, Eben L

2011-08-01

Head and neck squamous cell carcinoma tumors (HNSCC) contain a dense fibrous stroma which is known to promote tumor growth, although the mechanism of stroma-mediated growth remains unclear. As dysplastic mucosal epithelium progresses to cancer, there is incremental overexpression of extracellular matrix metalloprotease inducer (EMMPRIN) which is associated with tumor growth and metastasis. Here, we present evidence that gain of EMMPRIN expression allows tumor growth to be less dependent on fibroblasts by modulating fibroblast growth factor receptor-2 (FGFR2) signaling. We show that silencing EMMPRIN in FaDu and SCC-5 HNSCC cell lines inhibits cell growth, but when EMMPRIN-silenced tumor cells were cocultured with fibroblasts or inoculated with fibroblasts into severe combined immunodeficient mice, the growth inhibition by silencing EMMPRIN was blunted by the presence of fibroblasts. Coculture experiments showed fibroblast-dependent tumor cell growth occurred via a paracrine signaling. Analysis of tumor gene expression revealed expression of FGFR2 was inversely related to EMMPRIN expression. To determine the role of FGFR2 signaling in EMMPRIN-silenced tumor cells, ligands and inhibitors of FGFR2 were assessed. Both FGF1 and FGF2 enhanced tumor growth in EMMPRIN-silenced cells compared with control vector-transfected cells, whereas inhibition of FGFR2 with blocking antibody or with a synthetic inhibitor (PD173074) inhibited tumor cell growth in fibroblast coculture, suggesting the importance of FGFR2 signaling in fibroblast-mediated tumor growth. Analysis of xenografted tumors revealed that EMMPRIN-silenced tumors had a larger stromal compartment compared with control. Taken together, these results suggest that EMMPRIN acquired during tumor progression promotes fibroblast-independent tumor growth.

Fibroblast growth factor receptor mediates fibroblast-dependent growth in EMMPRIN depleted head and neck cancer tumor cells

PubMed Central

Liu, Zhiyong; Hartman, Yolanda E.; Warram, Jason M.; Knowles, Joseph A.; Sweeny, Larrisa; Zhou, Tong; Rosenthal, Eben L.

2011-01-01

Head and neck squamous cell carcinoma tumors (HNSCC) contain a dense fibrous stroma which is known to promote tumor growth, although the mechanism of stroma mediated growth remains unclear. As dysplastic mucosal epithelium progresses to cancer there is incremental overexpression of extracellular matrix metalloprotease inducer (EMMPRIN) which is associated with tumor growth and metastasis. Here we present evidence that gain of EMMPRIN expression allows tumor growth to be less dependent on fibroblasts by modulating fibroblast growth factor receptor-2 (FGFR2) signaling. We show that silencing EMMPRIN in FaDu and SCC-5 HNSCC cell lines inhibits cell growth, but when EMMPRIN-silenced tumor cells were co-cultured with fibroblasts or inoculated with fibroblasts into SCID mice, the growth inhibition by silencing EMMPRIN was blunted by the presence of fibroblasts. Co-culture experiments demonstrated fibroblast-dependent tumor cell growth occurred via a paracrine signaling. Analysis of tumor gene expression revealed expression of FGFR2 was inversely related to EMMPRIN expression. To determine the role of FGFR2 signaling in EMMPRIN silenced tumor cells, ligands and inhibitors of FGFR2 were assessed. Both FGF1 and FGF2 enhanced tumor growth in EMMPRIN silenced cells compared to control vector transfected cells, while inhibition of FGFR2 with blocking antibody or with a synthetic inhibitor (PD173074) inhibited tumor cell growth in fibroblast co-culture, suggesting the importance of FGFR2 signaling in fibroblast mediated tumor growth. Analysis of xenografted tumors revealed EMMPRIN silenced tumors had a larger stromal compartment compared to control. Taken together, these results suggest that EMMPRIN acquired during tumor progression promotes fibroblast independent tumor growth. PMID:21665938

FAP-overexpressing fibroblasts produce an extracellular matrix that enhances invasive velocity and directionality of pancreatic cancer cells

PubMed Central

2011-01-01

Background Alterations towards a permissive stromal microenvironment provide important cues for tumor growth, invasion, and metastasis. In this study, Fibroblast activation protein (FAP), a serine protease selectively produced by tumor-associated fibroblasts in over 90% of epithelial tumors, was used as a platform for studying tumor-stromal interactions. We tested the hypothesis that FAP enzymatic activity locally modifies stromal ECM (extracellular matrix) components thus facilitating the formation of a permissive microenvironment promoting tumor invasion in human pancreatic cancer. Methods We generated a tetracycline-inducible FAP overexpressing fibroblastic cell line to synthesize an in vivo-like 3-dimensional (3D) matrix system which was utilized as a stromal landscape for studying matrix-induced cancer cell behaviors. A FAP-dependent topographical and compositional alteration of the ECM was characterized by measuring the relative orientation angles of fibronectin fibers and by Western blot analyses. The role of FAP in the matrix-induced permissive tumor behavior was assessed in Panc-1 cells in assorted matrices by time-lapse acquisition assays. Also, FAP+ matrix-induced regulatory molecules in cancer cells were determined by Western blot analyses. Results We observed that FAP remodels the ECM through modulating protein levels, as well as through increasing levels of fibronectin and collagen fiber organization. FAP-dependent architectural/compositional alterations of the ECM promote tumor invasion along characteristic parallel fiber orientations, as demonstrated by enhanced directionality and velocity of pancreatic cancer cells on FAP+ matrices. This phenotype can be reversed by inhibition of FAP enzymatic activity during matrix production resulting in the disorganization of the ECM and impeded tumor invasion. We also report that the FAP+ matrix-induced tumor invasion phenotype is β1-integrin/FAK mediated. Conclusion Cancer cell invasiveness can be affected by

Understanding the "lethal" drivers of tumor-stroma co-evolution: emerging role(s) for hypoxia, oxidative stress and autophagy/mitophagy in the tumor micro-environment.

PubMed

Lisanti, Michael P; Martinez-Outschoorn, Ubaldo E; Chiavarina, Barbara; Pavlides, Stephanos; Whitaker-Menezes, Diana; Tsirigos, Aristotelis; Witkiewicz, Agnieszka; Lin, Zhao; Balliet, Renee; Howell, Anthony; Sotgia, Federica

2010-09-15

We have recently proposed a new model for understanding how tumors evolve. To achieve successful "Tumor-Stroma Co-Evolution", cancer cells induce oxidative stress in adjacent fibroblasts and possibly other stromal cells. Oxidative stress in the tumor stroma mimics the effects of hypoxia, under aerobic conditions, resulting in an excess production of reactive oxygen species (ROS). Excess stromal production of ROS drives the onset of an anti-oxidant defense in adjacent cancer cells, protecting them from apoptosis. Moreover, excess stromal ROS production has a "Bystander-Effect", leading to DNA damage and aneuploidy in adjacent cancer cells, both hallmarks of genomic instability. Finally, ROS-driven oxidative stress induces autophagy and mitophagy in the tumor micro-environment, leading to the stromal over-production of recycled nutrients (including energy-rich metabolites, such as ketones and L-lactate). These recycled nutrients or chemical building blocks then help drive mitochondrial biogenesis in cancer cells, thereby promoting the anabolic growth of cancer cells (via an energy imbalance). We also show that ketones and lactate help "fuel" tumor growth and cancer cell metastasis and can act as chemo-attractants for cancer cells. We have termed this new paradigm for accelerating tumor-stroma co-evolution, "The Autophagic Tumor Stroma Model of Cancer Cell Metabolism". Heterotypic signaling in cancer-associated fibroblasts activates the transcription factors HIF1alpha and NFκB, potentiating the onset of hypoxic and inflammatory response(s), which further upregulates the autophagic program in the stromal compartment. Via stromal autophagy, this hypoxic/inflammatory response may provide a new escape mechanism for cancer cells during anti-angiogenic therapy, further exacerbating tumor recurrence and metastasis.

Inhibition by rebamipide of cytokine-induced or lipopolysaccharide-induced chemokine synthesis in human corneal fibroblasts.

PubMed

Fukuda, Ken; Ishida, Waka; Tanaka, Hiroshi; Harada, Yosuke; Fukushima, Atsuki

2014-12-01

The dry-eye drug rebamipide has mucin secretagogue activity in and anti-inflammatory effects on corneal epithelial cells. Corneal stromal fibroblasts (transdifferentiated keratocytes) function as immune modulators in the pathogenesis of chronic ocular allergic inflammation and in innate immune responses at the ocular surface. The possible anti-inflammatory effects of rebamipide on human corneal stromal fibroblasts were examined. Serum-deprived cells were incubated for 1 h with rebamipide and then for various times in the additional absence or presence of cytokines or bacterial lipopolysaccharide (LPS). The release of chemokines into culture supernatants was determined with ELISAs. The intracellular abundance of chemokine mRNAs was quantitated by reverse transcription and real-time PCR analysis. Degradation of the nuclear factor κB (NFκB) inhibitor IκBα was detected by immunoblot analysis. Rebamipide suppressed the release of interleukin (IL)-8 and the upregulation of IL-8 mRNA induced by tumour necrosis factor α (TNF-α) or LPS in corneal fibroblasts. It also inhibited eotaxin-1 (CCL-11) expression at the protein and mRNA levels induced by the combination of TNF-α and IL-4. In addition, rebamipide attenuated the degradation of IκBα induced by TNF-α or LPS. Rebamipide inhibited the synthesis of chemokines by corneal fibroblasts in association with suppression of NFκB signalling. Rebamipide may therefore prove effective for the treatment of corneal stromal inflammation associated with allergy or bacterial infection. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Podoplanin-positive Cancer-associated Stromal Fibroblasts in Primary Tumor and Synchronous Lymph Node Metastases of HER2-overexpressing Breast Carcinomas.

PubMed

Niemiec, Joanna; Adamczyk, Agnieszka; Harazin-Lechowska, Agnieszka; Ambicka, Aleksandra; Grela-Wojewoda, Aleksandra; Majchrzyk, Kaja; Kruczak, Anna; Sas-Korczyńska, Beata; Ryś, Janusz

2018-04-01

We compared the status of stromal podoplanin-positive cancer-associated fibroblasts (ppCAFs) between primary tumors and paired synchronous lymph node metastases (LNMs) and analyzed the prognostic significance of tumoral ppCAFs in 203 patients with human epidermal growth factor receptor 2-positive breast carcinoma. ppCAFs were found in 167/203 and in 35/87 tumors and LNM, respectively. ppCAFs were most frequently found in tumors and corresponding LNM (n=52, 59.8%; p=0.001). However, for all LNMs (n=12) without ppCAFs, their paired tumors also lacked ppCAFs. In both tumors and LNMs, ppCAFs were α-smooth muscle actin-positive and cluster of differentiation 21 protein-negative, suggesting them not to be resident lymph node cells. Moreover, in our series, the presence of ppCAFs in tumors was borderline related to poor disease-free survival (p=0.058). These results speak in favor of a hypothesis suggesting ppCAFs accompany metastatic cancer cells migrating from tumor to LNMs. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Tumor grade and matrix metalloproteinase 2 expression in stromal fibroblasts help to stratify the high-risk group of patients with early breast cancer identified on the basis of st Gallen recommendations.

PubMed

Niemiec, Joanna; Adamczyk, Agnieszka; Małecki, Krzysztof; Ambicka, Aleksandra; Ryś, Janusz

2013-04-01

It is still being discussed if the assessment of basal markers or if adhesion molecules expression contributes additional prognostic information to the classic prognostic factors and hence should be included into standard morphologic reports. The aim of the study was to assess the prognostic significance of: (i) classification recommended by St Gallen experts (ii) tumor grade, expression of (iii) basal markers, (iv) adhesion molecules, and (v) matrix metalloproteinase 2 (MMP-2) in patients with T1-T2 N0M0 chemotherapy-naive ductal breast cancer. In 79 patients with tumors characterized by estrogen receptor (ER) and progesterone receptor (PgR) positive, human epidermal growth factor receptor 2 negative (HER2) phenotype and MIB-1 labeling index (MIB-l) LI ≤ 15% (low-risk group) cumulative 17-year breast cancer-specific survival probability was 100% and was significantly higher than in 95 patients from the high-risk group (ER(-)/PgR(-)/HER2(-) or HER2(+) or MIB-1 LI > 15%) (72.5%). We found that MMP-2 fibroblast expression indicated 2 subgroups with significantly different survival rates in women with grade 3 tumor (88.9% for MMP-2 positivity and 56.0% for negativity). Cox multivariate analysis revealed that both grade 3 combined with stromal fibroblast MMP-2(-) and a high-risk group according to St Gallen recommendations are independent negative prognostic factors that influence survival of patients with breast cancer. To the best of our knowledge, we have shown for the first time that MMP-2(-) in stromal fibroblasts might indicate poor survivors in the group of patients with grade 3 tumors and that the cumulative effect of both above-mentioned parameters might be helpful in selecting the high-risk individuals from the group of patients with luminal B subtype/HER2(+)/triple negative phenotype identified according to St Gallen recommendations. Copyright © 2013 Elsevier Inc. All rights reserved.

Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) promotes lung fibroblast proliferation, survival and differentiation to myofibroblasts.

PubMed

Hasaneen, Nadia A; Cao, Jian; Pulkoski-Gross, Ashleigh; Zucker, Stanley; Foda, Hussein D

2016-02-17

Idiopathic pulmonary fibrosis (IPF) is a chronic progressively fatal disease. Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) is a glycosylated transmembrane protein that induces the expression of some matrix metalloproteinase (MMP) in neighboring stromal cells through direct epithelial-stromal interactions. EMMPRIN is highly expressed in type II alveolar epithelial cells at the edges of the fibrotic areas in IPF lung sections. However, the exact role of EMMPRIN in IPF is unknown. To determine if EMMPRIN contributes to lung fibroblast proliferation, resistance to apoptosis, and differentiation to myofibroblasts, normal Human lung fibroblasts (NHLF) transiently transfected with either EMMPRIN/GFP or GFP were treated with TGF- β1 from 0 to 10 ng/ml for 48 h and examined for cell proliferation (thymidine incorporation), apoptosis (FACS analysis and Cell Death Detection ELISA assay), cell migration (Modified Boyden chamber) and differentiation to myofibroblasts using Western blot for α-smooth actin of cell lysates. The effect of EMMPRIN inhibition on NHLF proliferation, apoptosis, migration and differentiation to myofibroblasts after TGF- β1 treatment was examined using EMMPRIN blocking antibody. We examined the mechanism by which EMMPRIN induces its effects on fibroblasts by studying the β-catenin/canonical Wnt signaling pathway using Wnt luciferase reporter assays and Western blot for total and phosphorylated β-catenin. Human lung fibroblasts overexpressing EMMPRIN had a significant increase in cell proliferation and migration compared to control fibroblasts. Furthermore, EMMPRIN promoted lung fibroblasts resistance to apoptosis. Lung fibroblasts overexpressing EMMPRIN showed a significantly increased expression of α- smooth muscle actin, a marker of differentiation to myofibroblasts compared to control cells. TGF-β1 increased the expression of EMMPRIN in lung fibroblasts in a dose-dependent manner. Attenuation of EMMPRIN expression with the use of an

Visible red and infrared light alters gene expression in human marrow stromal fibroblast cells.

PubMed

Guo, J; Wang, Q; Wai, D; Zhang, Q Z; Shi, S H; Le, A D; Shi, S T; Yen, S L-K

2015-04-01

This study tested whether or not gene expression in human marrow stromal fibroblast (MSF) cells depends on light wavelength and energy density. Primary cultures of isolated human bone marrow stem cells (hBMSC) were exposed to visible red (VR, 633 nm) and infrared (IR, 830 nm) radiation wavelengths from a light emitting diode (LED) over a range of energy densities (0.5, 1.0, 1.5, and 2.0 Joules/cm2) Cultured cells were assayed for cell proliferation, osteogenic potential, adipogenesis, mRNA and protein content. mRNA was analyzed by microarray and compared among different wavelengths and energy densities. Mesenchymal and epithelial cell responses were compared to determine whether responses were cell type specific. Protein array analysis was used to further analyze key pathways identified by microarrays. Different wavelengths and energy densities produced unique sets of genes identified by microarray analysis. Pathway analysis pointed to TGF-beta 1 in the visible red and Akt 1 in the infrared wavelengths as key pathways to study. TGF-beta protein arrays suggested switching from canonical to non-canonical TGF-beta pathways with increases to longer IR wavelengths. Microarrays suggest RANKL and MMP 10 followed IR energy density dose-response curves. Epithelial and mesenchymal cells respond differently to stimulation by light suggesting cell type-specific response is possible. These studies demonstrate differential gene expression with different wavelengths, energy densities and cell types. These differences in gene expression have the potential to be exploited for therapeutic purposes and can help explain contradictory results in the literature when wavelengths, energy densities and cell types differ. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

High Numbers of Stromal Cancer-Associated Fibroblasts Are Associated With a Shorter Survival Time in Cats With Oral Squamous Cell Carcinoma.

PubMed

Klobukowska, H J; Munday, J S

2016-11-01

Cancer-associated fibroblasts (CAFs) are fibroblastic cells that express α-smooth muscle actin and have been identified in the stroma of numerous epithelial tumors. The presence of CAFs within the tumor stroma has been associated with a poorer prognosis in some human cancers, including oral squamous cell carcinomas (SCCs). Cats frequently develop oral SCCs, and although these are generally highly aggressive neoplasms, there is currently a lack of prognostic markers for these tumors. The authors investigated the prognostic value of the presence of CAFs within the stroma of oral SCC biopsy specimens from 47 cats. In addition, several epidemiologic, clinical, and histologic variables were also assessed for prognostic significance. A CAF-positive stroma was identified in 35 of 47 SCCs (74.5%), and the median survival time (ST) of cats with CAF-positive SCCs (35 days) was significantly shorter than that of cats with CAF-negative SCCs (48.5 days) (P = .031). ST was also associated with the location of the primary tumor (P = .0018): the median ST for oropharyngeal SCCs (179 days) was significantly longer than for maxillary (43.5 days; P = .047), mandibular (42 days; P = .022), and sublingual SCCs (22.5 days; P = .0005). The median ST of sublingual SCCs was also shorter compared with maxillary SCCs (P = .0017). Furthermore, a significant association was identified between site and the presence of stromal CAFs (P = .025). On the basis of this retrospective study, evaluating the tumor stroma for CAFs in feline oral SCC biopsy specimens may be of potential prognostic value. © The Author(s) 2016.

Cancer-cell-secreted exosomal miR-105 promotes tumour growth through the MYC-dependent metabolic reprogramming of stromal cells.

PubMed

Yan, Wei; Wu, Xiwei; Zhou, Weiying; Fong, Miranda Y; Cao, Minghui; Liu, Juan; Liu, Xiaojing; Chen, Chih-Hong; Fadare, Oluwole; Pizzo, Donald P; Wu, Jiawen; Liu, Liang; Liu, Xuxiang; Chin, Andrew R; Ren, Xiubao; Chen, Yuan; Locasale, Jason W; Wang, Shizhen Emily

2018-05-01

Cancer and other cells residing in the same niche engage various modes of interactions to synchronize and buffer the negative effects of environmental changes. Extracellular microRNAs (miRNAs) have recently been implicated in the intercellular crosstalk. Here we show a mechanistic model involving breast-cancer-secreted, extracellular-vesicle-encapsulated miR-105, which is induced by the oncoprotein MYC in cancer cells and, in turn, activates MYC signalling in cancer-associated fibroblasts (CAFs) to induce a metabolic program. This results in the capacity of CAFs to display different metabolic features in response to changes in the metabolic environment. When nutrients are sufficient, miR-105-reprogrammed CAFs enhance glucose and glutamine metabolism to fuel adjacent cancer cells. When nutrient levels are low and metabolic by-products accumulate, these CAFs detoxify metabolic wastes, including lactic acid and ammonium, by converting them into energy-rich metabolites. Thus, the miR-105-mediated metabolic reprogramming of stromal cells contributes to sustained tumour growth by conditioning the shared metabolic environment.

Crosstalk between stromal components and tumor cells of TNBC via secreted factors enhances tumor growth and metastasis

PubMed Central

Jin, Kideok; Pandey, Niranjan B.; Popel, Aleksander S.

2017-01-01

Triple negative breast cancer (TNBC) as a metastatic disease is currently incurable. Reliable and reproducible methods for testing drugs against metastasis are not available. Stromal cells may play a critical role in tumor progression and metastasis. In this study, we determined that fibroblasts and macrophages secreted IL-8 upon induction by tumor cell-conditioned media (TCM) from MDA-MB-231 cancer cells. Our data showed that the proliferation of MDA-MB-231 cells co-cultured with fibroblasts or macrophages was enhanced compared to the monoculture. Furthermore, TNBC cell migration, a key step in tumor metastasis, was promoted by conditioned media (CM) from TCM-induced fibroblasts or macrophages. Knockdown of the IL-8 receptor CXCR2 by CRISPR-Cas9 reduces MDA-MB-231 cell proliferation and migration compared to wild type. In a mouse xenograft tumor model, the growth of MDA-MB-231-CXCR2−/− tumor was significantly decreased compared to the growth of tumors from wild-type cells. In addition, the incidence of thoracic metastasis of MDA-MB-231-CXCR2−/− tumors was reduced compared to wild type. We found that the auto- and paracrine loop exists between TNBC cells and stroma, which results in enhanced IL-8 secretion from the stromal components. Significantly, inhibition of the IL-8 signaling pathway by reparixin, an inhibitor of the IL-8 receptor, CXCR1/2, reduced MDA-MB-231 tumor growth and metastasis. Taken together, these findings implicate IL-8 signaling as a critical event in TNBC tumor growth and metastasis via crosstalk with stromal components. PMID:28947965

Active Sonic Hedgehog Signaling between Androgen Independent Human Prostate Cancer Cells and Normal/Benign but Not Cancer-Associated Prostate Stromal Cells

PubMed Central

Shigemura, Katsumi; Huang, Wen-Chin; Li, Xiangyan; Zhau, Haiyen E.; Zhu, Guodong; Gotoh, Akinobu; Fujisawa, Masato; Xie, Jingwu; Marshall, Fray F.; Chung, Leland W. K.

2012-01-01

BACKGROUND Sonic hedgehog (Shh) signaling plays a pivotal role in stromal-epithelial interaction during normal development but its role in tumor-stromal interaction during carcinogenic progression is less well defined. Since hormone refractory prostate cancer with bone metastasis is difficult to treat, it is crucial to investigate how androgen independent (AI) human prostate cancer cells communicate with their associated stroma. METHODS Shh and its target transcription factor, Gli1 mRNA, were assessed by RT-PCR and/or quantitative RT-PCR in co-cultured cell recombinants comprised of AI C4-2 either with NPF (prostate fibroblasts from normal/benign prostate gland) or CPF cancer-associated stromal fibroblasts) under Shh/cyclopamine (a hedgehog signaling inhibitor) treatment. Human bone marrow stromal (HS27A) cells were used as controls. In vivo investigation was performed by checking serum PSA and immunohistochemical staining for the apoptosis-associated M30 gene in mice bearing chimeric C4-2/NPF tumors. RESULTS CONCLUSIONS Based on co-culture and chimeric tumor models, active Shh-mediated signaling was demonstrated between AI prostate cancer and NPF in a paracrine- and tumor progression-dependent manner. Our study suggests that drugs like cyclopamine that interfere with Shh signaling could be beneficial in preventing AI progression in prostate cancer cells. PMID:21520153

FAP-overexpressing fibroblasts produce an extracellular matrix that enhances invasive velocity and directionality of pancreatic cancer cells.

PubMed

Lee, Hyung-Ok; Mullins, Stefanie R; Franco-Barraza, Janusz; Valianou, Matthildi; Cukierman, Edna; Cheng, Jonathan D

2011-06-13

Alterations towards a permissive stromal microenvironment provide important cues for tumor growth, invasion, and metastasis. In this study, Fibroblast activation protein (FAP), a serine protease selectively produced by tumor-associated fibroblasts in over 90% of epithelial tumors, was used as a platform for studying tumor-stromal interactions. We tested the hypothesis that FAP enzymatic activity locally modifies stromal ECM (extracellular matrix) components thus facilitating the formation of a permissive microenvironment promoting tumor invasion in human pancreatic cancer. We generated a tetracycline-inducible FAP overexpressing fibroblastic cell line to synthesize an in vivo-like 3-dimensional (3D) matrix system which was utilized as a stromal landscape for studying matrix-induced cancer cell behaviors. A FAP-dependent topographical and compositional alteration of the ECM was characterized by measuring the relative orientation angles of fibronectin fibers and by Western blot analyses. The role of FAP in the matrix-induced permissive tumor behavior was assessed in Panc-1 cells in assorted matrices by time-lapse acquisition assays. Also, FAP+ matrix-induced regulatory molecules in cancer cells were determined by Western blot analyses. We observed that FAP remodels the ECM through modulating protein levels, as well as through increasing levels of fibronectin and collagen fiber organization. FAP-dependent architectural/compositional alterations of the ECM promote tumor invasion along characteristic parallel fiber orientations, as demonstrated by enhanced directionality and velocity of pancreatic cancer cells on FAP+ matrices. This phenotype can be reversed by inhibition of FAP enzymatic activity during matrix production resulting in the disorganization of the ECM and impeded tumor invasion. We also report that the FAP+ matrix-induced tumor invasion phenotype is β1-integrin/FAK mediated. Cancer cell invasiveness can be affected by alterations in the tumor

Stromalized microreactor supports murine hematopoietic progenitor enrichment.

PubMed

Khong, Danika; Li, Matthew; Singleton, Amy; Chin, Ling-Yee; Parekkadan, Biju

2018-01-20

There is an emerging need to process, expand, and even genetically engineer hematopoietic stem and progenitor cells (HSPCs) prior to administration for blood reconstitution therapy. A closed-system and automated solution for ex vivo HSC processing can improve adoption and standardize processing techniques. Here, we report a recirculating flow bioreactor where HSCs are stabilized and enriched for short-term processing by indirect fibroblast feeder coculture. Mouse 3 T3 fibroblasts were seeded on the extraluminal membrane surface of a hollow fiber micro-bioreactor and were found to support HSPC cell number compared to unsupported BMCs. CFSE analysis indicates that 3 T3-support was essential for the enhanced intrinsic cell cycling of HSPCs. This enhanced support was specific to the HSPC population with little to no effect seen with the Lineage positive and Lineage negative cells. Together, these data suggest that stromal-seeded hollow fiber micro-reactors represent a platform to screening various conditions that support the expansion and bioprocessing of HSPCs ex vivo.

Combining lymphovascular invasion with reactive stromal grade predicts prostate cancer mortality.

PubMed

Saeter, Thorstein; Vlatkovic, Ljiljana; Waaler, Gudmund; Servoll, Einar; Nesland, Jahn M; Axcrona, Karol; Axcrona, Ulrika

2016-09-01

Previous studies suggest that lymphovascular invasion (LVI) has a weak and variable effect on prognosis. It is uncertain whether LVI, determined by diagnostic prostate biopsy, predicts prostate cancer death. Data from experimental studies have indicated that carcinoma-associated fibroblasts in the reactive stroma could promote LVI and progression to metastasis. Thus, combining LVI with reactive stromal grade may identify prostate cancer patients at high risk of an unfavorable outcome. The purpose of the present study was to examine if LVI, determined by diagnostic biopsy, alone and in combination with reactive stromal grade could predict prostate cancer death. This population-based study included 283 patients with prostate cancer diagnosed by needle biopsy in Aust-Agder County (Norway) from 1991 to 1999. Clinical data were obtained by medical charts review. Two uropathologists evaluated LVI and reactive stromal grade. The endpoint was prostate cancer death. Patients with LVI had marginally higher risk of prostate cancer death compared to patients without LVI (hazard ratio: 1.8, P-value = 0.04). LVI had a stronger effect on prostate cancer death risk when a high reactive stromal grade was present (hazard ratio: 16.0, P-value <0.001). Therefore, patients with concomitant LVI and high reactive stromal grade were at particularly high risk for prostate cancer death. Evaluating LVI together with reactive stromal grade on diagnostic biopsies could be used to identify patients at high risk of death from prostate cancer. Prostate 76:1088-1094, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Tumour cells down-regulate CCN2 gene expression in co-cultured fibroblasts in a Smad7- and ERK-dependent manner.

PubMed

van Rooyen, Beverley A; Schäfer, Georgia; Leaner, Virna D; Parker, M Iqbal

2013-10-03

Recent studies have revealed that interactions between tumour cells and the surrounding stroma play an important role in facilitating tumour growth and invasion. Stromal fibroblasts produce most of the extracellular matrix components found in the stroma. The aim of this study was to investigate mechanisms involved in tumour cell-mediated regulation of extracellular matrix and adhesion molecules in co-cultured fibroblasts. To this end, microarray analysis was performed on CCD-1068SK human fibroblast cells after direct co-culture with MDA-MB-231 human breast tumour cells. We found that the expression of both connective tissue growth factor (CTGF/CCN2) and type I collagen was negatively regulated in CCD-1068SK fibroblast cells under direct co-culture conditions. Further analysis revealed that Smad7, a known negative regulator of the Smad signalling pathway involved in CCN2 promoter regulation, was increased in directly co-cultured fibroblasts. Inhibition of Smad7 expression in CCD-1068SK fibroblasts resulted in increased CCN2 expression, while Smad7 overexpression had the opposite effect. Silencing CCN2 gene expression in fibroblasts led, in turn, to a decrease in type I collagen mRNA and protein levels. ERK signalling was also shown to be impaired in CCD-1068SK fibroblasts after direct co-culture with MDA-MB-231 tumour cells, with Smad7 overexpression in fibroblasts leading to a similar decrease in ERK activity. These effects were not, however, seen in fibroblasts that were indirectly co-cultured with tumour cells. We therefore conclude that breast cancer cells require close contact with fibroblasts in order to upregulate Smad7 which, in turn, leads to decreased ERK signalling resulting in diminished expression of the stromal proteins CCN2 and type I collagen.

FGF7 supports hematopoietic stem and progenitor cells and niche-dependent myeloblastoma cells via autocrine action on bone marrow stromal cells in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ishino, Ruri; Minami, Kaori; Tanaka, Satowa

2013-10-11

Highlights: •FGF7 is downregulated in MED1-deficient mesenchymal cells. •FGF7 produced by mesenchymal stromal cells is a novel hematopoietic niche molecule. •FGF7 supports hematopoietic progenitor cells and niche-dependent leukemia cells. •FGF7 activates FGFR2IIIb of bone marrow stromal cells in an autocrine manner. •FGF7 indirectly acts on hematopoietic cells lacking FGFR2IIIb via stromal cells. -- Abstract: FGF1 and FGF2 support hematopoietic stem and progenitor cells (HSPCs) under stress conditions. In this study, we show that fibroblast growth factor (FGF7) may be a novel niche factor for HSPC support and leukemic growth. FGF7 expression was attenuated in mouse embryonic fibroblasts (MEFs) deficient formore » the MED1 subunit of the Mediator transcriptional coregulator complex. When normal mouse bone marrow (BM) cells were cocultured with Med1{sup +/+} MEFs or BM stromal cells in the presence of anti-FGF7 antibody, the growth of BM cells and the number of long-time culture-initiating cells (LTC-ICs) decreased significantly. Anti-FGF7 antibody also attenuated the proliferation and cobblestone formation of MB1 stromal cell-dependent myeloblastoma cells. The addition of recombinant FGF7 to the coculture of BM cells and Med1{sup −/−} MEFs increased BM cells and LTC-ICs. FGF7 and its cognate receptor, FGFR2IIIb, were undetectable in BM cells, but MEFs and BM stromal cells expressed both. FGF7 activated downstream targets of FGFR2IIIb in Med1{sup +/+} and Med1{sup −/−} MEFs and BM stromal cells. Taken together, we propose that FGF7 supports HSPCs and leukemia-initiating cells indirectly via FGFR2IIIb expressed on stromal cells.« less

Niche matters: The comparison between bone marrow stem cells and endometrial stem cells and stromal fibroblasts reveal distinct migration and cytokine profiles in response to inflammatory stimulus

PubMed Central

Sorjamaa, Anna; Kangasniemi, Marika; Sutinen, Meeri; Salo, Tuula; Liakka, Annikki; Lehenkari, Petri; Tapanainen, Juha S.; Vuolteenaho, Olli; Chen, Joseph C.; Lehtonen, Siri; Piltonen, Terhi T.

2017-01-01

Objective Intrinsic inflammatory characteristics play a pivotal role in stem cell recruitment and homing through migration where the subsequent change in niche has been shown to alter these characteristics. The bone marrow mesenchymal stem cells (bmMSCs) have been demonstrated to migrate to the endometrium contributing to the stem cell reservoir and regeneration of endometrial tissue. Thus, the aim of the present study was to compare the inflammation-driven migration and cytokine secretion profile of human bmMSCs to endometrial mesenchymal stem cells (eMSCs) and endometrial fibroblasts (eSFs). Materials and methods The bmMSCs were isolated from bone marrow aspirates through culturing, whereas eMSCs and eSFs were FACS-isolated. All cell types were tested for their surface marker, proliferation profiles and migration properties towards serum and inflammatory attractants. The cytokine/chemokine secretion profile of 35 targets was analysed in each cell type at basal level along with lipopolysaccharide (LPS)-induced state. Results Both stem cell types, bmMSCs and eMSCs, presented with similar stem cell surface marker profiles as well as possessed high proliferation and migration potential compared to eSFs. In multiplex assays, the secretion of 16 cytokine targets was detected and LPS stimulation expanded the cytokine secretion pattern by triggering the secretion of several targets. The bmMSCs exhibited higher cytokine secretion of vascular endothelial growth factor (VEGF)-A, stromal cell-derived factor-1 alpha (SDF)-1α, interleukin-1 receptor antagonist (IL-1RA), IL-6, interferon-gamma inducible protein (IP)-10, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)1α and RANTES compared to eMSCs and/or eSFs after stimulation with LPS. The basal IL-8 secretion was higher in both endometrial cell types compared to bmMSCs. Conclusion Our results highlight that similar to bmMSCs, the eMSCs possess high migration activity while the differentiation

Targeting Carcinoma-Associated Fibroblasts Within the Tumor Stroma With a Fibroblast Activation Protein-Activated Prodrug

PubMed Central

2012-01-01

Background Fibroblasts undergo a morphological transformation to a reactive phenotype in the tumor microenvironment characterized by the expression of proteins such as fibroblast activation protein (FAP), a post-prolyl endopeptidase with expression largely restricted to carcinoma-associated fibroblasts. Thapsigargin (TG) is a highly toxic natural plant product that triggers a rise in intracellular calcium levels and apoptosis. FAP is therefore a provocative target for the activation of prodrugs consisting of a FAP-specific peptide coupled to a potent cytotoxic analog of TG. Methods The efficacy of FAP-activated peptidyl-TG prodrugs was tested in vitro in cell proliferation assays and effects on intracellular calcium in human cancer cell lines. The effects of FAP-activated prodrugs on tumor growth and host toxicity were tested in Balb-C nude MCF-7 and LNCaP xenograft mice (n = 9–11 per group). P values were calculated using permutation tests based on 50 000 permutations. Mixed effects models were used to account for correlations among replicate measures. All statistical tests were two-sided. Results FAP-activated prodrugs killed human cancer cells at low nanomolar concentrations (MCF-7 cells: IC50 = 3.5nM). Amino acid-12ADT analogs from FAP-cleaved prodrugs, but not uncleaved prodrugs, produced a rapid rise in intracellular calcium within minutes of exposure. Immunohistochemical analysis of xenografts exposed to FAP-prodrugs documented stromal-selective cell death of fibroblasts, pericytes, and endothelial cells of sufficient magnitude to inhibit growth of MCF-7 and LNCaP xenografts with minimal systemic toxicity, whereas non-FAP cleavable prodrugs were inactive. MCF-7 and LNCaP xenografts treated with a FAP-activated prodrug had maximal treated-to-control tumor volume ratios of 0.36 (treated: mean = 0.206mm3, 95% CI = 0.068 to 0.344mm3; control: mean = 0.580mm3, 95% CI = 0.267 to 0.893mm3) and 0.24 (treated: mean = 0.131mm3, 95% CI = 0.09 to 0.180mm3; control

O-GlcNAcylation Negatively Regulates Cardiomyogenic Fate in Adult Mouse Cardiac Mesenchymal Stromal Cells

PubMed Central

Zafir, Ayesha; Bradley, James A.; Long, Bethany W.; Muthusamy, Senthilkumar; Li, Qianhong; Hill, Bradford G.; Wysoczynski, Marcin; Prabhu, Sumanth D.; Bhatnagar, Aruni; Bolli, Roberto; Jones, Steven P.

2015-01-01

In both preclinical and clinical studies, cell transplantation of several cell types is used to promote repair of damaged organs and tissues. Nevertheless, despite the widespread use of such strategies, there remains little understanding of how the efficacy of cell therapy is regulated. We showed previously that augmentation of a unique, metabolically derived stress signal (i.e., O-GlcNAc) improves survival of cardiac mesenchymal stromal cells; however, it is not known whether enhancing O-GlcNAcylation affects lineage commitment or other aspects of cell competency. In this study, we assessed the role of O-GlcNAc in differentiation of cardiac mesenchymal stromal cells. Exposure of these cells to routine differentiation protocols in culture increased markers of the cardiomyogenic lineage such as Nkx2.5 and connexin 40, and augmented the abundance of transcripts associated with endothelial and fibroblast cell fates. Differentiation significantly decreased the abundance of O-GlcNAcylated proteins. To determine if O-GlcNAc is involved in stromal cell differentiation, O-GlcNAcylation was increased pharmacologically during the differentiation protocol. Although elevated O-GlcNAc levels did not significantly affect fibroblast and endothelial marker expression, acquisition of cardiomyocyte markers was limited. In addition, increasing O-GlcNAcylation further elevated smooth muscle actin expression. In addition to lineage commitment, we also evaluated proliferation and migration, and found that increasing O-GlcNAcylation did not significantly affect either; however, we found that O-GlcNAc transferase—the protein responsible for adding O-GlcNAc to proteins—is at least partially required for maintaining cellular proliferative and migratory capacities. We conclude that O-GlcNAcylation contributes significantly to cardiac mesenchymal stromal cell lineage and function. O-GlcNAcylation and pathological conditions that may affect O-GlcNAc levels (such as diabetes) should be

Esophageal Squamous Cell Carcinoma Cells Modulate Chemokine Expression and Hyaluronan Synthesis in Fibroblasts*

PubMed Central

Kretschmer, Inga; Freudenberger, Till; Twarock, Sören; Yamaguchi, Yu; Grandoch, Maria; Fischer, Jens W.

2016-01-01

The aim of this study was to characterize the interaction of KYSE-410, an esophageal squamous cell carcinoma cell line, and fibroblasts with respect to the extracellular matrix component hyaluronan (HA) and chemokine expression. KYSE-410 cells induced the mRNA expression of HA synthase 2 (Has2) in normal skin fibroblasts (SF) only in direct co-cultures. Parallel to Has2 mRNA, Has2 antisense RNA (Has2os2) was up-regulated in co-cultures. Knockdown of LEF1, a downstream target of Wnt signaling, abrogated Has2 and Has2os2 induction. After knockdown of Has2 in SF, significantly less α-smooth muscle actin expression was detected in co-cultures. Moreover, it was investigated whether the phenotype of KYSE-410 was affected in co-culture with SF and whether Has2 knockdown in SF had an impact on KYSE-410 cells in co-culture. However, no effects on epithelial-mesenchymal transition markers, proliferation, and migration were detected. In addition to Has2 mRNA, the chemokine CCL5 was up-regulated and CCL11 was down-regulated in SF in co-culture. Furthermore, co-cultures of KYSE-410 cells and cancer-associated fibroblasts (CAF) were investigated. Similar to SF, Has2 and Ccl5 were up-regulated and Ccl11 was down-regulated in CAF in co-culture. Importantly and in contrast to SF, inhibiting HA synthesis by 4-methylumbelliferone abrogated the effect of co-culture on Ccl5 in CAF. Moreover, HA was found to promote adhesion of CD4+ but not CD8+ cells to xenogaft tumor tissues. In conclusion, direct co-culture of esophageal squamous cell carcinoma and fibroblasts induced stromal HA synthesis via Wnt/LEF1 and altered the chemokine profile of stromal fibroblasts, which in turn may affect the tumor immune response. PMID:26699196

In vitro inhibitory effects of imatinib mesylate on stromal cells and hematopoietic progenitors from bone marrow

PubMed Central

Soares, P.B.; Jeremias, T.S.; Alvarez-Silva, M.; Licínio, M.A.; Santos-Silva, M.C.; Vituri, C.L.

2012-01-01

Imatinib mesylate (IM) is used to treat chronic myeloid leukemia (CML) because it selectively inhibits tyrosine kinase, which is a hallmark of CML oncogenesis. Recent studies have shown that IM inhibits the growth of several non-malignant hematopoietic and fibroblast cells from bone marrow (BM). The aim of the present study was to evaluate the effects of IM on stromal and hematopoietic progenitor cells, specifically in the colony-forming units of granulocyte/macrophage (CFU-GM), using BM cultures from 108 1.5- to 2-month-old healthy Swiss mice. The results showed that low concentrations of IM (1.25 µM) reduced the growth of CFU-GM in clonogenic assays. In culture assays with stromal cells, fibroblast proliferation and α-SMA expression by immunocytochemistry analysis were also reduced in a concentration-dependent manner, with a survival rate of approximately 50% with a dose of 2.5 µM. Cell viability and morphology were analyzed using MTT and staining with acrydine orange/ethidium bromide. Most cells were found to be viable after treatment with 5 µM IM, although there was gradual growth inhibition of fibroblastic cells while the number of round cells (macrophage-like cells) increased. At higher concentrations (15 µM), the majority of cells were apoptotic and cell growth ceased completely. Oil red staining revealed the presence of adipocytes only in untreated cells (control). Cell cycle analysis of stromal cells by flow cytometry showed a blockade at the G0/G1 phases in groups treated with 5-15 µM. These results suggest that IM differentially inhibits the survival of different types of BM cells since toxic effects were achieved. PMID:23011404

Plasma rich in growth factors (PRGF) eye drops stimulates scarless regeneration compared to autologous serum in the ocular surface stromal fibroblasts.

PubMed

Anitua, E; de la Fuente, M; Muruzabal, F; Riestra, A; Merayo-Lloves, J; Orive, G

2015-06-01

Autologous serum (AS) eye drops was the first blood-derived product used for the treatment of corneal pathologies but nowadays PRGF arises as a novel interesting alternative to this type of diseases. The purpose of this study was to evaluate and compare the biological outcomes of autologous serum eye drops or Plasma rich in growth factors (PRGF) eye drops on corneal stromal keratocytes (HK) and conjunctival fibroblasts (HConF). To address this, blood from healthy donors was collected and processed to obtain autologous serum (AS) eye drops and plasma rich in growth factors (PRGF) eye drops. Blood-derivates were aliquoted and stored at -80°C until use. PDGF-AB, VEGF, EGF, FGFb and TGF-β1 were quantified. The potential of PRGF and AS in promoting wound healing was evaluated by means of proliferation and migration assays in HK and HConF. Fibroblast cells were induced to myofibroblast differentiation after treatment with 2.5ng/mL of TGF-β1. The capability of PRGF and AS to prevent and inhibit TGF-β1-induced differentiation was evaluated. Results showed significant higher levels of all growth factors analyzed in PRGF eye drops compared to AS. Moreover, PRGF eye drops enhanced significantly the biological outcomes of both HK and HConF, and reduced TGF-β1-induced myofibroblast differentiation in contrast to autologous serum eye drops (AS). In summary, these results suggest that PRGF exerts enhanced biological outcomes than AS. PRGF may improve the treatment of ocular surface wound healing minimizing the scar formation compared to AS. Results obtained herein suggest that PRGF protects and reverses the myofibroblast phenotype while promotes cell proliferation and migration. Copyright © 2015 Elsevier Ltd. All rights reserved.

WINGLESS (WNT) signaling is a progesterone target for rat uterine stromal cell proliferation

PubMed Central

Talbott, Alex; Bhusri, Anuradha; Krumsick, Zach; Foster, Sierra; Wormington, Joshua; Kimler, Bruce F

2016-01-01

Preparation of mammalian uterus for embryo implantation requires a precise sequence of cell proliferation. In rodent uterus, estradiol stimulates proliferation of epithelial cells. Progesterone operates as a molecular switch and redirects proliferation to the stroma by down-regulating glycogen synthase kinase-3β (GSK-3β) and stimulating β-catenin accumulation in the periluminal stromal cells. In this study, the WNT signal involved in the progesterone-dependent proliferative switch was investigated. Transcripts of four candidate Wnt genes were measured in the uteri from ovariectomized (OVX) rats, progesterone-pretreated (3 days of progesterone, 2mg/daily) rats, and progesterone-pretreated rats given a single dose (0.2µg) of estradiol. The spatial distribution of the WNT proteins was determined in the uteri after the same treatments. Wnt5a increased in response to progesterone and the protein emerged in the periluminal stromal cells of progesterone-pretreated rat uteri. To investigate whether WNT5A was required for proliferation, uterine stromal cell lines were stimulated with progesterone (1µM) and fibroblast growth factor (FGF, 50ng/mL). Proliferating stromal cells expressed a two-fold increase in WNT5A protein at 12h post stimulation. Stimulated stromal cells were cultured with actinomycin D (25µg/mL) to inhibit new RNA synthesis. Relative Wnt5a expression increased at 4 and 6 h of culture, suggesting that progesterone plus FGF preferentially increased Wnt5a mRNA stability. Knockdown of Wnt5a in uterine stromal cell lines inhibited stromal cell proliferation and decreased Wnt5a mRNA. The results indicate that progesterone initiates and synchronizes uterine stromal cell proliferation by increasing WNT5A expression and signaling. PMID:26975616

Differential effects of trichostatin A on gelatinase A expression in 3T3 fibroblasts and HT-1080 fibrosarcoma cells: implications for use of TSA in cancer therapy.

PubMed

Ailenberg, Menachem; Silverman, Mel

2003-03-07

Trichostatin A (TSA) is a histone deacetylase (HDAC) inhibitor with potential in cancer therapeutics. In a recent communication, we demonstrated that TSA is a selective, potent inhibitor of gelatinase A in 3T3 fibroblasts. In the present study, we extend these observations and examine the effects of TSA in 3T3 fibroblasts compared to HT-1080 fibrosarcoma cells with respect to gelatinase A expression, cell viability, and apoptosis. We find that while expression of gelatinase A in 3T3 fibroblasts is exquisitely sensitive to inhibition by TSA, expression of this enzyme in HT-1080 cells is minimally affected by this compound. Moreover, we show that TSA is pro-apoptotic in HT-1080 cells, but is anti-apoptotic in 3T3 cells. We propose a two-pronged model for the therapeutic action of TSA. On the one hand TSA selectively decreases cancer cell viability, while enhancing the viability of stromal cells. On the other hand, by selectively decreasing gelatinase A expression in stromal but not cancer cells, TSA acts to control metastatic potential by reducing the ability of metastatic cells to recruit stromal cells to secrete gelatinase A.

Suppressed prostate epithelial development with impaired branching morphogenesis in mice lacking stromal fibromuscular androgen receptor.

PubMed

Lai, Kuo-Pao; Yamashita, Shinichi; Vitkus, Spencer; Shyr, Chih-Rong; Yeh, Shuyuan; Chang, Chawnshang

2012-01-01

Using the cre-loxP system, we generated a new mouse model [double stromal androgen receptor knockout (dARKO)] with selectively deleted androgen receptor (AR) in both stromal fibroblasts and smooth muscle cells, and found the size of the anterior prostate (AP) lobes was significantly reduced as compared with those from wild-type littermate controls. The reduction in prostate size of the dARKO mouse was accompanied by impaired branching morphogenesis and partial loss of the infolding glandular structure. Further dissection found decreased proliferation and increased apoptosis of the prostate epithelium in the dARKO mouse AP. These phenotype changes were further confirmed with newly established immortalized prostate stromal cells (PrSC) from wild-type and dARKO mice. Mechanistically, IGF-1, placental growth factor, and secreted phosphoprotein-1 controlled by stromal AR were differentially expressed in PrSC-wt and PrSC-ARKO. Moreover, the conditioned media (CM) from PrSC-wt promoted prostate epithelium growth significantly as compared with CM from PrSC-dARKO. Finally, adding IGF-1/placental growth factor recombinant proteins into PrSC-dARKO CM was able to partially rescue epithelium growth. Together, our data concluded that stromal fibromuscular AR could modulate epithelium growth and maintain cellular homeostasis through identified growth factors.

FAP Promotes Immunosuppression by Cancer-Associated Fibroblasts in the Tumor Microenvironment via STAT3-CCL2 Signaling.

PubMed

Yang, Xuguang; Lin, Yuli; Shi, Yinghong; Li, Bingji; Liu, Weiren; Yin, Wei; Dang, Yongjun; Chu, Yiwei; Fan, Jia; He, Rui

2016-07-15

Cancer-associated fibroblasts (CAF) are components of the tumor microenvironment whose contributions to malignant progression are not fully understood. Here, we show that the fibroblast activation protein (FAP) triggers induction of a CAF subset with an inflammatory phenotype directed by STAT3 activation and inflammation-associated expression signature marked by CCL2 upregulation. Enforcing FAP expression in normal fibroblasts was sufficient to endow them with an inflammatory phenotype similar to FAP(+)CAFs. We identified FAP as a persistent activator of fibroblastic STAT3 through a uPAR-dependent FAK-Src-JAK2 signaling pathway. In a murine liver tumor model, we found that FAP(+)CAFs were a major source of CCL2 and that fibroblastic STAT3-CCL2 signaling in this setting promoted tumor growth by enhancing recruitment of myeloid-derived suppressor cells (MDSC). The CCL2 receptor CCR2 was expressed on circulating MDSCs in tumor-bearing subjects and FAP(+)CAF-mediated tumor promotion and MDSC recruitment was abrogated in Ccr2-deficient mice. Clinically, we observed a positive correlation between stromal expression of FAP, p-STAT3, and CCL2 in human intrahepatic cholangiocarcinoma, a highly aggressive liver cancer with dense desmoplastic stroma, where elevated levels of stromal FAP predicted a poor survival outcome. Taken together, our results showed how FAP-STAT3-CCL2 signaling in CAFs was sufficient to program an inflammatory component of the tumor microenvironment, which may have particular significance in desmoplasia-associated cancers. Cancer Res; 76(14); 4124-35. ©2016 AACR. ©2016 American Association for Cancer Research.

A co-culture system with three different primary human cell populations reveals that biomaterials and MSC modulate macrophage-driven fibroblast recruitment.

PubMed

Caires, Hugo R; Barros da Silva, Patrícia; Barbosa, Mário A; Almeida, Catarina R

2018-03-01

The biological response to implanted biomaterials is a complex and highly coordinated phenomenon involving many different cell types that interact within 3D microenvironments. Here, we increased the complexity of a 3D platform to include at least 3 cell types that play a role in the host response upon scaffold implantation. With this system, it was possible to address how immune responses triggered by 3D biomaterials mediate recruitment of stromal cells that promote tissue regeneration, mesenchymal stromal/stem cells (MSC), or a foreign body response, fibroblasts. Primary human macrophages yielded the highest fibroblast recruitment when interacting with chitosan scaffolds but not polylactic acid. Interestingly, when there were MSC and fibroblasts in the same environment, macrophages in chitosan scaffolds again promoted a significant increase on fibroblast recruitment, but not of MSC. However, macrophages that were firstly allowed to interact with MSC within the scaffolds were no longer able to recruit fibroblasts. This study illustrates the potential to use different scaffolds to regulate the dynamics of recruitment of proregenerative or fibrotic cell types through immunomodulation. Overall, this work strengths the idea that ex vivo predictive systems need to consider the different players involved in the biological response to biomaterials and that timing of arrival of specific cell types will affect the outcome. Copyright © 2017 John Wiley & Sons, Ltd.

Color-Coded Imaging of Syngeneic Orthotopic Malignant Lymphoma Interacting with Host Stromal Cells During Metastasis.

PubMed

Matsumoto, Takuro; Suetsugu, Atsushi; Hasegawa, Kosuke; Nakamura, Miki; Aoki, Hitomi; Kunisada, Takahiro; Tsurumi, Hisashi; Shimizu, Masahito; Hoffman, Robert M

2016-04-01

The EL4 cell line was previously derived from a lymphoma induced in a C57/BL6 mouse by 9,10-dimethyl-1,2-benzanthracene. In a previous study, EL4 lymphoma cells expressing red fluorescent protein (EL4-RFP) were established and injected into the tail vein of C57/BL6 green fluorescent protein (GFP) transgenic mice. Metastasis was observed at multiple sites which were also enriched with host GFP-expressing stromal cells. In the present study, our aim was to establish an orthotopic model of EL4-RFP. In the present study, EL4-RFP lymphoma cells were injected in the spleen of C57/BL6 GFP transgenic mice as an orthotopic model of lymphoma. Resultant primary tumor and metastases were imaged with the Olympus FV1000 scanning laser confocal microscope. EL4-RFP metastasis was observed 21 days later. EL4-RFP tumors in the spleen (primary injection site), liver, supra-mediastinum lymph nodes, abdominal lymph nodes, bone marrow, and lung were visualized by color-coded imaging. EL4-RFP metastases in the liver, lymph nodes, and bone marrow in C57/BL6 GFP mice were rich in GFP stromal cells such as macrophages, fibroblasts, dendritic cells, and normal lymphocytes derived from the host animal. Small tumors were observed in the spleen, which were rich in host stromal cells. In the lung, no mass formation of lymphoma cells occurred, but lymphoma cells circulated in lung peripheral blood vessels. Phagocytosis of EL4-RFP lymphoma cells by macrophages, as well as dendritic cells and fibroblasts, were observed in culture. Color-coded imaging of the lymphoma microenvironment suggests an important role of stromal cells in lymphoma progression and metastasis. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Enhancing proliferation and optimizing the culture condition for human bone marrow stromal cells using hypoxia and fibroblast growth factor-2.

PubMed

Lee, Jung-Seok; Kim, Seul Ki; Jung, Byung-Joo; Choi, Seong-Bok; Choi, Eun-Young; Kim, Chang-Sung

2018-04-01

This study aimed to determine the cellular characteristics and behaviors of human bone marrow stromal cells (hBMSCs) expanded in media in a hypoxic or normoxic condition and with or without fibroblast growth factor-2 (FGF-2) treatment. hBMSCs isolated from the vertebral body and expanded in these four groups were evaluated for cellular proliferation/migration, colony-forming units, cell-surface characterization, in vitro differentiation, in vivo transplantation, and gene expression. Culturing hBMSCs using a particular environmental factor (hypoxia) and with the addition of FGF-2 increased the cellular proliferation rate while enhancing the regenerative potential, modulated the multipotency-related processes (enhanced chondrogenesis-related processes/osteogenesis, but reduced adipogenesis), and increased cellular migration and collagen formation. The gene expression levels in the experimental samples showed activation of the hypoxia-inducible factor-1 pathway and glycolysis in the hypoxic condition, with this not being affected by the addition of FGF-2. The concurrent application of hypoxia and FGF-2 could provide a favorable condition for culturing hBMSCs to be used in clinical applications associated with bone tissue engineering, due to the enhancement of cellular proliferation and regenerative potential. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Role of Alpha-Smooth Muscle Actin and Fibroblast Activation Protein Alpha in Ovarian Neoplasms.

PubMed

da Silva, Ana Carolinne; Jammal, Millena Prata; Etchebehere, Renata Margarida; Murta, Eddie Fernando Candido; Nomelini, Rosekeila Simões

2018-04-05

Studies show that tumor growth is not just determined by the presence of malignant cells, since interactions between cancer cells and stromal microenvironment have important impacts on the cancer growth and progression. Cancer-associated fibroblasts play a prominent role in this process. The aims of the study were to investigate 2 cancer-associated fibroblasts markers, alpha-smooth muscle actin (α-SMA), and fibroblast activation protein alpha (FAP) in the stromal microenvironment of benign and malignant ovarian epithelial neoplasms, and to relate their tissue expression with prognostic factors in ovarian cancer. α-SMA and FAP were evaluated by immunohistochemistry in malignant (n = 28) and benign (n = 28) ovarian neoplasms. Fisher's exact test was used with a significance level lower than 0.05. FAP immunostaining was stronger in ovarian cancer when compared to benign neoplasms (p = 0.0366). There was no significant difference in relation to α-SMA expression between malignant and benign ovarian neoplasms as well as prognostic factors. In ovarian cancer, FAP stainings 2/3 was significantly related to histological grades 2 and 3 (p = 0.0183). FAP immunostaining is more intense in malignant neoplasms than in benign ovarian neoplasms, as well as in moderately differentiated and undifferentiated ovarian carcinomas compared to well-differentiated neoplasms, thus indicating that it can be used as a marker of worse prognosis. © 2018 S. Karger AG, Basel.

Consumptive hypothyroidism due to a gastrointestinal stromal tumor expressing type 3 iodothyronine deiodinase

PubMed Central

Yan, Lin-Hai; Mo, Xian-Wei; Qin, Yu-Zhou; Wang, Cheng; Chen, Zhi-Ning; Lin, Yuan; Chen, Jian-Si

2015-01-01

Context: Gastrointestinal stromal tumors (GISTs) are responsive to sunitinib (the tyrosine kinase inhibitor), this agent is widely used in prevention relapse of GISTs and neo-adjuvant chemotherapy in GIST patients without operation opportunity. The use of these agents has both advantages and disadvantages. On the one hand, it can improve the outcome for patient. On the other hand, it may lead to consumptive hypothyroidism, a rare syndrome caused by increased catabolism of T4 and T3 by increased type 3 iodothyronine deiodinase (D3) activity. D3 is the major physiologic inactivator of thyroid hormone, this selenoenzyme catalyzes the inner-ring deiodination of T(4) to reverse T(3) and T(3) to 3, 3;-diiodothyronine, both of which are biologically inactive [1]. Increased monitoring and supernormal thyroid hormone supplementation are required for affected patient. Objective: The aim of the study was to report the first case of consumptive hypothyroidism in an athyreotic patient after surgical resection of gastrointestinal stromal tumor. Design, setting, and patient: A 60-year-old athyreotic male was presented and he was euthyroid when receiving a stable therapeutic dose of thyroid hormone which was used to treat consumptive hypothyroidism resulting from the side effects of sunitinib, which is used for treatment of neo-adjuvant chemotherapy in gastrointestinal stromal tumor. With a discovery of large D3-expressing gastrointestinal stromal tumor, this patient suffered from marked Hyperthyrotropinemia, which instantly worsened after surgical resection of the gastrointestinal stromal tumor and then continued for 12 weeks after the surgical resection, in spite of further increases in levothyroxine therapy. The patient also had low serum T3 and elevated serum reverse T3 (rT3). Intervention: The patient’s consumptive hypothyroidism caused by marked overexpression of the thyroid hormone-inactivating D3 within the gastrointestinal stromal tumor and adjacent normal

Metabolic cooperation between co-cultured lung cancer cells and lung fibroblasts.

PubMed

Koukourakis, Michael I; Kalamida, Dimitra; Mitrakas, Achilleas G; Liousia, Maria; Pouliliou, Stamatia; Sivridis, Efthimios; Giatromanolaki, Alexandra

2017-11-01

Cooperation of cancer cells with stromal cells, such as cancer-associated fibroblasts (CAFs), has been revealed as a mechanism sustaining cancer cell survival and growth. In the current study, we focus on the metabolic interactions of MRC5 lung fibroblasts with lung cancer cells (A549 and H1299) using co-culture experiments and studying changes of the metabolic protein expression profile and of their growth and migration abilities. Using western blotting, confocal microscopy and RT-PCR, we observed that in co-cultures MRC5 respond by upregulating pyruvate dehydrogenase (PDH) and the monocarboxylate transporter MCT1. In contrast, cancer cells increase the expression of glucose transporters (GLUT1), LDH5, PDH kinase and the levels of phosphorylated/inactivated pPDH. H1299 cells growing in the same culture medium with fibroblasts exhibit a 'metastasis-like' phenomenon by forming nests within the fibroblast area. LDH5 and pPDH were drastically upregulated in these nests. The growth rate of both MRC5 and cancer cells increased in co-cultures. Suppression of LDHA or PDK1 in cancer cells abrogates the stimulatory signal from cancer cells to fibroblasts. Incubation of MRC5 fibroblasts with lactate resulted in an increase of LDHB and of PDH expression. Silencing of PDH gene in fibroblasts, or silencing of PDK1 or LDHA gene in tumor cells, impedes cancer cell's migration ability. Overall, a metabolic cooperation between lung cancer cells and fibroblasts has been confirmed in the context of direct Warburg effect, thus the fibroblasts reinforce aerobic metabolism to support the intensified anaerobic glycolytic pathways exploited by cancer cells.

Understanding the Impact of 2D and 3D Fibroblast Cultures on In Vitro Breast Cancer Models

PubMed Central

Sung, Kyung Eun; Su, Xiaojing; Berthier, Erwin; Pehlke, Carolyn; Friedl, Andreas; Beebe, David J.

2013-01-01

The utilization of 3D, physiologically relevant in vitro cancer models to investigate complex interactions between tumor and stroma has been increasing. Prior work has generally focused on the cancer cells and, the role of fibroblast culture conditions on tumor-stromal cell interactions is still largely unknown. Here, we focus on the stroma by comparing functional behaviors of human mammary fibroblasts (HMFs) cultured in 2D and 3D and their effects on the invasive progression of breast cancer cells (MCF10DCIS.com). We identified increased levels of several paracrine factors from HMFs cultured in 3D conditions that drive the invasive transition. Using a microscale co-culture model with improved compartmentalization and sensitivity, we demonstrated that HMFs cultured in 3D intensify the promotion of the invasive progression through the HGF/c-Met interaction. This study highlights the importance of the 3D stromal microenvironment in the development of multiple cell type in vitro cancer models. PMID:24124550

Anchored and soluble gangliosides contribute to myelosupportivity of stromal cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ziulkoski, Ana L.; Departamento de Bioquimica, ICBS, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS; Instituto de Ciencias da Saude, Centro Universitario Feevale, Novo Hamburgo, RS

2009-10-09

Stroma-mediated myelopoiesis depends upon growth factors and an appropriate intercellular microenvironment. Previous studies have demonstrated that gangliosides, produced by hepatic stromal cell types, are required for optimal myelosupportive function. Here, we compared the mielossuportive functions of a bone marrow stroma (S17) and skin fibroblasts (SF) regarding their ganglioside pattern of synthesis and shedding. The survival and proliferation of a myeloid precursor cell (FDC-P1) were used as reporter. Although the ganglioside synthesis of the two stromal cells was similar, their relative content and shedding were distinct. The ganglioside requirement for mielossuportive function was confirmed by the decreased proliferation of FDC-P1 cellsmore » in ganglioside synthesis-inhibited cultures and in presence of an antibody to GM3 ganglioside. The distinct mielossuportive activities of the S17 and SF stromata may be related to differences on plasma membrane ganglioside concentrations or to differences on the gangliosides shed and their subsequent uptake by myeloid cells, specially, GM3 ganglioside.« less

CXCL12 overexpression and secretion by aging fibroblasts enhance human prostate epithelial proliferation in vitro.

PubMed

Begley, Lesa; Monteleon, Christine; Shah, Rajal B; Macdonald, James W; Macoska, Jill A

2005-12-01

The direct relationship between the aging process and the incidence and prevalence of both benign prostatic hyperplasia (BPH) and prostate cancer (PCa) implies that certain risk factors associated with the development of both diseases increase with the aging process. In particular, both diseases share an overly proliferative phenotype, suggesting that mechanisms that normally act to suppress cellular proliferation are disrupted or rendered dysfunctional as a consequence of the aging process. We propose that one such mechanism involves changes in the prostate microenvironment, which 'evolves' during the aging process and disrupts paracrine interactions between epithelial and associated stromal fibroblasts. We show that stromal fibroblasts isolated from the prostates of men 63-81 years of age at the time of surgery express and secrete higher levels of the CXCL12 chemokine compared with those isolated from younger men, and stimulate CXCR4-mediated signaling pathways that induce cellular proliferation. These studies represent an important first step towards a mechanistic elucidation of the role of aging in the etiology of benign and malignant prostatic diseases.

Transformation of human mesenchymal cells and skin fibroblasts into hematopoietic cells.

PubMed

Harris, David M; Hazan-Haley, Inbal; Coombes, Kevin; Bueso-Ramos, Carlos; Liu, Jie; Liu, Zhiming; Li, Ping; Ravoori, Murali; Abruzzo, Lynne; Han, Lin; Singh, Sheela; Sun, Michael; Kundra, Vikas; Kurzrock, Razelle; Estrov, Zeev

2011-01-01

Patients with prolonged myelosuppression require frequent platelet and occasional granulocyte transfusions. Multi-donor transfusions induce alloimmunization, thereby increasing morbidity and mortality. Therefore, an autologous or HLA-matched allogeneic source of platelets and granulocytes is needed. To determine whether nonhematopoietic cells can be reprogrammed into hematopoietic cells, human mesenchymal stromal cells (MSCs) and skin fibroblasts were incubated with the demethylating agent 5-azacytidine (Aza) and the growth factors (GF) granulocyte-macrophage colony-stimulating factor and stem cell factor. This treatment transformed MSCs to round, non-adherent cells expressing T-, B-, myeloid-, or stem/progenitor-cell markers. The transformed cells engrafted as hematopoietic cells in bone marrow of immunodeficient mice. DNA methylation and mRNA array analysis suggested that Aza and GF treatment demethylated and activated HOXB genes. Indeed, transfection of MSCs or skin fibroblasts with HOXB4, HOXB5, and HOXB2 genes transformed them into hematopoietic cells. Further studies are needed to determine whether transformed MSCs or skin fibroblasts are suitable for therapy.

Transformation of Human Mesenchymal Cells and Skin Fibroblasts into Hematopoietic Cells

PubMed Central

Harris, David M.; Hazan-Haley, Inbal; Coombes, Kevin; Bueso-Ramos, Carlos; Liu, Jie; Liu, Zhiming; Li, Ping; Ravoori, Murali; Abruzzo, Lynne; Han, Lin; Singh, Sheela; Sun, Michael; Kundra, Vikas; Kurzrock, Razelle; Estrov, Zeev

2011-01-01

Patients with prolonged myelosuppression require frequent platelet and occasional granulocyte transfusions. Multi-donor transfusions induce alloimmunization, thereby increasing morbidity and mortality. Therefore, an autologous or HLA-matched allogeneic source of platelets and granulocytes is needed. To determine whether nonhematopoietic cells can be reprogrammed into hematopoietic cells, human mesenchymal stromal cells (MSCs) and skin fibroblasts were incubated with the demethylating agent 5-azacytidine (Aza) and the growth factors (GF) granulocyte-macrophage colony-stimulating factor and stem cell factor. This treatment transformed MSCs to round, non-adherent cells expressing T-, B-, myeloid-, or stem/progenitor-cell markers. The transformed cells engrafted as hematopoietic cells in bone marrow of immunodeficient mice. DNA methylation and mRNA array analysis suggested that Aza and GF treatment demethylated and activated HOXB genes. Indeed, transfection of MSCs or skin fibroblasts with HOXB4, HOXB5, and HOXB2 genes transformed them into hematopoietic cells. Further studies are needed to determine whether transformed MSCs or skin fibroblasts are suitable for therapy. PMID:21731684

Transcriptional profile of fibroblasts obtained from the primary site, lymph node and bone marrow of breast cancer patients.

PubMed

Del Valle, Paulo Roberto; Milani, Cintia; Brentani, Maria Mitzi; Katayama, Maria Lucia Hirata; de Lyra, Eduardo Carneiro; Carraro, Dirce Maria; Brentani, Helena; Puga, Renato; Lima, Leandro A; Rozenchan, Patricia Bortman; Nunes, Bárbara Dos Santos; Góes, João Carlos Guedes Sampaio; Azevedo Koike Folgueira, Maria Aparecida

2014-09-01

Cancer-associated fibroblasts (CAF) influence tumor development at primary as well as in metastatic sites, but there have been no direct comparisons of the transcriptional profiles of stromal cells from different tumor sites. In this study, we used customized cDNA microarrays to compare the gene expression profile of stromal cells from primary tumor (CAF, n = 4), lymph node metastasis (N+, n = 3) and bone marrow (BM, n = 4) obtained from breast cancer patients. Biological validation was done in another 16 samples by RT-qPCR. Differences between CAF vs N+, CAF vs BM and N+ vs BM were represented by 20, 235 and 245 genes, respectively (SAM test, FDR < 0.01). Functional analysis revealed that genes related to development and morphogenesis were overrepresented. In a biological validation set, NOTCH2 was confirmed to be more expressed in N+ (vs CAF) and ADCY2, HECTD1, HNMT, LOX, MACF1, SLC1A3 and USP16 more expressed in BM (vs CAF). Only small differences were observed in the transcriptional profiles of fibroblasts from the primary tumor and lymph node of breast cancer patients, whereas greater differences were observed between bone marrow stromal cells and the other two sites. These differences may reflect the activities of distinct differentiation programs.

Transcriptional profile of fibroblasts obtained from the primary site, lymph node and bone marrow of breast cancer patients

PubMed Central

Del Valle, Paulo Roberto; Milani, Cintia; Brentani, Maria Mitzi; Katayama, Maria Lucia Hirata; de Lyra, Eduardo Carneiro; Carraro, Dirce Maria; Brentani, Helena; Puga, Renato; Lima, Leandro A.; Rozenchan, Patricia Bortman; Nunes, Bárbara dos Santos; Góes, João Carlos Guedes Sampaio; Azevedo Koike Folgueira, Maria Aparecida

2014-01-01

Cancer-associated fibroblasts (CAF) influence tumor development at primary as well as in metastatic sites, but there have been no direct comparisons of the transcriptional profiles of stromal cells from different tumor sites. In this study, we used customized cDNA microarrays to compare the gene expression profile of stromal cells from primary tumor (CAF, n = 4), lymph node metastasis (N+, n = 3) and bone marrow (BM, n = 4) obtained from breast cancer patients. Biological validation was done in another 16 samples by RT-qPCR. Differences between CAF vs N+, CAF vs BM and N+ vs BM were represented by 20, 235 and 245 genes, respectively (SAM test, FDR < 0.01). Functional analysis revealed that genes related to development and morphogenesis were overrepresented. In a biological validation set, NOTCH2 was confirmed to be more expressed in N+ (vs CAF) and ADCY2, HECTD1, HNMT, LOX, MACF1, SLC1A3 and USP16 more expressed in BM (vs CAF). Only small differences were observed in the transcriptional profiles of fibroblasts from the primary tumor and lymph node of breast cancer patients, whereas greater differences were observed between bone marrow stromal cells and the other two sites. These differences may reflect the activities of distinct differentiation programs. PMID:25249769

Irradiated fibroblasts promote epithelial–mesenchymal transition and HDGF expression of esophageal squamous cell carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bao, Ci-Hang; Wang, Xin-Tong; Ma, Wei

2015-03-06

Recent evidence suggested that nonirradiated cancer-associated fibroblasts (CAFs) promoted aggressive phenotypes of cancer cells through epithelial–mesenchymal transition (EMT). Hepatoma-derived growth factor (HDGF) is a radiosensitive gene of esophageal squamous cell carcinoma (ESCC). This study aimed to investigate the effect of irradiated fibroblasts on EMT and HDGF expression of ESCC. Our study demonstrated that coculture with nonirradiated fibroblasts significantly increased the invasive ability of ESCC cells and the increased invasiveness was further accelerated when they were cocultured with irradiated fibroblasts. Scattering of ESCC cells was also accelerated by the supernatant from irradiated fibroblasts. Exposure of ESCC cells to supernatant from irradiatedmore » fibroblasts resulted in decreased E-cadherin, increased vimentin in vitro and β-catenin was demonstrated to localize to the nucleus in tumor cells with irradiated fibroblasts in vivo models. The expression of HDGF and β-catenin were increased in both fibroblasts and ESCC cells of irradiated group in vitro and in vivo models. Interestingly, the tumor cells adjoining the stromal fibroblasts displayed strong nuclear HDGF immunoreactivity, which suggested the occurrence of a paracrine effect of fibroblasts on HDGF expression. These data suggested that irradiated fibroblasts promoted invasion, growth, EMT and HDGF expression of ESCC. - Highlights: • Irradiated CAFs accelerated invasiveness and scattering of ESCC cell lines. • Irradiated CAFs promoted EMT of ESCC cells. • Irradiated fibroblasts induced nuclear β-catenin relocalization in ESCC cells. • Irradiated fibroblasts increased HDGF expression in vitro and in vivo.« less

Stromal deletion of the APC tumor suppressor in mice triggers development of endometrial cancer

PubMed Central

Tanwar, Pradeep S.; Zhang, LiHua; Roberts, Drucilla J.; Teixeira, Jose M.

2011-01-01

The contribution of the stromal microenvironment to the progression of endometrial cancer (EC) has not been well explored. We have conditionally expressed a mutant allele of adenomatous polyposis coli (APCcKO) in murine uterine stroma cells to study its effect on uterine development and function. In addition to metrorrhagia, the mice develop complex atypical endometrial gland hyperplasia that progresses to endometrial carcinoma in situ and endometrial adenocarcinoma as evidenced by myometrial invasion. Stromal cells subjacent to the carcinoma cells express αSMA with fewer cells expressing PDGFR-α compared to normal stromal cells suggesting that the mutant stromal cells have acquired a more myofibroblastic phenotype, which have been described as cancer-associated fibroblasts and have been shown to induce carcinogenesis in other organ systems. Analyses of human EC specimens showed substantial αSMA expression in the stroma compared with normal endometrial stroma cells. We also show that APCcKO mutant uteri and human EC have decreased stromal levels of TGFβ and BMP activities and that the mutant uteri failed to respond to exogenous estradiol stimulation. The mutant stroma cells also had higher levels of VEGF and SDF signaling components and diminished expression of ERα and PR which is common in advanced stages of human EC and is an indicator of poor prognosis. Our results indicate that de novo mutation or loss of heterozygosity in stromal APC is sufficient to induce endometrial hyperplasia and endometrial carcinogenesis by mechanisms that are consistent with unopposed estrogen signaling in the endometrial epithelium. PMID:21363919

Esophageal Squamous Cell Carcinoma Cells Modulate Chemokine Expression and Hyaluronan Synthesis in Fibroblasts.

PubMed

Kretschmer, Inga; Freudenberger, Till; Twarock, Sören; Yamaguchi, Yu; Grandoch, Maria; Fischer, Jens W

2016-02-19

The aim of this study was to characterize the interaction of KYSE-410, an esophageal squamous cell carcinoma cell line, and fibroblasts with respect to the extracellular matrix component hyaluronan (HA) and chemokine expression. KYSE-410 cells induced the mRNA expression of HA synthase 2 (Has2) in normal skin fibroblasts (SF) only in direct co-cultures. Parallel to Has2 mRNA, Has2 antisense RNA (Has2os2) was up-regulated in co-cultures. Knockdown of LEF1, a downstream target of Wnt signaling, abrogated Has2 and Has2os2 induction. After knockdown of Has2 in SF, significantly less α-smooth muscle actin expression was detected in co-cultures. Moreover, it was investigated whether the phenotype of KYSE-410 was affected in co-culture with SF and whether Has2 knockdown in SF had an impact on KYSE-410 cells in co-culture. However, no effects on epithelial-mesenchymal transition markers, proliferation, and migration were detected. In addition to Has2 mRNA, the chemokine CCL5 was up-regulated and CCL11 was down-regulated in SF in co-culture. Furthermore, co-cultures of KYSE-410 cells and cancer-associated fibroblasts (CAF) were investigated. Similar to SF, Has2 and Ccl5 were up-regulated and Ccl11 was down-regulated in CAF in co-culture. Importantly and in contrast to SF, inhibiting HA synthesis by 4-methylumbelliferone abrogated the effect of co-culture on Ccl5 in CAF. Moreover, HA was found to promote adhesion of CD4(+) but not CD8(+) cells to xenogaft tumor tissues. In conclusion, direct co-culture of esophageal squamous cell carcinoma and fibroblasts induced stromal HA synthesis via Wnt/LEF1 and altered the chemokine profile of stromal fibroblasts, which in turn may affect the tumor immune response. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

TβRIII Expression in Human Breast Cancer Stroma and the Role of Soluble TβRIII in Breast Cancer Associated Fibroblasts.

PubMed

Jovanović, Bojana; Pickup, Michael W; Chytil, Anna; Gorska, Agnieszka E; Johnson, Kimberly C; Moses, Harold L; Owens, Philip

2016-11-04

The TGF-β pathway plays a major role in tumor progression through regulation of epithelial and stromal cell signaling. Dysfunction of the pathway can lead to carcinoma progression and metastasis. To gain insight into the stromal role of the TGF-β pathway in breast cancer, we performed laser capture microdissection (LCM) from breast cancer patients and reduction mammoplasty patients. Microdissected tumor stroma and normal breast stroma were examined for gene expression. Expression of the TGF-β type III receptor ( TGFBR3 ) was greatly decreased in the tumor stroma compared to control healthy breast tissue. These results demonstrated a 44-fold decrease in TGFBR3 mRNA in tumor stroma in comparison to control tissue. We investigated publicly available databases, and have identified that TGFBR3 mRNA levels are decreased in tumor stroma. We next investigated fibroblast cell lines derived from cancerous and normal breast tissue and found that in addition to mRNA levels, TβRIII protein levels were significantly reduced. Having previously identified that cancer-associated fibroblasts secrete greater levels of tumor promoting cytokines, we investigated the consequences of soluble-TβRIII (sTβRIII) on fibroblasts. Fibroblast conditioned medium was analyzed for 102 human secreted cytokines and distinct changes in response to sTβRIII were observed. Next, we used the fibroblast-conditioned medium to stimulate human monocyte cell line THP-1. These results indicate a distinct transcriptional response depending on sTβRIII treatment and whether it was derived from normal or cancerous breast tissue. We conclude that the effect of TβRIII has distinct roles not only in cancer-associated fibroblasts but that sTβRIII has distinct paracrine functions in the tumor microenvironment.

Fibroblast-derived CXCL12/SDF-1α promotes CXCL6 secretion and co-operatively enhances metastatic potential through the PI3K/Akt/mTOR pathway in colon cancer

PubMed Central

Ma, Jia-Chi; Sun, Xiao-Wen; Su, He; Chen, Quan; Guo, Tian-Kang; Li, Yuan; Chen, Xiao-Chang; Guo, Jin; Gong, Zhen-Qiang; Zhao, Xiao-Dan; Qi, Jian-Bo

2017-01-01

AIM To investigate the underlying mechanism by which CXCL12 and CXCL6 influences the metastatic potential of colon cancer and internal relation of colon cancer and stromal cells. METHODS Western blotting was used to detect the expression of CXCL12 and CXCL6 in colon cancer cells and stromal cells. The co-operative effects of CXCL12 and CXCL6 on proliferation and invasion of colon cancer cells and human umbilical vein endothelial cells (HUVECs) were determined by enzyme-linked immunosorbent assay, and proliferation and invasion assays. The angiogenesis of HUVECs through interaction with cancer cells and stromal cells was examined by angiogenesis assay. We eventually investigated activation of PI3K/Akt/mTOR signaling by CXCL12 involved in the metastatic process of colon cancer. RESULTS CXCL12 was expressed in DLD-1 cancer cells and fibroblasts. The secretion level of CXCL6 by colon cancer cells and HUVECs were significantly promoted by fibroblasts derived from CXCL12. CXCL6 and CXCL2 could significantly enhance HUVEC proliferation and migration (P < 0.01). CXCL6 and CXCL2 enhanced angiogenesis by HUVECs when cultured with fibroblast cells and colon cancer cells (P < 0.01). CXCL12 also enhanced the invasion of colon cancer cells. Stromal cell-derived CXCL12 promoted the secretion level of CXCL6 and co-operatively promoted metastasis of colon carcinoma through activation of the PI3K/Akt/mTOR pathway. CONCLUSION Fibroblast-derived CXCL12 enhanced the CXCL6 secretion of colon cancer cells, and both CXCL12 and CXCL6 co-operatively regulated the metastasis via the PI3K/Akt/mTOR signaling pathway. Blocking this pathway may be a potential anti-metastatic therapeutic target for patients with colon cancer. PMID:28811711

Rapid Selection of Mesenchymal Stem and Progenitor Cells in Primary Prostate Stromal Cultures

PubMed Central

Brennen, W. Nathaniel; Kisteman, L. Nelleke; Isaacs, John T.

2016-01-01

BACKGROUND Carcinoma-associated fibroblasts (CAFs) are a dominant component of the tumor microenvironment with pro-tumorigenic properties. Despite this knowledge, their physiologic origins remain poorly understood. Mesenchymal stem cells (MSCs) can be recruited from the bone marrow to areas of tissue damage and inflammation, including prostate cancer. MSCs can generate and have many overlapping properties with CAFs in preclinical models. METHODS Multiparameter flow cytometry and multipotent differentiation assays used to define MSCs in primary prostate stromal cultures derived from young (>25 yrs) organ donors and prostate cancer patients compared with bone marrow-derived stromal cultures. Population doubling times, population doublings, cell size, and differentiation potential determined under multiple culture conditions, including normoxia, hypoxia, and a variety of media. TGF-β measured by ELISA. RESULTS MSCs and stromal progenitors are not only present in normal and malignant prostate tissue, but are quickly selected for in primary stromal cultures derived from these tissues; becoming the dominant population within just a few passages. Growth potential inversely associated with TGF-β concentrations. All conditions generated populations with an average cell diameter >15 μm. All cultures tested had the ability to undergo osteogenic and chondrogenic differentiation, but unlike bone marrow-derived MSCs, primary stromal cultures derived from normal prostate tissue lack adipogenic differentiation potential. In contrast, a subset of stromal cultures derived from prostate cancer patients retain the ability to differentiate into adipocytes; a property that is significantly suppressed under hypoxic conditions in both bone marrow- and prostate-derived MSCs. CONCLUSIONS Primary prostate stromal cultures are highly enriched in cells with an MSC or stromal progenitor phenotype. The use of primary cultures such as these to study CAFs raises interesting implications when

Rapid selection of mesenchymal stem and progenitor cells in primary prostate stromal cultures.

PubMed

Brennen, W Nathaniel; Kisteman, L Nelleke; Isaacs, John T

2016-05-01

Carcinoma-associated fibroblasts (CAFs) are a dominant component of the tumor microenvironment with pro-tumorigenic properties. Despite this knowledge, their physiologic origins remain poorly understood. Mesenchymal stem cells (MSCs) can be recruited from the bone marrow to areas of tissue damage and inflammation, including prostate cancer. MSCs can generate and have many overlapping properties with CAFs in preclinical models. Multiparameter flow cytometry and multipotent differentiation assays used to define MSCs in primary prostate stromal cultures derived from young (<25 yrs) organ donors and prostate cancer patients compared with bone marrow-derived stromal cultures. Population doubling times, population doublings, cell size, and differentiation potential determined under multiple culture conditions, including normoxia, hypoxia, and a variety of media. TGF-β measured by ELISA. MSCs and stromal progenitors are not only present in normal and malignant prostate tissue, but are quickly selected for in primary stromal cultures derived from these tissues; becoming the dominant population within just a few passages. Growth potential inversely associated with TGF-β concentrations. All conditions generated populations with an average cell diameter >15 µm. All cultures tested had the ability to undergo osteogenic and chondrogenic differentiation, but unlike bone marrow-derived MSCs, primary stromal cultures derived from normal prostate tissue lack adipogenic differentiation potential. In contrast, a subset of stromal cultures derived from prostate cancer patients retain the ability to differentiate into adipocytes; a property that is significantly suppressed under hypoxic conditions in both bone marrow- and prostate-derived MSCs. Primary prostate stromal cultures are highly enriched in cells with an MSC or stromal progenitor phenotype. The use of primary cultures such as these to study CAFs raises interesting implications when considering their overlapping

Stromal Cells Positively and Negatively Modulate the Growth of Cancer Cells: Stimulation via the PGE2-TNFα-IL-6 Pathway and Inhibition via Secreted GAPDH-E-Cadherin Interaction

PubMed Central

Kawada, Manabu; Inoue, Hiroyuki; Ohba, Shun-ichi; Yoshida, Junjiro; Masuda, Tohru; Yamasaki, Manabu; Usami, Ihomi; Sakamoto, Shuichi; Abe, Hikaru; Watanabe, Takumi; Yamori, Takao; Shibasaki, Masakatsu; Nomoto, Akio

2015-01-01

Fibroblast-like stromal cells modulate cancer cells through secreted factors and adhesion, but those factors are not fully understood. Here, we have identified critical stromal factors that modulate cancer growth positively and negatively. Using a cell co-culture system, we found that gastric stromal cells secreted IL-6 as a growth and survival factor for gastric cancer cells. Moreover, gastric cancer cells secreted PGE2 and TNFα that stimulated IL-6 secretion by the stromal cells. Furthermore, we found that stromal cells secreted glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Extracellular GAPDH, or its N-terminal domain, inhibited gastric cancer cell growth, a finding confirmed in other cell systems. GAPDH bound to E-cadherin and downregulated the mTOR-p70S6 kinase pathway. These results demonstrate that stromal cells could regulate cancer cell growth through the balance of these secreted factors. We propose that negative regulation of cancer growth using GAPDH could be a new anti-cancer strategy. PMID:25785838

Time-dependent micromechanical responses of breast cancer cells and adjacent fibroblasts to electric treatment.

PubMed

Yizraeli, Maayan Lia; Weihs, Daphne

2011-12-01

Direct-current, low-intensity, electric fields were suggested as a minimally invasive treatment for various cancers. The tumor microenvironment may affect treatment efficacy, albeit it has not generally been considered when evaluating novel anti-cancer treatments. We evaluate the effects of electric treatment on epithelial, breast-cancer cells, co-cultured with non-cancerous fibroblasts, a simplified model for the tumor-microenvironment. We evaluate changes in morphology, cytoskeleton, and focus on dynamic intracellular structure and mechanics. Multiple-particle tracking was used within living cells to quantify time-dependent structural and mechanical changes. Cancer cells suffer severe cell death and exhibit transient rounding and changes in internal structural and mechanics. Interestingly, treating cancer cells in co-culture with fibroblasts delays and reduces their responses to treatment. Our particle-tracking data indicates a mechanism relating the observed changes in intracellular transport to transient changes in the microtubule network and its motors. In contrast, fibroblasts are only minimally affected by treatment, separately or in co-culture. To conclude, intracellular mechanics reveal time-dependent responses after treatment, unavailable by bulk measurements. This time-dependence could provide a window of opportunity for continued treatment. We demonstrate the importance of evaluating anti-cancer treatments within their microenvironment, which can affect response magnitude and time-course.

Progesterone receptor expression during prostate cancer progression suggests a role of this receptor in stromal cell differentiation.

PubMed

Yu, Yue; Yang, Ou; Fazli, Ladan; Rennie, Paul S; Gleave, Martin E; Dong, Xuesen

2015-07-01

The progesterone receptor, like the androgen receptor, belongs to the steroid receptor superfamily. Our previous studies have reported that the PR is expressed specifically in prostate stroma. PR inhibits proliferation of, and regulates cytokine secretion by stromal cells. However, PR protein expression in cancer-associated stroma during prostate cancer progression has not been profiled. Since the phenotypes of prostate stromal cells change dynamically as tumors progress, whether the PR plays a role in regulating stromal cell differentiation needs to be investigated. Immunohistochemistry assays measured PR protein levels on human prostate tissue microarrays containing 367 tissue cores from benign prostate, prostate tumors with different Gleason scores, tumors under various durations of castration therapy, and tumors at the castration-resistant stage. Immunoblotting assays determined whether PR regulated the expression of alpha smooth muscle actin (α-SMA), vimentin, and fibroblast specific protein (FSP) in human prostate stromal cells. PR protein levels decreased in cancer-associated stroma when compared with that in benign prostate stroma. This reduction in PR expression was not correlated with Gleason scores. PR protein levels were elevated by castration therapy, but reduced to pre-castration levels when tumors progressed to the castration-resistant stage. Enhanced PR expression in human prostate stromal cells increased α-SMA, but decreased vimentin and FSP protein levels ligand-independently. These results suggest that PR plays an active role in regulating stromal cell phenotypes during prostate cancer progression. © 2015 Wiley Periodicals, Inc.

Carcinoma associated fibroblasts (CAFs) promote breast cancer motility by suppressing mammalian Diaphanous-related formin-2 (mDia2).

PubMed

Dvorak, Kaitlyn M; Pettee, Krista M; Rubinic-Minotti, Kaitlin; Su, Robin; Nestor-Kalinoski, Andrea; Eisenmann, Kathryn M

2018-01-01

The tumor microenvironment (TME) promotes tumor cell invasion and metastasis. An important step in the shift to a pro-cancerous microenvironment is the transformation of normal stromal fibroblasts to carcinoma-associated fibroblasts (CAFs). CAFs are present in a majority of solid tumors and can directly promote tumor cell motility via cytokine, chemokine and growth factor secretion into the TME. The exact effects that the TME has upon cytoskeletal regulation in motile tumor cells remain enigmatic. The conserved formin family of cytoskeleton regulating proteins plays an essential role in the assembly and/or bundling of unbranched actin filaments. Mammalian Diaphanous-related formin 2 (mDia2/DIAPH3/Drf3/Dia) assembles a dynamic F-actin cytoskeleton that underlies tumor cell migration and invasion. We therefore sought to understand whether CAF-derived chemokines impact breast tumor cell motility through modification of the formin-assembled F-actin cytoskeleton. In MDA-MB-231 cells, conditioned media (CM) from WS19T CAFs, a human breast tumor-adjacent CAF line, significantly and robustly increased wound closure and invasion relative to normal human mammary fibroblast (HMF)-CM. WS19T-CM also promoted proteasome-mediated mDia2 degradation in MDA-MB-231 cells relative to control HMF-CM and WS21T CAF-CM, a breast CAF cell line that failed to promote robust MDA-MB-231 migration. Cytokine array analysis of CM identified up-regulated secreted factors in WS19T relative to control WS21T CM. We identified CXCL12 as a CM factor influencing loss of mDia2 protein while increasing MDA-MB-231 cell migration. Our data suggest a mechanism whereby CAFs promote tumor cell migration and invasion through CXCL12 secretion to regulate the mDia2-directed cytoskeleton in breast tumor cells.

Response of human corneal fibroblasts on silk film surface patterns.

PubMed

Gil, Eun Seok; Park, Sang-Hyug; Marchant, Jeff; Omenetto, Fiorenzo; Kaplan, David L

2010-06-11

Transparent, biodegradable, mechanically robust, and surface-patterned silk films were evaluated for the effect of surface morphology on human corneal fibroblast (hCF) cell proliferation, orientation, and ECM deposition and alignment. A series of dimensionally different surface groove patterns were prepared from optically graded glass substrates followed by casting poly(dimethylsiloxane) (PDMS) replica molds. The features on the patterned silk films showed an array of asymmetric triangles and displayed 37-342 nm depths and 445-3 582 nm widths. hCF DNA content on all patterned films were not significantly different from that on flat silk films after 4 d in culture. However, the depth and width of the grooves influenced cell alignment, while the depth differences affected cell orientation; overall, deeper and narrower grooves induced more hCF orientation. Over 14 d in culture, cell layers and actin filament organization demonstrated that confluent hCFs and their cytoskeletal filaments were oriented along the direction of the silk film patterned groove axis. Collagen type V and proteoglycans (decorin and biglycan), important markers of corneal stromal tissue, were highly expressed with alignment. Understanding corneal stromal fibroblast responses to surface features on a protein-based biomaterial applicable in vivo for corneal repair potential suggests options to improve corneal tissue mimics. Further, the approaches provide fundamental biomaterial designs useful for bioengineering oriented tissue layers, an endemic feature in most biological tissue structures that lead to critical tissue functions.

Fibroblast drug scavenging increases intratumoural gemcitabine accumulation in murine pancreas cancer

PubMed Central

Hessmann, E; Patzak, M S; Klein, L; Chen, N; Kari, V; Ramu, I; Bapiro, T E; Frese, K K; Gopinathan, A; Richards, F M; Jodrell, D I; Verbeke, C; Li, X; Heuchel, R; Löhr, J M; Johnsen, S A; Gress, T M; Ellenrieder, V; Neesse, A

2018-01-01

Objective Desmoplasia and hypovascularity are thought to impede drug delivery in pancreatic ductal adenocarcinoma (PDAC). However, stromal depletion approaches have failed to show clinical responses in patients. Here, we aimed to revisit the role of the tumour microenvironment as a physical barrier for gemcitabine delivery. Design Gemcitabine metabolites were analysed in LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) murine tumours and matched liver metastases, primary tumour cell lines, cancer-associated fibroblasts (CAFs) and pancreatic stellate cells (PSCs) by liquid chromatography-mass spectrometry/mass spectrometry. Functional and preclinical experiments, as well as expression analysis of stromal markers and gemcitabine metabolism pathways were performed in murine and human specimen to investigate the preclinical implications and the mechanism of gemcitabine accumulation. Results Gemcitabine accumulation was significantly enhanced in fibroblast-rich tumours compared with liver metastases and normal liver. In vitro, significantly increased concentrations of activated 2′,2′-difluorodeoxycytidine-5′-triphosphate (dFdCTP) and greatly reduced amounts of the inactive gemcitabine metabolite 2′,2′-difluorodeoxyuridine were detected in PSCs and CAFs. Mechanistically, key metabolic enzymes involved in gemcitabine inactivation such as hydrolytic cytosolic 5′-nucleotidases (Nt5c1A, Nt5c3) were expressed at low levels in CAFs in vitro and in vivo, and recombinant expression of Nt5c1A resulted in decreased intracellular dFdCTP concentrations in vitro. Moreover, gemcitabine treatment in KPC mice reduced the number of liver metastases by >50%. Conclusions Our findings suggest that fibroblast drug scavenging may contribute to the clinical failure of gemcitabine in desmoplastic PDAC. Metabolic targeting of CAFs may thus be a promising strategy to enhance the antiproliferative effects of gemcitabine. PMID:28077438

Fibroblast drug scavenging increases intratumoural gemcitabine accumulation in murine pancreas cancer.

PubMed

Hessmann, E; Patzak, M S; Klein, L; Chen, N; Kari, V; Ramu, I; Bapiro, T E; Frese, K K; Gopinathan, A; Richards, F M; Jodrell, D I; Verbeke, C; Li, X; Heuchel, R; Löhr, J M; Johnsen, S A; Gress, T M; Ellenrieder, V; Neesse, A

2018-03-01

Desmoplasia and hypovascularity are thought to impede drug delivery in pancreatic ductal adenocarcinoma (PDAC). However, stromal depletion approaches have failed to show clinical responses in patients. Here, we aimed to revisit the role of the tumour microenvironment as a physical barrier for gemcitabine delivery. Gemcitabine metabolites were analysed in LSL-Kras G12D/+ ; LSL-Trp53 R172H/+ ; Pdx-1-Cre (KPC) murine tumours and matched liver metastases, primary tumour cell lines, cancer-associated fibroblasts (CAFs) and pancreatic stellate cells (PSCs) by liquid chromatography-mass spectrometry/mass spectrometry. Functional and preclinical experiments, as well as expression analysis of stromal markers and gemcitabine metabolism pathways were performed in murine and human specimen to investigate the preclinical implications and the mechanism of gemcitabine accumulation. Gemcitabine accumulation was significantly enhanced in fibroblast-rich tumours compared with liver metastases and normal liver. In vitro, significantly increased concentrations of activated 2',2'-difluorodeoxycytidine-5'-triphosphate (dFdCTP) and greatly reduced amounts of the inactive gemcitabine metabolite 2',2'-difluorodeoxyuridine were detected in PSCs and CAFs. Mechanistically, key metabolic enzymes involved in gemcitabine inactivation such as hydrolytic cytosolic 5'-nucleotidases (Nt5c1A, Nt5c3) were expressed at low levels in CAFs in vitro and in vivo, and recombinant expression of Nt5c1A resulted in decreased intracellular dFdCTP concentrations in vitro. Moreover, gemcitabine treatment in KPC mice reduced the number of liver metastases by >50%. Our findings suggest that fibroblast drug scavenging may contribute to the clinical failure of gemcitabine in desmoplastic PDAC. Metabolic targeting of CAFs may thus be a promising strategy to enhance the antiproliferative effects of gemcitabine. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a

Influence of the interaction between nodal fibroblast and breast cancer cells on gene expression.

PubMed

Santos, Rosângela Portilho Costa; Benvenuti, Ticiana Thomazine; Honda, Suzana Terumi; Del Valle, Paulo Roberto; Katayama, Maria Lucia Hirata; Brentani, Helena Paula; Carraro, Dirce Maria; Rozenchan, Patrícia Bortman; Brentani, Maria Mitzi; de Lyra, Eduardo Carneiro; Torres, César Henrique; Salzgeber, Marcia Batista; Kaiano, Jane Haruko Lima; Góes, João Carlos Sampaio; Folgueira, Maria Aparecida Azevedo Koike

2011-02-01

Our aim was to evaluate the interaction between breast cancer cells and nodal fibroblasts, by means of their gene expression profile. Fibroblast primary cultures were established from negative and positive lymph nodes from breast cancer patients and a similar gene expression pattern was identified, following cell culture. Fibroblasts and breast cancer cells (MDA-MB231, MDA-MB435, and MCF7) were cultured alone or co-cultured separated by a porous membrane (which allows passage of soluble factors) for comparison. Each breast cancer lineage exerted a particular effect on fibroblasts viability and transcriptional profile. However, fibroblasts from positive and negative nodes had a parallel transcriptional behavior when co-cultured with a specific breast cancer cell line. The effects of nodal fibroblasts on breast cancer cells were also investigated. MDA MB-231 cells viability and migration were enhanced by the presence of fibroblasts and accordingly, MDA-MB435 and MCF7 cells viability followed a similar pattern. MDA-MB231 gene expression profile, as evaluated by cDNA microarray, was influenced by the fibroblasts presence, and HNMT, COMT, FN3K, and SOD2 were confirmed downregulated in MDA-MB231 co-cultured cells with fibroblasts from both negative and positive nodes, in a new series of RT-PCR assays. In summary, transcriptional changes induced in breast cancer cells by fibroblasts from positive as well as negative nodes are very much alike in a specific lineage. However, fibroblasts effects are distinct in each one of the breast cancer lineages, suggesting that the inter-relationships between stromal and malignant cells are dependent on the intrinsic subtype of the tumor.

Targeting Inhibition of Fibroblast Activation Protein-α and Prolyl Oligopeptidase Activities on Cells Common to Metastatic Tumor Microenvironments1

PubMed Central

Christiansen, Victoria J; Jackson, Kenneth W; Lee, Kyung N; Downs, Tamyra D; McKee, Patrick A

2013-01-01

Fibroblast activation protein (FAP), a membrane prolyl-specific proteinase with both dipeptidase and endopeptidase activities, is overexpressed by reactive stromal fibroblasts during epithelial-derived cancer growth. FAP digests extracellular matrix as tissue is remodeled during cancer expansion and may also promote an immunotolerant tumor microenvironment. Recent studies suggest that nonspecific FAP inhibitors suppress human cancer xenografts in mouse models. Prolyl oligopeptidase (POP), another prolyl-specific serine proteinase, is also elevated in many cancers and may have a regulatory role in angiogenesis promotion. FAP and POP cell-associated activities may be targets for diagnosis and treatment of various cancers, but their accessibilities to highly effective specific inhibitors have not been shown for cells important to cancer growth. Despite their frequent simultaneous expression in many cancers and their overlapping activities toward commonly used substrates, precise, separate measurement of FAP or POP activity has largely been ignored. To distinguish each of the two activities, we synthesized highly specific substrates and inhibitors for FAP or POP based on amino acid sequences surrounding the scissile bonds of their respective putative substrates. We found varying amounts of FAP and POP protein and activities on activated fibroblasts, mesenchymal cells, normal breast cells, and one breast cancer cell line, with some cells exhibiting more POP than FAP activity. Replicating endothelial cells (ECs) expressed POP but not FAP until tubulogenesis began. Targeting FAP-positive cells, especially mesenchymal stem cells and cancer-associated fibroblasts for inactivation or destruction, and inhibiting POP-producing EC may abrogate stromal invasion and angiogenesis simultaneously and thereby diminish cancer growth. PMID:23555181

Silibinin prevents prostate cancer cell-mediated differentiation of naïve fibroblasts into cancer-associated fibroblast phenotype by targeting TGF β2.

PubMed

Ting, Harold J; Deep, Gagan; Jain, Anil K; Cimic, Adela; Sirintrapun, Joseph; Romero, Lina M; Cramer, Scott D; Agarwal, Chapla; Agarwal, Rajesh

2015-09-01

Tumor microenvironment (TM) is an essential element in prostate cancer (PCA), offering unique opportunities for its prevention. TM includes naïve fibroblasts that are recruited by nascent neoplastic lesion and altered into 'cancer-associated fibroblasts' (CAFs) that promote PCA. A better understanding and targeting of interaction between PCA cells and fibroblasts and inhibiting CAF phenotype through non-toxic agents are novel approaches to prevent PCA progression. One well-studied cancer chemopreventive agent is silibinin, and thus, we examined its efficacy against PCA cells-mediated differentiation of naïve fibroblasts into a myofibroblastic-phenotype similar to that found in CAFs. Silibinin's direct inhibitory effect on the phenotype of CAFs derived directly from PCA patients was also assessed. Human prostate stromal cells (PrSCs) exposed to control conditioned media (CCM) from human PCA PC3 cells showed more invasiveness, with increased alpha-smooth muscle actin (α-SMA) and vimentin expression, and differentiation into a phenotype we identified in CAFs. Importantly, silibinin (at physiologically achievable concentrations) inhibited α-SMA expression and invasiveness in differentiated fibroblasts and prostate CAFs directly, as well as indirectly by targeting PCA cells. The observed increase in α-SMA and CAF-like phenotype was transforming growth factor (TGF) β2 dependent, which was strongly inhibited by silibinin. Furthermore, induction of α-SMA and CAF phenotype by CCM were also strongly inhibited by a TGFβ2-neutralizing antibody. The inhibitory effect of silibinin on TGFβ2 expression and CAF-like biomarkers was also observed in PC3 tumors. Together, these findings highlight the potential usefulness of silibinin in PCA prevention through targeting the CAF phenotype in the prostate TM. © 2014 Wiley Periodicals, Inc.

In vitro hepatic differentiation of human endometrial stromal stem cells.

PubMed

Yang, Xin-yuan; Wang, Wei; Li, Xu

2014-02-01

Human endometrial stromal stem cells (hESSCs) can differentiate into mesodermal and ectodermal cellular lineages in the endometrium. However, whether hESSCs can differentiate into functional hepatic-like cells is unknown. In this study, we developed a multiple-step induction protocol to differentiate hESSCs into functional hepatic-like cells in vitro. Endometrial stromal cells were isolated by magnetic affinity sorting using anti-epithelial cell adhesion molecule-coated Dynabeads. The enriched hESSCs were analyzed by flow cytometry and were able to differentiate into osteoblasts or adipocytes under proper induction media. To differentiate into hepatic-like cells, hESSCs were cultured in a stepwise system containing hepatocyte growth factor, fibroblast growth factor-4, oncostatin M, and trichostatin A for a total of 24 d. The hepatic-like cell differentiation was analyzed by confocal microscopy and immunocytochemical staining. Glycogen storage, cellular urea synthesis, and ammonia concentrations were measured. Hepatic-like cells were successfully generated from hESSCs and were identified by their epithelial-like shape characteristics and expression of specific biomarkers albumin and cytokeratin 8 accompanied with a reduction of alpha-fetoprotein and alpha-smooth muscle actin expression. The hepatic-like cells generated were functional as evidenced by urea synthesis and glycogen storage. Our study demonstrated that hESSCs were able to differentiate into hepatic-like cells in vitro. Thus, endometrial stromal cells may be used as an easily accessible alternative source of stem cells for potential therapeutic applications in liver disease.

Nerve growth factor effect on human primary fibroblastic-keratocytes: possible mechanism during corneal healing.

PubMed

Micera, Alessandra; Lambiase, Alessandro; Puxeddu, Ilaria; Aloe, Luigi; Stampachiacchiere, Barbara; Levi-Schaffer, Francesca; Bonini, Sergio; Bonini, Stefano

2006-10-01

In response to corneal injury, cytokines and growth factors play a crucial role by influencing epithelial-stromal interaction during the healing and reparative processes which may resolve in tissue remodeling and fibrosis. While transforming growth factor-beta1 (TGF-beta1) is considered the main profibrogenic modulator of these process, recently the nerve growth factor (NGF) appears as a pleiotropic modulator of wound-healing and inflammatory responses. Interestingly in the cornea, where NGF, trkA(NGFR) and p75(NTR) are expressed by epithelial cells and keratocytes, the NGF eye-drop induces the healing of neurotrophic or autoimmune corneal ulcers. During corneal healing, quiescent keratocytes are replaced by active fibroblast-like keratocytes/myofibroblasts. While the NGF effect on epithelial cells has been investigated, no data are reported for NGF effects on fibroblastic-keratocytes, during corneal healing. NGF, trkA(NGFR) and p75(NTR) were found expressed by fibroblastic-keratocytes. NGF was able to induce fibroblastic-keratocyte differentiation into myofibroblasts, migration, Metalloproteinase-9 expression/activity and contraction of a 3D collagen gel, without affecting their proliferation and collagen production. These data also show a two-directional control of fibroblastic-keratocytes by NGF and TGF-beta1. To sum up, the findings of this study indicate that NGF can modulate some functional activities of fibroblastic-keratocytes, thus substantiating the healing effects of NGF on corneal wound-healing.

Direct Melanoma Cell Contact Induces Stromal Cell Autocrine Prostaglandin E2-EP4 Receptor Signaling That Drives Tumor Growth, Angiogenesis, and Metastasis*

PubMed Central

Inada, Masaki; Takita, Morichika; Yokoyama, Satoshi; Watanabe, Kenta; Tominari, Tsukasa; Matsumoto, Chiho; Hirata, Michiko; Maru, Yoshiro; Maruyama, Takayuki; Sugimoto, Yukihiko; Narumiya, Shuh; Uematsu, Satoshi; Akira, Shizuo; Murphy, Gillian; Nagase, Hideaki; Miyaura, Chisato

2015-01-01

The stromal cells associated with tumors such as melanoma are significant determinants of tumor growth and metastasis. Using membrane-bound prostaglandin E synthase 1 (mPges1−/−) mice, we show that prostaglandin E2 (PGE2) production by host tissues is critical for B16 melanoma growth, angiogenesis, and metastasis to both bone and soft tissues. Concomitant studies in vitro showed that PGE2 production by fibroblasts is regulated by direct interaction with B16 cells. Autocrine activity of PGE2 further regulates the production of angiogenic factors by fibroblasts, which are key to the vascularization of both primary and metastatic tumor growth. Similarly, cell-cell interactions between B16 cells and host osteoblasts modulate mPGES-1 activity and PGE2 production by the osteoblasts. PGE2, in turn, acts to stimulate receptor activator of NF-κB ligand expression, leading to osteoclast differentiation and bone erosion. Using eicosanoid receptor antagonists, we show that PGE2 acts on osteoblasts and fibroblasts in the tumor microenvironment through the EP4 receptor. Metastatic tumor growth and vascularization in soft tissues was abrogated by an EP4 receptor antagonist. EP4-null Ptger4−/− mice do not support B16 melanoma growth. In vitro, an EP4 receptor antagonist modulated PGE2 effects on fibroblast production of angiogenic factors. Our data show that B16 melanoma cells directly influence host stromal cells to generate PGE2 signals governing neoangiogenesis and metastatic growth in bone via osteoclast erosive activity as well as angiogenesis in soft tissue tumors. PMID:26475855

Local application of periodontal ligament stromal cells promotes soft tissue regeneration.

PubMed

Baik, H S; Park, J; Lee, K J; Chung, C

2014-09-01

To test the potential stimulatory effect of local application of periodontal ligament (PDL) stromal cells on soft tissue regeneration. Fluorescently labeled PDL cells outgrown from extracted human premolars or phosphate-buffered saline were locally injected to the cutaneous wounds created on mice. Soft tissue regeneration was evaluated for 14 days using photographs and histomorphometry. PDL cell engraftment was tracked with confocal microscopy. To detect the paracrine effect of the PDL cells on soft tissue regeneration, PDL cell-conditioned medium (CM) was evaluated for the concentration of secretory factors, transforming growth factor-beta 1 (TGFβ1). The effect of PDL CM on the proliferation and migration of dermal fibroblast and keratinocyte was tested using MTT assay and migration assay. The application of PDL cells significantly promoted soft tissue regeneration compared with the application of PBS. Self-replicating PDL cells were engrafted into the hair follicles of the host tissue. Dermal fibroblast proliferation and keratinocyte migration were significantly enhanced by the treatment with PDL CM. Physiologically significant amount of TGFβ1 was secreted from PDL cells into the CM. Local injection of PDL cells promoted soft tissue regeneration in part by the enhancement of fibroblast proliferation and keratinocyte migration through a paracrine mechanism. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Influence of the interaction between nodal fibroblast and breast cancer cells on gene expression

PubMed Central

Santos, Rosângela Portilho Costa; Benvenuti, Ticiana Thomazine; Honda, Suzana Terumi; Del Valle, Paulo Roberto; Katayama, Maria Lucia Hirata; Brentani, Helena Paula; Carraro, Dirce Maria; Rozenchan, Patrícia Bortman; Brentani, Maria Mitzi; de Lyra, Eduardo Carneiro; Torres, César Henrique; Salzgeber, Marcia Batista; Kaiano, Jane Haruko Lima; Góes, João Carlos Sampaio

2010-01-01

Our aim was to evaluate the interaction between breast cancer cells and nodal fibroblasts, by means of their gene expression profile. Fibroblast primary cultures were established from negative and positive lymph nodes from breast cancer patients and a similar gene expression pattern was identified, following cell culture. Fibroblasts and breast cancer cells (MDA-MB231, MDA-MB435, and MCF7) were cultured alone or co-cultured separated by a porous membrane (which allows passage of soluble factors) for comparison. Each breast cancer lineage exerted a particular effect on fibroblasts viability and transcriptional profile. However, fibroblasts from positive and negative nodes had a parallel transcriptional behavior when co-cultured with a specific breast cancer cell line. The effects of nodal fibroblasts on breast cancer cells were also investigated. MDA MB-231 cells viability and migration were enhanced by the presence of fibroblasts and accordingly, MDA-MB435 and MCF7 cells viability followed a similar pattern. MDA-MB231 gene expression profile, as evaluated by cDNA microarray, was influenced by the fibroblasts presence, and HNMT, COMT, FN3K, and SOD2 were confirmed downregulated in MDA-MB231 co-cultured cells with fibroblasts from both negative and positive nodes, in a new series of RT-PCR assays. In summary, transcriptional changes induced in breast cancer cells by fibroblasts from positive as well as negative nodes are very much alike in a specific lineage. However, fibroblasts effects are distinct in each one of the breast cancer lineages, suggesting that the inter-relationships between stromal and malignant cells are dependent on the intrinsic subtype of the tumor. Electronic supplementary material The online version of this article (doi:10.1007/s13277-010-0108-7) contains supplementary material, which is available to authorized users. PMID:20820980

Molecular cloning and chromosomal mapping of bone marrow stromal cell surface gene, BST2, that may be involved in pre-B-cell growth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ishikawa, Jun; Kaisho, Tsuneyasu; Tomizawa, Hitoshi

1995-04-10

Bone marrow stromal cells regulate B-cell growth and development through their surface molecules and cytokines. In this study, we generated a mAb, RS38, that recognized a novel human membrane protein, BST-2, expressed on bone marrow stromal cell lines and synovial cell lines. We cloned a cDNA encoding BST-2 from a rheumatoid arthritis-derived synovial cell line. BST-2 is a 30- to 36-kDa type II transmembrane protein, consisting of 180 amino acids. The BST-2 gene (HGMW-approved symbol BST2) is located on chromosome 19p13.2. BST-2 is expressed not only on certain bone marrow stromal cell lines but also on various normal tissues, althoughmore » its expression pattern is different from that of another bone marrow stromal cell surface molecule, BST-1. BST-2 surface expression on fibroblast cell lines facilitated the stromal cell-dependent growth of a murine bone marrow-derived pre-B-cell line, DW34. The results suggest that BST-2 may be involved in pre-B-cell growth. 45 refs., 7 figs., 2 tabs.« less

Comparative characterization of stromal vascular cells derived from three types of vascular wall and adipose tissue.

PubMed

Yang, Santsun; Eto, Hitomi; Kato, Harunosuke; Doi, Kentaro; Kuno, Shinichiro; Kinoshita, Kahori; Ma, Hsu; Tsai, Chi-Han; Chou, Wan-Ting; Yoshimura, Kotaro

2013-12-01

Multipotent stem/progenitor cells localize perivascularly in many organs and vessel walls. These tissue-resident stem/progenitor cells differentiate into vascular endothelial cells, pericytes, and other mesenchymal lineages, and participate in physiological maintenance and repair of vasculatures. In this study, we characterized stromal vascular cells obtained through the explant culture method from three different vessel walls in humans: arterial wall (ART; >500 μm in diameter), venous wall (VN; >500 μm in diameter), and small vessels in adipose tissue (SV; arterioles and venules, <100 μm in diameter). These were examined for functionality and compared with adipose-derived stem/stromal cells (ASCs). All stromal vascular cells of different origins presented fibroblast-like morphology and we could not visually discriminate one population from another. Flow cytometry showed that the cultured population heterogeneously expressed a variety of surface antigens associated with stem/progenitor cells, but CD105 was expressed by most cells in all groups, suggesting that the cells generally shared the characteristics of mesenchymal stem cells. Our histological and flow cytometric data suggested that the main population of vessel wall-derived stromal vascular cells were CD34(+)/CD31(-) and came from the tunica adventitia and areola tissue surrounding the adventitia. CD271 (p75NTR) was expressed by the vasa vasorum in the VN adventitia and by a limited population in the adventitia of SV. All three populations differentiated into multiple lineages as did ASCs. ART cells induced the largest quantity of calcium formation in the osteogenic medium, whereas ASCs showed the greatest adipogenic differentiation. SV and VN stromal cells had greater potency for network formation than did ART stromal cells. In conclusion, the three stromal vascular populations exhibited differential functional properties. Our results have clinical implications for vascular diseases such as

Latrunculin B and substratum stiffness regulate corneal fibroblast to myofibroblast transformation.

PubMed

Thomasy, Sara M; Raghunathan, Vijay Krishna; Miyagi, Hidetaka; Evashenk, Alexander T; Sermeno, Jasmyne C; Tripp, Geneva K; Morgan, Joshua T; Murphy, Christopher J

2018-05-01

The transformation of keratocytes and fibroblasts to myofibroblasts is important to corneal wound healing as well as formation of stromal haze. The purpose of this study was to determine the effect of latrunculin B, an actin cytoskeleton disruptor in conjunction with a fundamental biophysical cue, substrate stiffness, on myofibroblast transformation in vitro and in vivo. Rabbit corneal fibroblasts were cultured on substrates of differing compliance (1.5, 22, and 71 kPa) and tissue culture plastic (TCP; > 1 GPa) in media containing 0 or 10 ng/ml TGFβ1 for 72 h. Cells were treated with 0.4 μM Lat-B or DMSO for 30 min every 24 h for 72 h. RNA was collected from cells and expression of alpha-smooth muscle actin (α-SMA), keratocan, and ALDH1A1 determined using qPCR; immunocytochemistry was used to assess α-SMA protein expression. A rabbit phototherapeutic keratectomy (PTK) model was used to assess the impact of 0.1% Lat-B (n = 3) or 25% DMSO (vehicle control, n = 3) on corneal wound healing by assessment of epithelial wound size with fluorescein stain and semi-quantitative stromal haze scoring by an observer masked to treatment group as well as Fourier-domain optical coherence tomography (FD-OCT) at set time points. Statistical analysis was completed using one-way or two-way analysis of variance. Treatment with Lat-B versus DMSO resulted in significantly less αSMA mRNA (P ≤ 0.007) for RCF cells grown on 22 and 71 kPa substrates as well as TCP without or with TGFβ1, and significantly decreased α-SMA protein expression in RCFs cultured on the intermediate (22 kPa) stiffness in the absence (P = 0.028) or presence (P = 0.018) of TGFβ1. Treatment with Lat-B versus DMSO but did not significantly alter expression of keratocan or ALDH1A1 mRNA in RCFs (P > 0.05) in the absence or presence of TGFβ1, but RCFs grown on stiff hydrogels (71 kPa) had significantly more keratocan mRNA expression versus the 22 kPa hydrogel or

Stromal matrix metalloproteinase 2 regulates collagen expression and promotes the outgrowth of experimental metastases.

PubMed

Bates, Andreia L; Pickup, Michael W; Hallett, Miranda A; Dozier, E Ashley; Thomas, Stacy; Fingleton, Barbara

2015-04-01

Breast cancer survival rates decrease from 99% for patients with local disease to 25% for those with distant metastases. Matrix metalloproteinases (MMPs), including MMP2, are associated with metastatic progression. We found that loss of host MMP2 reduces the proliferation of experimental metastases in the lungs and identified fibroblasts in tumour-bearing lungs as the major source of MMP2. In vitro, spheroidal mammary tumour growth was increased by co-culture with control fibroblasts isolated from tumour-bearing lungs, but not when fibroblasts with stable Mmp2 knockdown were used. This result prompted us to assess whether MMP2 was responsible for a tumour-proliferative, activated fibroblast phenotype. To test this, we evaluated: (a) fibroblasts from wild-type tumour-bearing lungs, with or without shRNA-mediated MMP2 knockdown; and (b) normal, quiescent fibroblasts isolated from either WT or Mmp2(-/-) mice. Quantitative PCR revealed that Mmp2 knockdown attenuated expression of two markers of activation (α-smooth muscle actin and vimentin), but there was minimal expression in quiescent WT or Mmp2(-/-) fibroblasts, as expected. Placing quiescent fibroblasts under activating conditions led to increases in activation-associated transcripts in WT but not Mmp2(-/-) fibroblasts. Additionally, Mmp2 knockdown fibroblasts showed significantly decreased expression of the matrix transcripts collagen I, collagen IV and fibronectin. Addition of active TGFβ was sufficient to rescue the MMP2-dependent collagen I and IV expression, while MMP2-induced collagen expression was blocked by the addition of TGFβ1-neutralizing antibody. Gene expression data in stromal cells of human breast cancers reveal that MMP2 expression is also positively correlated with activation and matrix transcripts. Thus, we present a model whereby MMP2 production in tumour fibroblasts is important for TGFβ1 activity and subsequent activation of fibroblasts to a matrix-producing, proliferation

Dataset on transcriptional profiles and the developmental characteristics of PDGFRα expressing lung fibroblasts.

PubMed

Endale, Mehari; Ahlfeld, Shawn; Bao, Erik; Chen, Xiaoting; Green, Jenna; Bess, Zach; Weirauch, Matthew; Xu, Yan; Perl, Anne Karina

2017-08-01

The following data are derived from key stages of acinar lung development and define the developmental role of lung interstitial fibroblasts expressing platelet-derived growth factor alpha (PDGFRα). This dataset is related to the research article entitled "Temporal, spatial, and phenotypical changes of PDGFRα expressing fibroblasts during late lung development" (Endale et al., 2017) [1]. At E16.5 (canalicular), E18.5 (saccular), P7 (early alveolar) and P28 (late alveolar), PDGFRα GFP mice, in conjunction with immunohistochemical markers, were utilized to define the spatiotemporal relationship of PDGFRα + fibroblasts to endothelial, stromal and epithelial cells in both the proximal and distal acinar lung. Complimentary analysis with flow cytometry was employed to determine changes in cellular proliferation, define lipofibroblast and myofibroblast populations via the presence of intracellular lipid or alpha smooth muscle actin (αSMA), and evaluate the expression of CD34, CD29, and Sca-1. Finally, PDGFRα + cells isolated at each stage of acinar lung development were subjected to RNA-Seq analysis, data was subjected to Bayesian timeline analysis and transcriptional factor promoter enrichment analysis.

Oncogenic functions of tumour suppressor p21(Waf1/Cip1/Sdi1): association with cell senescence and tumour-promoting activities of stromal fibroblasts.

PubMed

Roninson, Igor B

2002-05-08

p21(Waf1/Cip1/Sdi1) is best known as a broad-specificity inhibitor of cyclin/cyclin-dependent kinase complexes, but p21 also interacts with many other regulators of transcription or signal transduction. p21 induction, which is mediated by p53 and by p53-independent mechanisms, is essential for the onset of cell cycle arrest in damage response and cell senescence. The effects of p21 knockout in mice and its expression patterns in human cancer are consistent with a role for p21 as both a tumour suppressor and an oncogene. Several functions of p21 are likely to promote carcinogenesis and tumour progression. These include endoreduplication and abnormal mitosis that develop in tumour cells after release from p21-induced growth arrest, the ability of p21 to inhibit apoptosis through several different mechanisms, and its ability to stimulate transcription of secreted factors with mitogenic and anti-apoptotic activities. The latter effects of p21 show close resemblance to paracrine activities of senescent cells and to tumour-promoting functions of stromal fibroblasts. Therapeutic strategies targeting the oncogenic consequences of p21 expression may provide a new approach to chemoprevention and treatment of cancer.

Specific inhibition of fibroblast activation protein (FAP)-alpha prevents tumor progression in vitro.

PubMed

Teichgräber, Volker; Monasterio, Carmen; Chaitanya, Krishna; Boger, Regina; Gordon, Katrin; Dieterle, Thomas; Jäger, Dirk; Bauer, Stefan

2015-09-01

Solid tumors modulate their environment to keep non-malignant stromal cells in a tumor-promoting state. The main cells in the stroma of epithelial derived tumors are cancer associated fibroblasts (CAF) that are critical to tumorigenesis and angiogenesis. CAFs also supply the tumor cells with growth factors and extracellular matrix (ECM) degrading enzymes. They are thus essential for tumor initiation as well as tumor progression and metastasis, suggesting that they represent an ideal cellular target of an integrative tumor therapy. Fibroblast activation protein (FAP) is a well-defined marker, expressed at high levels on the cell surface of CAFs. FAP, a constitutively active serine peptidase with both dipeptidyl peptidase IV (DPP IV) and collagenase/gelatinase activity, promotes malignant and invasive behavior of epithelial cancers. High stromal expression levels of FAP correlate with poor prognosis. FAP is difficult to detect in non-diseased adult tissue, but it is generally expressed at sites of tissue remodeling. In our experiments, we aimed for a reduction of the pro-tumorigenic activities of CAFs by depleting FAP from fibroblasts growing in a composite environment with epithelial tumor cells. FAP depletion was achieved by two therapeutically relevant approaches: a novel internalizing anti-FAP IgG1 antibody and FAP gene knock-down by siRNA delivery. The antibody effectively removed FAP from the cell surface and was capable of reversing the FAP mediated migratory and invasive capacity. FAP RNA interference was equally effective when compared to the antibody. Thus, targeting FAP on CAF suppresses pro-tumorigenic activities and may result in a clinically effective reduction of tumor progression and dissemination. Copyright © 2015 Medical University of Bialystok. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Cancer-associated fibroblasts enact field cancerization by promoting extratumoral oxidative stress.

PubMed

Chan, Jeremy Soon Kiat; Tan, Ming Jie; Sng, Ming Keat; Teo, Ziqiang; Phua, Terri; Choo, Chee Chong; Li, Liang; Zhu, Pengcheng; Tan, Nguan Soon

2017-01-19

Histological inspection of visually normal tissue adjacent to neoplastic lesions often reveals multiple foci of cellular abnormalities. This suggests the presence of a regional carcinogenic signal that spreads oncogenic transformation and field cancerization. We observed an abundance of mutagenic reactive oxygen species in the stroma of cryosectioned patient tumor biopsies, indicative of extratumoral oxidative stress. Diffusible hydrogen peroxide (H 2 O 2 ) was elevated in the conditioned medium of cultured skin epithelia at various stages of oncogenic transformation, and H 2 O 2 production increased with greater tumor-forming and metastatic capacity of the studied cell lines. Explanted cancer-associated fibroblasts (CAFs) also had higher levels of H 2 O 2 secretion compared with normal fibroblasts (FIBs). These results suggest that extracellular H 2 O 2 acts as a field effect carcinogen. Indeed, H 2 O 2 -treated keratinocytes displayed decreased phosphatase and tensin homolog (PTEN) and increased Src activities because of oxidative modification. Furthermore, treating FIBs with CAF-conditioned medium or exogenous H 2 O 2 resulted in the acquisition of an oxidative, CAF-like state. In vivo, the proliferative potential and invasiveness of composite tumor xenografts comprising cancerous or non-tumor-forming epithelia with CAFs and FIBs could be attenuated by the presence of catalase. Importantly, we showed that oxidatively transformed FIBs isolated from composite tumor xenografts retained their ability to promote tumor growth and aggressiveness when adoptively transferred into new xenografts. Higher H 2 O 2 production by CAFs was contingent on impaired TGFβ signaling leading to the suppression of the antioxidant enzyme glutathione peroxidase 1 (GPX1). Finally, we detected a reduction in Smad3, TAK1 and TGFβRII expression in a cohort of 197 clinical squamous cell carcinoma (SCC) CAFs, suggesting that impaired stromal TGFβ signaling may be a clinical feature of SCC

Cancer-associated fibroblasts enact field cancerization by promoting extratumoral oxidative stress

PubMed Central

Chan, Jeremy Soon Kiat; Tan, Ming Jie; Sng, Ming Keat; Teo, Ziqiang; Phua, Terri; Choo, Chee Chong; LI, Liang; Zhu, Pengcheng; Tan, Nguan Soon

2017-01-01

Histological inspection of visually normal tissue adjacent to neoplastic lesions often reveals multiple foci of cellular abnormalities. This suggests the presence of a regional carcinogenic signal that spreads oncogenic transformation and field cancerization. We observed an abundance of mutagenic reactive oxygen species in the stroma of cryosectioned patient tumor biopsies, indicative of extratumoral oxidative stress. Diffusible hydrogen peroxide (H2O2) was elevated in the conditioned medium of cultured skin epithelia at various stages of oncogenic transformation, and H2O2 production increased with greater tumor-forming and metastatic capacity of the studied cell lines. Explanted cancer-associated fibroblasts (CAFs) also had higher levels of H2O2 secretion compared with normal fibroblasts (FIBs). These results suggest that extracellular H2O2 acts as a field effect carcinogen. Indeed, H2O2-treated keratinocytes displayed decreased phosphatase and tensin homolog (PTEN) and increased Src activities because of oxidative modification. Furthermore, treating FIBs with CAF-conditioned medium or exogenous H2O2 resulted in the acquisition of an oxidative, CAF-like state. In vivo, the proliferative potential and invasiveness of composite tumor xenografts comprising cancerous or non-tumor-forming epithelia with CAFs and FIBs could be attenuated by the presence of catalase. Importantly, we showed that oxidatively transformed FIBs isolated from composite tumor xenografts retained their ability to promote tumor growth and aggressiveness when adoptively transferred into new xenografts. Higher H2O2 production by CAFs was contingent on impaired TGFβ signaling leading to the suppression of the antioxidant enzyme glutathione peroxidase 1 (GPX1). Finally, we detected a reduction in Smad3, TAK1 and TGFβRII expression in a cohort of 197 clinical squamous cell carcinoma (SCC) CAFs, suggesting that impaired stromal TGFβ signaling may be a clinical feature of SCC. Our study indicated

Direct Melanoma Cell Contact Induces Stromal Cell Autocrine Prostaglandin E2-EP4 Receptor Signaling That Drives Tumor Growth, Angiogenesis, and Metastasis.

PubMed

Inada, Masaki; Takita, Morichika; Yokoyama, Satoshi; Watanabe, Kenta; Tominari, Tsukasa; Matsumoto, Chiho; Hirata, Michiko; Maru, Yoshiro; Maruyama, Takayuki; Sugimoto, Yukihiko; Narumiya, Shuh; Uematsu, Satoshi; Akira, Shizuo; Murphy, Gillian; Nagase, Hideaki; Miyaura, Chisato

2015-12-11

The stromal cells associated with tumors such as melanoma are significant determinants of tumor growth and metastasis. Using membrane-bound prostaglandin E synthase 1 (mPges1(-/-)) mice, we show that prostaglandin E2 (PGE2) production by host tissues is critical for B16 melanoma growth, angiogenesis, and metastasis to both bone and soft tissues. Concomitant studies in vitro showed that PGE2 production by fibroblasts is regulated by direct interaction with B16 cells. Autocrine activity of PGE2 further regulates the production of angiogenic factors by fibroblasts, which are key to the vascularization of both primary and metastatic tumor growth. Similarly, cell-cell interactions between B16 cells and host osteoblasts modulate mPGES-1 activity and PGE2 production by the osteoblasts. PGE2, in turn, acts to stimulate receptor activator of NF-κB ligand expression, leading to osteoclast differentiation and bone erosion. Using eicosanoid receptor antagonists, we show that PGE2 acts on osteoblasts and fibroblasts in the tumor microenvironment through the EP4 receptor. Metastatic tumor growth and vascularization in soft tissues was abrogated by an EP4 receptor antagonist. EP4-null Ptger4(-/-) mice do not support B16 melanoma growth. In vitro, an EP4 receptor antagonist modulated PGE2 effects on fibroblast production of angiogenic factors. Our data show that B16 melanoma cells directly influence host stromal cells to generate PGE2 signals governing neoangiogenesis and metastatic growth in bone via osteoclast erosive activity as well as angiogenesis in soft tissue tumors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

[Primary culture and characteristics of colorectal cancer-associated fibroblasts].

PubMed

Wen, Huan; Nie, Qianqian; Jiang, Zhinong; Deng, Hong

2015-10-01

To compare the biological characteristics of colorectal cancer associated fibroblasts (CAFs) with normal fibroblasts (NFs). CAFs and NFs were isolated from fresh specimens of colorectal cancer and their paired normal colon tissue and cultured by tissue explant method. Light microscopy, quantitative polymerase chain reaction (qPCR), Western blot, immunofluorescence microscopy, electron microscopy and flow cytometry were used to identify isolated fibroblasts and to explore their characteristics of activation and growth. Primary colorectal CAFs and NFs were isolated and cultured successfully. NFs showed spindled morphology and were arranged in interlacing or spiral bundles. CAFs were polygonal or spindle, but were fatter than NFs. They were distributed randomly and arranged irregularly, and had obvious actin expression. CAFs and NFs both expressed fibronectin, but not E-cadherin, CD31 and caldesmon. qPCR showed that CAFs expressed more fibroblast activation protein (FAP) and less fibroblast specific protein 1 (FSP1) than that of NFs. There was no difference in the expression of α-SMA between NFs and CAFs by Western blot. α-SMA was bundled in parallel to the long axis of the cell by immunofluorescence. By electron microscopy, CAFs but not NFs showed dense myofilament that was arranged regularly. Flow cytometry showed that the percentage of S- and G2-phase in CAFs were significantly lower than that in NFs. mRNA expression of transforming growth factor β1, stromal derived factor 1 (SDF-1) and platelet derived growth factor (PDGF)-D in CAFs were lower while that for PDGFC was higher than that in NFs. That indicated the proliferation of CAFs was inhibited and the secretion of some cytokines was different when compared with NFs. CAFs show differences with NFs in morphology, characteristics of activation and secretion of some cytokines. The proliferation of CAFs is down regulated as compared with NFs.

The application of the fibroblast activation protein α-targeted immunotherapy strategy

PubMed Central

Du, Jun; Zhang, Kun-Shui; Zhang, Qiu-Gui; Wang, Xiao-Wei; Liu, Zhi-Gang; Liu, Shuang-Quan; Xie, Wan-Ying; Liu, Hui-Fang; Liu, Jing-Shi; Wu, Bai-Ping

2016-01-01

Cancer immunotherapy has primarily been focused on attacking tumor cells. However, given the close interaction between tumor cells and cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME), CAF-targeted strategies could also contribute to an integrated cancer immunotherapy. Fibroblast activation protein α (FAP α) is not detectible in normal tissues, but is overexpressed by CAFs and is the predominant component of the stroma in most types of cancer. FAP α has both dipeptidyl peptidase and endopeptidase activities, cleaving substrates at a post-proline bond. When all FAP α-expressing cells (stromal and cancerous) are destroyed, tumors rapidly die. Furthermore, a FAP α antibody, FAP α vaccine, and modified vaccine all inhibit tumor growth and prolong survival in mouse models, suggesting FAP α is an adaptive tumor-associated antigen. This review highlights the role of FAP α in tumor development, explores the relationship between FAP α and immune suppression in the TME, and discusses FAP α as a potential immunotherapeutic target. PMID:26985769

A Role for Fibroblasts in Mediating the Effects of Tobacco-Induced Epithelial Cell Growth and Invasion

PubMed Central

Coppe, Jean-Philippe; Boysen, Megan; Ho Sun, Chung; Wong, Brian J.F.; Kang, Mo K.; Park, No-Hee; Desprez, Pierre-Yves; Campisi, Judith; Krtolica, Ana

2009-01-01

Cigarette smoke and smokeless tobacco extracts contain multiple carcinogenic compounds, but little is known about the mechanisms by which tumors develop and progress upon chronic exposure to carcinogens such as those present in tobacco products. Here, we examine the effects of smokeless tobacco extracts on human oral fibroblasts. We show that smokeless tobacco extracts elevated the levels of intracellular reactive oxygen, oxidative DNA damage, and DNA double-strand breaks in a dose-dependent manner. Extended exposure to extracts induced fibroblasts to undergo a senescence-like growth arrest, with striking accompanying changes in the secretory phenotype. Using cocultures of smokeless tobacco extracts–exposed fibroblasts and immortalized but nontumorigenic keratinocytes, we further show that factors secreted by extracts-modified fibroblasts increase the proliferation and invasiveness of partially transformed epithelial cells, but not their normal counterparts. In addition, smokeless tobacco extracts–exposed fibroblasts caused partially transformed keratinocytes to lose the expression of E-cadherin and ZO-1, as well as involucrin, changes that are indicative of compromised epithelial function and commonly associated with malignant progression. Together, our results suggest that fibroblasts may contribute to tumorigenesis indirectly by increasing epithelial cell aggressiveness. Thus, tobacco may not only initiate mutagenic changes in epithelial cells but also promote the growth and invasion of mutant cells by creating a procarcinogenic stromal environment. PMID:18644973

Fibrocytes: A Novel Stromal Cells to Regulate Resistance to Anti-Angiogenic Therapy and Cancer Progression.

PubMed

Goto, Hisatsugu; Nishioka, Yasuhiko

2017-12-29

An adequate blood supply is essential for cancer cells to survive and grow; thus, the concept of inhibiting tumor angiogenesis has been applied to cancer therapy, and several drugs are already in clinical use. It has been shown that treatment with those anti-angiogenic drugs improved the response rate and prolonged the survival of patients with various types of cancer; however, it is also true that the effect was mostly limited. Currently, the disappointing clinical results are explained by the existence of intrinsic or acquired resistance to the therapy mediated by both tumor cells and stromal cells. This article reviews the mechanisms of resistance mediated by stromal cells such as endothelial cells, pericytes, fibroblasts and myeloid cells, with an emphasis on fibrocytes, which were recently identified as the cell type responsible for regulating acquired resistance to anti-angiogenic therapy. In addition, the other emerging role of fibrocytes as mediator-producing cells in tumor progression is discussed.

Single cell sequencing reveals heterogeneity within ovarian cancer epithelium and cancer associated stromal cells.

PubMed

Winterhoff, Boris J; Maile, Makayla; Mitra, Amit Kumar; Sebe, Attila; Bazzaro, Martina; Geller, Melissa A; Abrahante, Juan E; Klein, Molly; Hellweg, Raffaele; Mullany, Sally A; Beckman, Kenneth; Daniel, Jerry; Starr, Timothy K

2017-03-01

The purpose of this study was to determine the level of heterogeneity in high grade serous ovarian cancer (HGSOC) by analyzing RNA expression in single epithelial and cancer associated stromal cells. In addition, we explored the possibility of identifying subgroups based on pathway activation and pre-defined signatures from cancer stem cells and chemo-resistant cells. A fresh, HGSOC tumor specimen derived from ovary was enzymatically digested and depleted of immune infiltrating cells. RNA sequencing was performed on 92 single cells and 66 of these single cell datasets passed quality control checks. Sequences were analyzed using multiple bioinformatics tools, including clustering, principle components analysis, and geneset enrichment analysis to identify subgroups and activated pathways. Immunohistochemistry for ovarian cancer, stem cell and stromal markers was performed on adjacent tumor sections. Analysis of the gene expression patterns identified two major subsets of cells characterized by epithelial and stromal gene expression patterns. The epithelial group was characterized by proliferative genes including genes associated with oxidative phosphorylation and MYC activity, while the stromal group was characterized by increased expression of extracellular matrix (ECM) genes and genes associated with epithelial-to-mesenchymal transition (EMT). Neither group expressed a signature correlating with published chemo-resistant gene signatures, but many cells, predominantly in the stromal subgroup, expressed markers associated with cancer stem cells. Single cell sequencing provides a means of identifying subpopulations of cancer cells within a single patient. Single cell sequence analysis may prove to be critical for understanding the etiology, progression and drug resistance in ovarian cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

Cancer associated fibroblasts (CAFs) are activated in cutaneous basal cell carcinoma and in the peritumoural skin.

PubMed

Omland, Silje Haukali; Wettergren, Erika Elgstrand; Mollerup, Sarah; Asplund, Maria; Mourier, Tobias; Hansen, Anders Johannes; Gniadecki, Robert

2017-10-07

Cutaneous basal cell carcinoma (BCC) is the commonest cancer worldwide. BCC is locally invasive and the surrounding stromal microenvironment is pivotal for tumourigenesis. Cancer associated fibroblasts (CAFs) in the microenvironment are essential for tumour growth in a variety of neoplasms but their role in BCC is poorly understood. Material included facial BCC and control skin from the peritumoural area and from the buttocks. With next-generation sequencing (NGS) we compared mRNA expression between BCC and peritumoural skin. qRT-PCR, immunohistochemical and immunofluorescent staining were performed to validate the NGS results and to investigate CAF-related cyto-and chemokines. NGS revealed upregulation of 65 genes in BCC coding for extracellular matrix components pointing at CAF-related matrix remodeling. qRT-PCR showed increased mRNA expression of CAF markers FAP-α, PDGFR-β and prolyl-4-hydroxylase in BCC. Peritumoural skin (but not buttock skin) also exhibited high expression of PDGFR-β and prolyl-4-hydroxylase but not FAP-α. We found a similar pattern for the CAF-associated chemokines CCL17, CCL18, CCL22, CCL25, CXCL12 and IL6 with high expression in BCC and peritumoural skin but absence in buttock skin. Immunofluorescence revealed correlation between FAP-α and PDGFR-β and CXCL12 and CCL17. Matrix remodeling is the most prominent molecular feature of BCC. CAFs are present within BCC stroma and associated with increased expression of chemokines involved in tumour progression and immunosuppression (CXCL12, CCL17). Fibroblasts from chronically sun-exposed skin near tumours show gene expression patterns resembling that of CAFs, indicating that stromal fibroblasts in cancer-free surgical BCC margins exhibit a tumour promoting phenotype.

A 3D Fibroblast-Epithelium Co-culture Model for Understanding Microenvironmental Role in Branching Morphogenesis of the Mammary Gland.

PubMed

Koledova, Zuzana; Lu, Pengfei

2017-01-01

The mammary gland consists of numerous tissue compartments, including mammary epithelium, an array of stromal cells, and the extracellular matrix (ECM). Bidirectional interactions between the epithelium and its surrounding stroma are essential for proper mammary gland development and homeostasis, whereas their deregulation leads to developmental abnormalities and cancer. To study the relationships between the epithelium and the stroma, development of models that could recapitulate essential aspects of these interacting systems in vitro has become necessary. Here we describe a three-dimensional (3D) co-culture assay and show that the addition of fibroblasts to mammary organoid cultures promotes the epithelium to undergo branching morphogenesis, thus allowing the role of the stromal microenvironment to be examined in this essential developmental process.

Stromal Fibroblasts from the Interface Zone of Triple Negative Breast Carcinomas Induced Epithelial-Mesenchymal Transition and its Inhibition by Emodin

PubMed Central

Wang, Hao-Yu; Hung, Chao-Ming; Lin, Ying-Chao; Ho, Chi-Tang; Way, Tzong-Der

2017-01-01

“Triple negative breast cancer” (TNBC) is associated with a higher rate and earlier time of recurrence and worse prognosis after recurrence. In this study, we aimed to examine the crosstalk between fibroblasts and TNBC cells. The fibroblasts were isolated from TNBC patients’ tissue in tumor burden zones, distal normal zones and interface zones. The fibroblasts were indicated as cancer-associated fibroblasts (CAFs), normal zone fibroblasts (NFs) and interface zone fibroblasts (INFs). Our study found that INFs grew significantly faster than NFs and CAFs in vitro. The epithelial BT20 cells cultured with the conditioned medium of INFs (INFs-CM) and CAFs (CAFs-CM) showed more spindle-like shape and cell scattering than cultured with the conditioned medium of NFs (NFs-CM). These results indicated that factors secreted by INFs-CM or CAFs-CM could induce the epithelial-mesenchymal transition (EMT) phenotype in BT20 cells. Using an in vitro co-culture model, INFs or CAFs induced EMT and promoted cancer cell migration in BT20 cells. Interestingly, we found that emodin inhibited INFs-CM or CAFs-CM-induced EMT programming and phenotype in BT20 cells. Previous studies reported that CAFs and INFs-secreted TGF-β promoted human breast cancer cell proliferation, here; our results indicated that TGF-β initiated EMT in BT20 cells. Pretreatment with emodin significantly suppressed the TGF-β-induced EMT and cell migration in BT20 cells. These results suggest that emodin may be used as a novel agent for the treatment of TNBC. PMID:28060811

Exosome-mediated delivery of miR-9 induces cancer-associated fibroblast-like properties in human breast fibroblasts

PubMed Central

Baroni, S; Romero-Cordoba, S; Plantamura, I; Dugo, M; D'Ippolito, E; Cataldo, A; Cosentino, G; Angeloni, V; Rossini, A; Daidone, M G; Iorio, M V

2016-01-01

It is established that the interaction between microenvironment and cancer cells has a critical role in tumor development, given the dependence of neoplastic cells on stromal support. However, how this communication promotes the activation of normal (NFs) into cancer-associated fibroblasts (CAFs) is still not well understood. Most microRNA (miRNA) studies focused on tumor cell, but there is increasing evidence of their involvement in reprogramming NFs into CAFs. Here we show that miR-9, upregulated in various breast cancer cell lines and identified as pro-metastatic miRNA, affects the properties of human breast fibroblasts, enhancing the switch to CAF phenotype, thus contributing to tumor growth. Expressed at higher levels in primary triple-negative breast CAFs versus NFs isolated from patients, miR-9 improves indeed migration and invasion capabilities when transfected in immortalized NFs; viceversa, these properties are strongly impaired in CAFs upon miR-9 inhibition. We also demonstrate that tumor-secreted miR-9 can be transferred via exosomes to recipient NFs and this uptake results in enhanced cell motility. Moreover, we observed that this miRNA is also secreted by fibroblasts and in turn able to alter tumor cell behavior, by modulating its direct target E-cadherin, and NFs themselves. Consistently with the biological effects observed, gene expression profiles of NFs upon transient transfection with miR-9 show the modulation of genes mainly involved in cell motility and extracellular matrix remodeling pathways. Finally, we were able to confirm the capability of NFs transiently transfected with miR-9 to promote in vivo tumor growth. Taken together, these data provide new insights into the role of miR-9 as an important player in the cross-talk between cancer cells and stroma. PMID:27468688

Exosome-mediated delivery of miR-9 induces cancer-associated fibroblast-like properties in human breast fibroblasts.

PubMed

Baroni, S; Romero-Cordoba, S; Plantamura, I; Dugo, M; D'Ippolito, E; Cataldo, A; Cosentino, G; Angeloni, V; Rossini, A; Daidone, M G; Iorio, M V

2016-07-28

It is established that the interaction between microenvironment and cancer cells has a critical role in tumor development, given the dependence of neoplastic cells on stromal support. However, how this communication promotes the activation of normal (NFs) into cancer-associated fibroblasts (CAFs) is still not well understood. Most microRNA (miRNA) studies focused on tumor cell, but there is increasing evidence of their involvement in reprogramming NFs into CAFs. Here we show that miR-9, upregulated in various breast cancer cell lines and identified as pro-metastatic miRNA, affects the properties of human breast fibroblasts, enhancing the switch to CAF phenotype, thus contributing to tumor growth. Expressed at higher levels in primary triple-negative breast CAFs versus NFs isolated from patients, miR-9 improves indeed migration and invasion capabilities when transfected in immortalized NFs; viceversa, these properties are strongly impaired in CAFs upon miR-9 inhibition. We also demonstrate that tumor-secreted miR-9 can be transferred via exosomes to recipient NFs and this uptake results in enhanced cell motility. Moreover, we observed that this miRNA is also secreted by fibroblasts and in turn able to alter tumor cell behavior, by modulating its direct target E-cadherin, and NFs themselves. Consistently with the biological effects observed, gene expression profiles of NFs upon transient transfection with miR-9 show the modulation of genes mainly involved in cell motility and extracellular matrix remodeling pathways. Finally, we were able to confirm the capability of NFs transiently transfected with miR-9 to promote in vivo tumor growth. Taken together, these data provide new insights into the role of miR-9 as an important player in the cross-talk between cancer cells and stroma.

Microencapsulated equine mesenchymal stromal cells promote cutaneous wound healing in vitro.

PubMed

Bussche, Leen; Harman, Rebecca M; Syracuse, Bethany A; Plante, Eric L; Lu, Yen-Chun; Curtis, Theresa M; Ma, Minglin; Van de Walle, Gerlinde R

2015-04-11

The prevalence of impaired cutaneous wound healing is high and treatment is difficult and often ineffective, leading to negative social and economic impacts for our society. Innovative treatments to improve cutaneous wound healing by promoting complete tissue regeneration are therefore urgently needed. Mesenchymal stromal cells (MSCs) have been reported to provide paracrine signals that promote wound healing, but (i) how they exert their effects on target cells is unclear and (ii) a suitable delivery system to supply these MSC-derived secreted factors in a controlled and safe way is unavailable. The present study was designed to provide answers to these questions by using the horse as a translational model. Specifically, we aimed to (i) evaluate the in vitro effects of equine MSC-derived conditioned medium (CM), containing all factors secreted by MSCs, on equine dermal fibroblasts, a cell type critical for successful wound healing, and (ii) explore the potential of microencapsulated equine MSCs to deliver CM to wounded cells in vitro. MSCs were isolated from the peripheral blood of healthy horses. Equine dermal fibroblasts from the NBL-6 (horse dermal fibroblast cell) line were wounded in vitro, and cell migration and expression levels of genes involved in wound healing were evaluated after treatment with MSC-CM or NBL-6-CM. These assays were repeated by using the CM collected from MSCs encapsulated in core-shell hydrogel microcapsules. Our salient findings were that equine MSC-derived CM stimulated the migration of equine dermal fibroblasts and increased their expression level of genes that positively contribute to wound healing. In addition, we found that equine MSCs packaged in core-shell hydrogel microcapsules had similar effects on equine dermal fibroblast migration and gene expression, indicating that microencapsulation of MSCs does not interfere with the release of bioactive factors. Our results demonstrate that the use of CM from MSCs might be a promising

Tumor-promoting desmoplasia is disrupted by depleting FAP-expressing stromal cells

PubMed Central

Scholler, John; Monslow, James; Avery, Diana; Newick, Kheng; O'Brien, Shaun; Evans, Rebecca A.; Bajor, David J.; Clendenin, Cynthia; Durham, Amy C; Buza, Elizabeth L; Vonderheide, Robert H; June, Carl H

2015-01-01

Malignant cells drive the generation of a desmoplastic and immunosuppressive tumor microenvironment. Cancer-associated stromal cells (CASCs) are a heterogeneous population that provides both negative and positive signals for tumor cell growth and metastasis. Fibroblast activation protein (FAP) is a marker of a major subset of CASCs in virtually all carcinomas. Clinically, FAP expression serves as an independent negative prognostic factor for multiple types of human malignancies. Prior studies established that depletion of FAP+ cells inhibits tumor growth by augmenting anti-tumor immunity. However, the potential for immune-independent effects on tumor growth have not been defined. Herein, we demonstrate that FAP+ CASCs are required for maintenance of the provisional tumor stroma since depletion of these cells, by adoptive transfer of FAP-targeted chimeric antigen receptor (CAR) T cells, reduced extracellular matrix proteins and glycosaminoglycans. Adoptive transfer of FAP-CAR T cells also decreased tumor vascular density and restrained growth of desmoplastic human lung cancer xenografts and syngeneic murine pancreatic cancers in an immune-independent fashion. Adoptive transfer of FAP-CAR T cells also restrained autochthonous pancreatic cancer growth. These data distinguish the function of FAP+ CASCs from other CASC subsets and provide support for further development of FAP+ stromal cell-targeted therapies for the treatment of solid tumors. PMID:25979873

Tumor-Promoting Desmoplasia Is Disrupted by Depleting FAP-Expressing Stromal Cells.

PubMed

Lo, Albert; Wang, Liang-Chuan S; Scholler, John; Monslow, James; Avery, Diana; Newick, Kheng; O'Brien, Shaun; Evans, Rebecca A; Bajor, David J; Clendenin, Cynthia; Durham, Amy C; Buza, Elizabeth L; Vonderheide, Robert H; June, Carl H; Albelda, Steven M; Puré, Ellen

2015-07-15

Malignant cells drive the generation of a desmoplastic and immunosuppressive tumor microenvironment. Cancer-associated stromal cells (CASC) are a heterogeneous population that provides both negative and positive signals for tumor cell growth and metastasis. Fibroblast activation protein (FAP) is a marker of a major subset of CASCs in virtually all carcinomas. Clinically, FAP expression serves as an independent negative prognostic factor for multiple types of human malignancies. Prior studies established that depletion of FAP(+) cells inhibits tumor growth by augmenting antitumor immunity. However, the potential for immune-independent effects on tumor growth have not been defined. Herein, we demonstrate that FAP(+) CASCs are required for maintenance of the provisional tumor stroma because depletion of these cells, by adoptive transfer of FAP-targeted chimeric antigen receptor (CAR) T cells, reduced extracellular matrix proteins and glycosaminoglycans. Adoptive transfer of FAP-CAR T cells also decreased tumor vascular density and restrained growth of desmoplastic human lung cancer xenografts and syngeneic murine pancreatic cancers in an immune-independent fashion. Adoptive transfer of FAP-CAR T cells also restrained autochthonous pancreatic cancer growth. These data distinguish the function of FAP(+) CASCs from other CASC subsets and provide support for further development of FAP(+) stromal cell-targeted therapies for the treatment of solid tumors. ©2015 American Association for Cancer Research.

Suppression of SRC Signaling Is Effective in Reducing Synergy between Glioblastoma and Stromal Cells.

PubMed

Calgani, Alessia; Vignaroli, Giulia; Zamperini, Claudio; Coniglio, Federica; Festuccia, Claudio; Di Cesare, Ernesto; Gravina, Giovanni Luca; Mattei, Claudia; Vitale, Flora; Schenone, Silvia; Botta, Maurizio; Angelucci, Adriano

2016-07-01

Glioblastoma cells efficiently interact with and infiltrate the surrounding normal tissue, rendering surgical resection and adjuvant chemo/radiotherapy ineffective. New therapeutic targets, able to interfere with glioblastoma's capacity to synergize with normal brain tissue, are currently under investigation. The compound Si306, a pyrazolo[3,4-d]pyrimidine derivative, selected for its favorable activity against SRC, was tested in vitro and in vivo on glioblastoma cell lines. In vivo, combination treatment with Si306 and radiotherapy was strongly active in reducing U-87 xenograft growth with respect to control and single treatments. The histology revealed a significant difference in the stromal compartment of tumoral tissue derived from control or radiotherapy-treated samples with respect to Si306-treated samples, showing in the latter a reduced presence of collagen and α-SMA-positive cells. This effect was paralleled in vitro by the capacity of Si306 to interfere with myofibroblastic differentiation of normal fibroblasts induced by U-87 cells. In the presence of Si306, TGF-β released by U-87 cells, mainly in hypoxia, was ineffective in upregulating α-SMA and β-PDGFR in fibroblasts. Si306 efficiently reached the brain and significantly prolonged the survival of mice orthotopically injected with U-87 cells. Drugs that target SRC could represent an effective therapeutic strategy in glioblastoma, able to block positive paracrine loop with stromal cells based on the β-PDGFR axis and the formation of a tumor-promoting microenvironment. This approach could be important in combination with conventional treatments in the effort to reduce tumor resistance to therapy. Mol Cancer Ther; 15(7); 1535-44. ©2016 AACR. ©2016 American Association for Cancer Research.

Pericyte–fibroblast transition promotes tumor growth and metastasis

PubMed Central

Hosaka, Kayoko; Yang, Yunlong; Seki, Takahiro; Fischer, Carina; Dubey, Olivier; Fredlund, Erik; Hartman, Johan; Religa, Piotr; Ishii, Yoko; Sasahara, Masakiyo; Larsson, Ola; Cossu, Giulio; Cao, Renhai; Lim, Sharon; Cao, Yihai

2016-01-01

Vascular pericytes, an important cellular component in the tumor microenvironment, are often associated with tumor vasculatures, and their functions in cancer invasion and metastasis are poorly understood. Here we show that PDGF-BB induces pericyte–fibroblast transition (PFT), which significantly contributes to tumor invasion and metastasis. Gain- and loss-of-function experiments demonstrate that PDGF-BB-PDGFRβ signaling promotes PFT both in vitro and in in vivo tumors. Genome-wide expression analysis indicates that PDGF-BB–activated pericytes acquire mesenchymal progenitor features. Pharmacological inhibition and genetic deletion of PDGFRβ ablate the PDGF-BB–induced PFT. Genetic tracing of pericytes with two independent mouse strains, TN-AP-CreERT2:R26R-tdTomato and NG2-CreERT2:R26R-tdTomato, shows that PFT cells gain stromal fibroblast and myofibroblast markers in tumors. Importantly, coimplantation of PFT cells with less-invasive tumor cells in mice markedly promotes tumor dissemination and invasion, leading to an increased number of circulating tumor cells and metastasis. Our findings reveal a mechanism of vascular pericytes in PDGF-BB–promoted cancer invasion and metastasis by inducing PFT, and thus targeting PFT may offer a new treatment option of cancer metastasis. PMID:27608497

Human mesenchymal stromal cells transplanted into mice stimulate renal tubular cells and enhance mitochondrial function.

PubMed

Perico, Luca; Morigi, Marina; Rota, Cinzia; Breno, Matteo; Mele, Caterina; Noris, Marina; Introna, Martino; Capelli, Chiara; Longaretti, Lorena; Rottoli, Daniela; Conti, Sara; Corna, Daniela; Remuzzi, Giuseppe; Benigni, Ariela

2017-10-17

Mesenchymal stromal cells (MSCs) are renoprotective and drive regeneration following injury, although cellular targets of such an effect are still ill-defined. Here, we show that human umbilical cord (UC)-MSCs transplanted into mice stimulate tubular cells to regain mitochondrial mass and function, associated with enhanced microtubule-rich projections that appear to mediate mitochondrial trafficking to create a reparative dialogue among adjacent tubular cells. Treatment with UC-MSCs in mice with cisplatin-induced acute kidney injury (AKI) regulates mitochondrial biogenesis in proximal tubuli by enhancing PGC1α expression, NAD + biosynthesis and Sirtuin 3 (SIRT3) activity, thus fostering antioxidant defenses and ATP production. The functional role of SIRT3 in tubular recovery is highlighted by data that in SIRT3-deficient mice with AKI, UC-MSC treatment fails to induce renoprotection. These data document a previously unrecognized mechanism through which UC-MSCs facilitate renal repair, so as to induce global metabolic reprogramming of damaged tubular cells to sustain energy supply.Mesenchymal stromal cells drive renal regeneration following injury. Here, the authors show that human mesenchymal stromal cells, when transplanted into mice with acute kidney injury, stimulate renal tubular cell growth and enhance mitochondrial function via SIRT3.

A subset of high Gleason grade prostate carcinomas contain a large burden of prostate cancer syndecan-1 positive stromal cells.

PubMed

Sharpe, Benjamin; Alghezi, Dhafer A; Cattermole, Claire; Beresford, Mark; Bowen, Rebecca; Mitchard, John; Chalmers, Andrew D

2017-05-01

There is a pressing need to identify prognostic and predictive biomarkers for prostate cancer to aid treatment decisions in both early and advanced disease settings. Syndecan-1, a heparan sulfate proteoglycan, has been previously identified as a potential prognostic biomarker by multiple studies at the tissue and serum level. However, other studies have questioned its utility. Anti-Syndecan-1 immunohistochemistry was carried out on 157 prostate tissue samples (including cancerous, adjacent normal tissue, and non-diseased prostate) from three independent cohorts of patients. A population of Syndecan-1 positive stromal cells was identified and the number and morphological parameters of these cells quantified. The identity of the Syndecan-1-positive stromal cells was assessed by multiplex immunofluorescence using a range of common cell lineage markers. Finally, the burden of Syndecan-1 positive stromal cells was tested for association with clinical parameters. We identified a previously unreported cell type which is marked by Syndecan-1 expression and is found in the stroma of prostate tumors and adjacent normal tissue but not in non-diseased prostate. We call these cells Prostate Cancer Syndecan-1 Positive (PCSP) cells. Immunofluorescence analysis revealed that the PCSP cell population did not co-stain with markers of common prostate epithelial, stromal, or immune cell populations. However, morphological analysis revealed that PCSP cells are often elongated and displayed prominent lamellipodia, suggesting they are an unidentified migratory cell population. Analysis of clinical parameters showed that PCSP cells were found with a frequency of 20-35% of all tumors evaluated, but were not present in non-diseased normal tissue. Interestingly, a subset of primary Gleason 5 prostate tumors had a high burden of PCSP cells. The current study identifies PCSP cells as a novel, potentially migratory cell type, which is marked by Syndecan-1 expression and is found in the stroma

In vitro characterization of human hair follicle dermal sheath mesenchymal stromal cells and their potential in enhancing diabetic wound healing.

PubMed

Ma, Dongrui; Kua, Jonah Ee Hsiang; Lim, Wee Keng; Lee, Seng Teik; Chua, Alvin Wen Choong

2015-08-01

Little is published on the characterization and therapeutic potential of human mesenchymal cells derived from hair follicle (HF) dermal sheath (DS). In this study, we isolated and characterized HF DS-mesenchymal stromal cells (DS-MSCs) with respect to the bone marrow mesenchymal stromal cells (BM-MSCs). We further tested if DS-MSC-conditioned medium (CM), like what was previously reported for BM-MSC CM, has superior wound-healing properties, in both in vitro and in vivo wound models compared with skin fibroblast CM. DS-MSCs were isolated from HF and cultured in vitro to assess long-term growth potential, colony-forming efficiency (CFE), expression of CD surface markers and differentiation potential. The cytokine expression of DS-MSC CM was determined through an antibody-based protein array analysis. The wound-healing effects of the CM were tested in vitro with the use of human cell cultures and in vivo with the use of a diabetic mouse wound model. In vitro results revealed that DS-MSCs have high growth capacity and CFE while displaying some phenotypes similar to BM-MSCs. DS-MSCs strongly expressed many surface markers expressed in BM-MSCs and could also differentiate into osteoblasts, chondrocytes and adipocytes. DS-MSCs secreted significantly higher proportions of paracrine factors such as interleukin-6 (IL-6), IL-8 and growth-related oncogene. DS-MSC-CM demonstrated enhanced wound-healing effects on human skin keratinocytes, fibroblasts and endothelial cells in vitro, and the wound-healing time in diabetic mice was found to be shorter, compared with vehicle controls. Human HF DS stromal cells demonstrated MSC-like properties and might be an alternative source for therapeutic use in wound healing. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Isolation and characterization of canine perivascular stem/stromal cells for bone tissue engineering.

PubMed

James, Aaron W; Zhang, Xinli; Crisan, Mihaela; Hardy, Winters R; Liang, Pei; Meyers, Carolyn A; Lobo, Sonja; Lagishetty, Venu; Childers, Martin K; Asatrian, Greg; Ding, Catherine; Yen, Yu-Hsin; Zou, Erin; Ting, Kang; Peault, Bruno; Soo, Chia

2017-01-01

For over 15 years, human subcutaneous adipose tissue has been recognized as a rich source of tissue resident mesenchymal stem/stromal cells (MSC). The isolation of perivascular progenitor cells from human adipose tissue by a cell sorting strategy was first published in 2008. Since this time, the interest in using pericytes and related perivascular stem/stromal cell (PSC) populations for tissue engineering has significantly increased. Here, we describe a set of experiments identifying, isolating and characterizing PSC from canine tissue (N = 12 canine adipose tissue samples). Results showed that the same antibodies used for human PSC identification and isolation are cross-reactive with canine tissue (CD45, CD146, CD34). Like their human correlate, canine PSC demonstrate characteristics of MSC including cell surface marker expression, colony forming unit-fibroblast (CFU-F) inclusion, and osteogenic differentiation potential. As well, canine PSC respond to osteoinductive signals in a similar fashion as do human PSC, such as the secreted differentiation factor NEL-Like Molecule-1 (NELL-1). Nevertheless, important differences exist between human and canine PSC, including differences in baseline osteogenic potential. In summary, canine PSC represent a multipotent mesenchymogenic cell source for future translational efforts in tissue engineering.

Transforming growth factor-β synthesized by stromal cells and cancer cells participates in bone resorption induced by oral squamous cell carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakamura, Ryosuke; Department of Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo; Kayamori, Kou

Transforming growth factor beta (TGF-β) plays a significant role in the regulation of the tumor microenvironment. To explore the role of TGF-β in oral cancer-induced bone destruction, we investigated the immunohistochemical localization of TGF-β and phosphorylated Smad2 (p-Smad2) in 12 surgical specimens of oral squamous cell carcinoma (OSCC). These studies revealed TGF-β and p-Smad2 expression in cancer cells in all tested cases. Several fibroblasts located between cancer nests and resorbing bone expressed TGF-β in 10 out of 12 cases and p-Smad2 in 11 out of 12 cases. Some osteoclasts also exhibited p ∼ Smad2 expression. The OSCC cell line, HSC3, and themore » bone marrow-derived fibroblastic cell line, ST2, synthesized substantial levels of TGF-β. Culture media derived from HSC3 cells could stimulate Tgf-β1 mRNA expression in ST2 cells. Recombinant TGF-β1 could stimulate osteoclast formation induced by receptor activator of nuclear factor kappa-B ligand (RANKL) in RAW264 cells. TGF-β1 could upregulate the expression of p-Smad2 in RAW264 cells, and this action was suppressed by the addition of a neutralizing antibody against TGF-β or by SB431542. Transplantation of HSC3 cells onto the calvarial region of athymic mice caused bone destruction, associated with the expression of TGF-β and p-Smad2 in both cancer cells and stromal cells. The bone destruction was substantially inhibited by the administration of SB431542. The present study demonstrated that TGF-β synthesized by both cancer cells and stromal cells participates in the OSCC-induced bone destruction. - Highlights: • Cancer cell, fibroblastic cells, and osteoclasts at bone resorbing area by oral cancer exhibited TGF-β and p-Smad2. • TGF-β1 stimulated osteoclastogenesis induced by RAKL in RAW264 cell. • Xenograft model of oral cancer-induced bone resorption was substantially inhibited by SB431542. • TGF-β synthesized by both cancer cells and stromal cells participates in the OSCC

Auto-transplanted mesenchymal stromal cell fate in periodontal tissue of beagle dogs.

PubMed

Wei, Na; Gong, Ping; Liao, Dapeng; Yang, Xingmei; Li, Xiaoyu; Liu, Yurong; Yuan, Quan; Tan, Zhen

2010-07-01

Mesenchymal stromal cells (MSC) possess multilineage differentiation potential and characteristics of self-renewal. It has been reported that MSC can acquire characteristics of cells in the periodontal ligament (PDL) in vitro. Moreover, the transplantation of MSC has been shown to be a promising strategy for treating periodontal defects. However, little is known about the fate of MSC in periodontal tissue in vivo. The aim of this study was to trace the paths of MSC after transplantation into periodontal tissues in vivo. MSC labeled with bromodeoxyuridine (BrdU) were transplanted into periodontal defects of beagle dogs. Six weeks after surgery, the animals were killed and decalcified specimens were prepared. Migration and differentiation of MSC were detected by single/double immunohistochemistry and a combination of immunohistochemistry and in situ hybridization. BrdU-labeled MSC were observed distributing into periodontal tissue that included alveolar bone, PDL, cementum and blood vessels and expressing surface markers typical of osteoblasts and fibroblasts. Cumulatively, our data suggest that MSC migrate throughout periodontal tissue and differentiate into osteoblasts and fibroblasts after transplantation into periodontal defects at 6 weeks in vivo, and have the potential to regenerate periodontal tissue.

Effects of biomaterial-derived fibroblast conditioned medium on the α-amylase expression of parotid gland acinar cells.

PubMed

Chou, Ya-Shuan; Young, Tai-Horng; Lou, Pei-Jen

2015-11-01

Salivary gland cells are surrounded by a complex stromal environment, in which fibroblasts are the main cells in proximity to the gland cells. In this study, the interaction between parotid gland acinar cells (PGACs), fibroblasts, and biomaterials was investigated. We prepared different biomaterials, including chitosan, polyvinyl alcohol (PVA), poly (ethylene-co-vinyl alcohol) (EVAL), polyvinylidene fluoride (PVDF), and tissue culture polystyrene (TCPS) to culture fibroblasts and then collect their conditioned media to culture PGACs. We observed no difference in AQP3, AQP5, and E-cadherin expression among different fibroblast conditioned medium treatments. Interestingly, α-amylase expression was obviously enhanced in PGACs cultured in the presence of conditioned medium from fibroblasts cultured on PVDF. Higher neurotrophin-4 (NT-4) expression was observed in PVDF-derived fibroblast conditioned medium using a growth factor protein array assay. In addition, directly adding NT-4 into the culture medium significantly promoted α-amylase expression by PGACs. Finally, nestin and βIII-tubulin expression by fibroblasts cultured on PVDF was also enhanced. Together, these results suggest that PVDF could promote α-amylase expression by PGACs via the NT-4 produced by fibroblasts. To date, there is no effective therapy for patients with dry mouth with persistent salivary hypofunction. The study made use of different biomaterials to culture fibroblasts and then collect their conditioned media to culture PGACs. It was found that the effect of fibroblast conditioned medium from PVDF on the α-amylase expression of PGACs was obviously enhanced and higher neurotrophin-4 (NT-4) expression was found in PVDF-derived fibroblast conditioned medium. In addition, directly adding NT-4 into the culture medium significantly promoted the expression of α-amylase by PGACs and the expression of nestin and βIII-tubulin of fibroblasts after being cultured on PVDF was enhanced. Therefore, the

Role of Corneal Stromal Cells on Epithelial Cell Function during Wound Healing

PubMed Central

Kowtharapu, Bhavani S.; Murín, Radovan; Jünemann, Anselm G. M.; Stachs, Oliver

2018-01-01

Following injury, corneal stromal keratocytes transform into repair-phenotype of activated stromal fibroblasts (SFs) and participate in wound repair. Simultaneously, ongoing bi-directional communications between corneal stromal-epithelial cells also play a vital role in mediating the process of wound healing. Factors produced by stromal cells are known to induce proliferation, differentiation, and motility of corneal epithelial cells, which are also subsequently the main processes that occur during wound healing. In this context, the present study aims to investigate the effect of SFs conditioned medium (SFCM) on corneal epithelial cell function along with substance P (SP). Antibody microarrays were employed to profile differentially expressed cell surface markers and cytokines in the presence of SFCM and SP. Antibody microarray data revealed enhanced expression of the ITGB1 in corneal epithelial cells following stimulation with SP whereas SFCM induced abundant expression of IL-8, ITGB1, PD1L1, PECA1, IL-15, BDNF, ICAM1, CD8A, CD44 and NTF4. All these proteins have either direct or indirect roles in epithelial cell growth, movement and adhesion related signaling cascades during tissue regeneration. We also observed activation of MAPK signaling pathway along with increased expression of focal adhesion kinase (FAK), paxillin, vimentin, β-catenin and vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Additionally, epithelial-to-mesenchymal transition (EMT) regulating transcription factors Slug and ZEB1 expression were enhanced in the presence of SFCM. SP enriched the expression of integrin subunits α4, α5, αV, β1 and β3 whereas SFCM increased α4, α5, αV, β1 and β5 integrin subunits. We also observed increased expression of Serpin E1 following SP and SFCM treatment. Wound healing scratch assay revealed enhanced migration of epithelial cells following the addition of SFCM. Taken together, we conclude that SFCM-mediated sustained activation of ZEB1

Effect of Metformin on Viability, Morphology, and Ultrastructure of Mouse Bone Marrow-Derived Multipotent Mesenchymal Stromal Cells and Balb/3T3 Embryonic Fibroblast Cell Line

PubMed Central

Czyrek, Aleksandra; Basinska, Katarzyna; Trynda, Justyna; Skaradzińska, Aneta; Siudzińska, Anna; Marycz, Krzysztof

2015-01-01

Metformin, a popular drug used to treat diabetes, has recently gained attention as a potentially useful therapeutic agent for treating cancer. In our research metformin was added to in vitro cultures of bone marrow-derived multipotent mesenchymal stromal cells (BMSCs) and Balb/3T3 fibroblast at concentration of 1 mM, 5 mM, and 10 mM. Obtained results indicated that metformin negatively affected proliferation activity of investigated cells. The drug triggered the formation of autophagosomes and apoptotic bodies in all tested cultures. Additionally, we focused on determination of expression of genes involved in insulin-like growth factor 2 (IGF2) signaling pathway. The most striking finding was that the mRNA level of IGF2 was constant in both BMSCs and Balb/3T3. Further, the analysis of IGF2 concentration in cell supernatants showed that it decreased in BMSC cultures after 5 and 10 mM metformin treatments. In case of Balb/3T3 the concentration of IGF2 in culture supernatants decreased after 1 and 5 mM and increased after 10 mM of metformin. Our results suggest that metformin influences the cytophysiology of somatic cells in a dose- and time-dependent manner causing inhibition of proliferation and abnormalities of their morphology and ultrastructure. PMID:26064951

Prostate cancer cells specifically reorganize epithelial cell-fibroblast communication through proteoglycan and junction pathways.

PubMed

Suhovskih, Anastasia V; Kashuba, Vladimir I; Klein, George; Grigorieva, Elvira V

2017-01-02

Microenvironment and stromal fibroblasts are able to inhibit tumor cell proliferation both through secreted signaling molecules and direct cell-cell interactions but molecular mechanisms of these effects remain unclear. In this study, we investigated a role of cell-cell contact-related molecules (protein ECM components, proteoglycans (PGs) and junction-related molecules) in intercellular communications between the human TERT immortalized fibroblasts (BjTERT fibroblasts) and normal (PNT2) or cancer (LNCaP, PC3, DU145) prostate epithelial cells. It was shown that BjTERT-PNT2 cell coculture resulted in significant decrease of both BjTERT and PNT2 proliferation rates and reorganization of transcriptional activity of cell-cell contact-related genes in both cell types. Immunocytochemical staining revealed redistribution of DCN and LUM in PNT2 cells and significant increase of SDC1 at the intercellular contact zones between BjTERT and PNT2 cells, suggesting active involvement of the PGs in cell-cell contacts and contact inhibition of cell proliferation. Unlike to PNT2 cells, PC3 cells did not respond to BjTERT in terms of PGs expression, moderately increased transcriptional activity of junctions-related genes (especially tight junction) and failed to establish PC3-BjTERT contacts. At the same time, PC3 cells significantly down-regulated junctions-related genes (especially focal adhesions and adherens junctions) in BjTERT fibroblasts resulting in visible preference for homotypic PC3-PC3 over heterotypic PC3-BjTERT contacts and autonomous growth of PC3 clones. Taken together, the results demonstrate that an instructing role of fibroblasts to normal prostate epithelial cells is revoked by cancer cells through deregulation of proteoglycans and junction molecules expression and overall disorganization of fibroblast-cancer cell communication.

GFPT2-expressing cancer-associated fibroblasts mediate metabolic reprogramming in human lung adenocarcinoma.

PubMed

Zhang, Weiruo; Bouchard, Gina; Yu, Alice; Shafiq, Majid; Jamali, Mehran; Shrager, Joseph B; Ayers, Kelsey; Bakr, Shaimaa; Gentles, Andrew J; Diehn, Maximilian; Quon, Andrew; West, Robert B; Nair, Viswam; van de Rijn, Matt; Napel, Sandy; Plevritis, Sylvia K

2018-05-14

Metabolic reprogramming of the tumor microenvironment is recognized as a cancer hallmark. To identify new molecular processes associated with tumor metabolism, we analyzed the transcriptome of bulk and flow-sorted human primary non-small cell lung cancer (NSCLC) together with 18FDG-positron emission tomography scans, which provide a clinical measure of glucose uptake. Tumors with higher glucose uptake were functionally enriched for molecular processes associated with invasion in adenocarcinoma (AD) and cell growth in squamous cell carcinoma (SCC). Next, we identified genes correlated to glucose uptake that were predominately overexpressed in a single cell-type comprising the tumor microenvironment. For SCC, most of these genes were expressed by malignant cells, whereas in AD they were predominately expressed by stromal cells, particularly cancer-associated fibroblasts (CAFs). Among these AD genes correlated to glucose uptake, we focused on Glutamine-Fructose-6-Phosphate Transaminase 2 (GFPT2), which codes for the Glutamine-Fructose-6-Phosphate Aminotransferase 2 (GFAT2), a rate-limiting enzyme of the hexosamine biosynthesis pathway (HBP), which is responsible for glycosylation. GFPT2 was predictive of glucose uptake independent of GLUT1, the primary glucose transporter, and was prognostically significant at both gene and protein level. We confirmed that normal fibroblasts transformed to CAF-like cells, following TGF-β treatment, upregulated HBP genes, including GFPT2, with less change in genes driving glycolysis, pentose phosphate pathway and TCA cycle. Our work provides new evidence of histology-specific tumor-stromal properties associated with glucose uptake in NSCLC and identifies GFPT2 as a critical regulator of tumor metabolic reprogramming in AD. Copyright ©2018, American Association for Cancer Research.

In Vitro and In Vivo Development of Horse Cloned Embryos Generated with iPSCs, Mesenchymal Stromal Cells and Fetal or Adult Fibroblasts as Nuclear Donors.

PubMed

Olivera, Ramiro; Moro, Lucia Natalia; Jordan, Roberto; Luzzani, Carlos; Miriuka, Santiago; Radrizzani, Martin; Donadeu, F Xavier; Vichera, Gabriel

2016-01-01

The demand for equine cloning as a tool to preserve high genetic value is growing worldwide; however, nuclear transfer efficiency is still very low. To address this issue, we first evaluated the effects of time from cell fusion to activation (<1h, n = 1261; 1-2h, n = 1773; 2-3h, n = 1647) on in vitro and in vivo development of equine embryos generated by cloning. Then, we evaluated the effects of using different nuclear donor cell types in two successive experiments: I) induced pluripotent stem cells (iPSCs) vs. adult fibroblasts (AF) fused to ooplasts injected with the pluripotency-inducing genes OCT4, SOX2, MYC and KLF4, vs. AF alone as controls; II) umbilical cord-derived mesenchymal stromal cells (UC-MSCs) vs. fetal fibroblasts derived from an unborn cloned foetus (FF) vs. AF from the original individual. In the first experiment, both blastocyst production and pregnancy rates were higher in the 2-3h group (11.5% and 9.5%, respectively), respect to <1h (5.2% and 2%, respectively) and 1-2h (5.6% and 4.7%, respectively) groups (P<0.05). However, percentages of born foals/pregnancies were similar when intervals of 2-3h (35.2%) or 1-2h (35.7%) were used. In contrast to AF, the iPSCs did not generate any blastocyst-stage embryos. Moreover, injection of oocytes with the pluripotency-inducing genes did not improve blastocyst production nor pregnancy rates respect to AF controls. Finally, higher blastocyst production was obtained using UC-MSC (15.6%) than using FF (8.9%) or AF (9.3%), (P<0.05). Despite pregnancy rates were similar for these 3 groups (17.6%, 18.2% and 22%, respectively), viable foals (two) were obtained only by using FF. In summary, optimum blastocyst production rates can be obtained using a 2-3h interval between cell fusion and activation as well as using UC-MSCs as nuclear donors. Moreover, FF line can improve the efficiency of an inefficient AF line. Overall, 24 healthy foals were obtained from a total of 29 born foals.

Highly Tumorigenic Diffuse Large B Cell Lymphoma Cells Are Produced by Coculture with Stromal Cells.

PubMed

Lin, Zhiguang; Chen, Bobin; Wu, Ting; Xu, Xiaoping

2018-05-23

Diffuse large B cell lymphoma (DLBCL) is heterogeneous. We aimed to explore how tumor microenvironment promotes lymphoma cell aggressiveness and heterogeneity. We created a coculture system using human DLBCL cells and mouse bone marrow stromal cells. Proliferative capacity, drug resistance, clonogenicity, and tumorigenicity were compared in lymphoma cells from the coculture system and lymphoma cells cultured alone. Expression of Notch signaling associated genes was evaluated using real-time reverse transcriptase PCR and Western blot. Lymphoma cells in the coculture system differentiated into a suspended cell group and an adherent cell group. They acquired a stronger proliferative capacity and drug resistance than lymphoma cells cultured alone, and differences existed between the adherent cell and suspended cell groups. The suspended cell group acquired the most powerful clonogenic and tumorigenic potential. However, Notch3 was exclusively expressed in the adherent lymphoma cell group and the use of N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester, an inhibitor of Notch pathway, could abolish the emergence of highly aggressive lymphoma cells. Highly tumorigenic lymphoma cells could be generated by coculture with stromal cells, and it was dependent on Notch3 expression in the adjacent lymphoma cells through interaction with stromal cells. © 2018 S. Karger AG, Basel.

The intriguing role of fibroblasts and c-Jun in the chemopreventive and therapeutic effect of finasteride on xenograft models of prostate cancer

PubMed Central

Niu, Yi-Nong; Wang, Kai; Jin, Song; Fan, Dong-Dong; Wang, Ming-Shuai; Xing, Nian-Zeng; Xia, Shu-Jie

2016-01-01

In a large clinical trial, finasteride reduced the rate of low-grade prostate cancer (PCa) while increasing the incidence of high-grade cancer. Whether finasteride promotes the development of high-grade tumors remains controversial. We demonstrated the role of fibroblasts and c-Jun in chemopreventive and therapeutic effect of finasteride on xenograft models of PCa. LNCaP (PC3) cells or recombinants of cancer cells and fibroblasts were implanted in male athymic nude mice treated with finasteride. Tumor growth, cell proliferation, apoptosis, p-Akt, and p-ERK1/2 were evaluated. In LNCaP (PC3) mono-grafted models, finasteride did not change the tumor growth. In recombinant-grafted models, fibroblasts and c-Jun promoted tumor growth; finasteride induced proliferation of LNCaP cells and repressed PC3 cell apoptosis. When c-Jun was knocked out, fibroblasts and/or finasteride did not promote the tumor growth. Finasteride inhibited p-Akt and p-ERK1/2 in mono-culture cancer cells while stimulating the same signaling molecules in the presence of fibroblasts. Reduced p-Akt and p-ERK1/2 were noted in the presence of c-Jun−/− fibroblasts. Fibroblasts and c-Jun promote PCa growth; finasteride further stimulates tumor growth with promoted proliferation, repressed apoptosis, and up-regulated pro-proliferative molecular pathway in the presence of fibroblasts and c-Jun. Stromal-epithelial interactions play critical roles in finasteride's therapeutic effects on PCa. Our findings have preliminary implications in using finasteride as a chemopreventive or therapeutic agent for PCa patients. PMID:26698232

Fibroblast activation protein augments progression and metastasis of pancreatic ductal adenocarcinoma

PubMed Central

Li, Chung-Pin; Buza, Elizabeth L.; Blomberg, Rachel; Govindaraju, Priya; Avery, Diana; Monslow, James; Hsiao, Michael

2017-01-01

Pancreatic ductal adenocarcinomas (PDAs) are desmoplastic and can undergo epithelial-to-mesenchymal transition to confer metastasis and chemoresistance. Studies have demonstrated that phenotypically and functionally distinct stromal cell populations exist in PDAs. Fibroblast activation protein–expressing (FAP-expressing) cells act to enhance PDA progression, while α–smooth muscle actin myofibroblasts can restrain PDA. Thus, identification of precise molecular targets that mediate the protumorigenic activity of FAP+ cells will guide development of therapy for PDA. Herein, we demonstrate that FAP overexpression in the tumor microenvironment correlates with poor overall and disease-free survival of PDA patients. Genetic deletion of FAP delayed onset of primary tumor and prolonged survival of mice in the KPC mouse model of PDA. While genetic deletion of FAP did not affect primary tumor weight in advanced disease, FAP deficiency increased tumor necrosis and impeded metastasis to multiple organs. Lineage-tracing studies unexpectedly showed that FAP is not only expressed by stromal cells, but can also be detected in a subset of CD90+ mesenchymal PDA cells, representing up to 20% of total intratumoral FAP+ cells. These data suggest that FAP may regulate PDA progression and metastasis in cell-autonomous and/or non-cell-autonomous fashions. Together, these data support pursuing FAP as a therapeutic target in PDA. PMID:28978805

Protective role of vitamin E preconditioning of human dermal fibroblasts against thermal stress in vitro.

PubMed

Butt, Hira; Mehmood, Azra; Ali, Muhammad; Tasneem, Saba; Anjum, Muhammad Sohail; Tarar, Moazzam N; Khan, Shaheen N; Riazuddin, Sheikh

2017-09-01

Oxidative microenvironment of burnt skin restricts the outcome of cell based therapies of thermal skin injuries. The aim of this study was to precondition human dermal fibroblasts with an antioxidant such as vitamin E to improve their survival and therapeutic abilities in heat induced oxidative in vitro environment. Fibroblasts were treated with 100μM vitamin E for 24h at 37°C followed by heat shock for 10min at 51°C in fresh serum free medium. Preconditioning with vitamin E reduced cell injury as demonstrated by decreased expression of annexin-V, cytochrome p450 (CYP450) mediated oxidative reactions, senescence and release of lactate dehydrogenase (LDH) accomplished by down-regulated expression of pro-apoptotic BAX gene. Vitamin E preconditioned cells exhibited remarkable improvement in cell viability, release of paracrine factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), stromal derived factor-1alpha (SDF-1α) and also showed significantly up-regulated levels of PCNA, VEGF, BCL-XL, FGF7, FGF23, FLNβ and Col7α genes presumably through activation of phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. The results suggest that pretreatment of fibroblasts with vitamin E prior to transplantation in burnt skin speeds up the wound healing process by improving the antioxidant scavenging responses in oxidative environment of transplanted burn wounds. Copyright © 2017 Elsevier Inc. All rights reserved.

Role of fibroblast growth factor receptor signaling in kidney development.

PubMed

Bates, Carlton M

2007-03-01

Fibroblast growth factor receptors (Fgfrs) are expressed in the ureteric bud and metanephric mesenchyme of the developing kidney. Furthermore, in vitro and in vivo studies have shown that exogenous fibroblast growth factors (Fgfs) increase growth and maturation of the metanephric mesenchyme and ureteric bud. Deletion of fgf7, fgf10, and fgfr2IIIb (the receptor isoform that binds Fgf7 and Fgf10) in mice lead to smaller kidneys with fewer collecting ducts and nephrons. Overexpression of a dominant negative receptor isoform in transgenic mice has revealed more striking defects including renal aplasia or severe dysplasia. Moreover, deletion of many fgf ligands and receptors in mice results in early embryonic lethality, making it difficult to determine their roles in kidney development. Recently, conditional targeting approaches revealed that deletion of fgf8 from the metanephric mesenchyme interrupts nephron formation. Furthermore, deletion of fgfr2 from the ureteric bud resulted in both ureteric bud branching and stromal mesenchymal patterning defects. Deletion of both fgfr1 and fgfr2 in the metanephric mesenchyme resulted in renal aplasia, characterized by defects in metanephric mesenchyme formation and initial ureteric bud elongation and branching. Thus, Fgfr signaling is critical for growth and patterning of all renal lineages at early and later stages of kidney development.

Role of fibroblast growth factor receptor signaling in kidney development.

PubMed

Bates, Carlton M

2011-09-01

Fibroblast growth factor receptors (Fgfrs) are expressed throughout the developing kidney. Several early studies have shown that exogenous fibroblast growth factors (Fgfs) affect growth and maturation of the metanephric mesenchyme (MM) and ureteric bud (UB). Transgenic mice that over-express a dominant negative receptor isoform develop renal aplasia/severe dysplasia, confirming the importance of Fgfrs in renal development. Furthermore, global deletion of Fgf7, Fgf10, and Fgfr2IIIb (isoform that binds Fgf7 and Fgf10) in mice leads to small kidneys with fewer collecting ducts and nephrons. Deletion of Fgfrl1, a receptor lacking intracellular signaling domains, causes severe renal dysgenesis. Conditional targeting of Fgf8 from the MM interrupts nephron formation. Deletion of Fgfr2 from the UB results in severe ureteric branching and stromal mesenchymal defects, although loss of Frs2α (major signaling adapter for Fgfrs) in the UB causes only mild renal hypoplasia. Deletion of both Fgfr1 and Fgfr2 in the MM results in renal aplasia with defects in MM formation and initial UB elongation and branching. Loss of Fgfr2 in the MM leads to many renal and urinary tract anomalies as well as vesicoureteral reflux. Thus, Fgfr signaling is critical for patterning of virtually all renal lineages at early and later stages of development.

Chemical Burn-Induced Stromal Demarcation Line.

PubMed

Brosh, Koby; Rozenman, Yaacov

2016-02-01

A stromal demarcation line is a well-known sign after collagen cross-linking. It has been proposed that this line is the transition zone between cellular and acellular stroma, and thus it might reveal the depth of photochemical changes in the corneal stroma. We report 2 cases of a similar demarcation line after chemical alkali burns. To the best of our knowledge, this is the first report of a stromal demarcation line after a chemical burn. Two patients presented to the emergency department after an ocular alkali burn. At presentation, both had total corneal erosion, corneal edema, and limbal ischemia. After 12 to 15 days, a stromal line was apparent by both slit-lamp examination and anterior segment optical coherence tomography. The stromal demarcation lines disappeared approximately 3 months after the injury. A stromal demarcation line may appear not only after collagen cross-linking but also after a chemical burn. The line depth may be associated with the severity of the injury, and therefore, may have prognostic significance. Patients with chemical burns should be examined for evidence of a stromal line in the cornea.

Stromal haze, myofibroblasts, and surface irregularity after PRK.

PubMed

Netto, Marcelo V; Mohan, Rajiv R; Sinha, Sunilima; Sharma, Ajay; Dupps, William; Wilson, Steven E

2006-05-01

The aim of this study was to investigate the relationship between the level of stromal surface irregularity after photorefractive keratectomy (PRK) and myofibroblast generation along with the development of corneal haze. Variable levels of stromal surface irregularity were generated in rabbit corneas by positioning a fine mesh screen in the path of excimer laser during ablation for a variable percentage of the terminal pulses of the treatment for myopia that does not otherwise generate significant opacity. Ninety-six rabbits were divided into eight groups: [see table in text]. Slit lamp analysis and haze grading were performed in all groups. Rabbits were sacrificed at 4 hr or 4 weeks after surgery and histochemical analysis was performed on corneas for apoptosis (TUNEL assay), myofibroblast marker alpha-smooth muscle actin (SMA), and integrin alpha4 to delineate the epithelial basement membrane. Slit-lamp grading revealed severe haze formation in corneas in groups IV and VI, with significantly less haze in groups II, III, and VII and insignificant haze compared with the unwounded control in groups I and V. Analysis of SMA staining at 4 weeks after surgery, the approximate peak of haze formation in rabbits, revealed low myofibroblast formation in group I (1.2+/-0.2 cells/400x field) and group V (1.8+/-0.4), with significantly more in groups II (3.5+/-1.8), III (6.8+/-1.6), VII (7.9+/-3.8), IV (12.4+/-4.2) and VI (14.6+/-5.1). The screened groups were significantly different from each other (p < 0.05), with myofibroblast generation increasing with higher surface irregularity in the -4.5 diopter PRK groups. The -9.0 diopter PRK group VI had significantly more myofibroblast generation than the -9.0 diopter PRK with PTK-smoothing group VII (p < 0.01). Areas of basement membrane disruption were demonstrated by staining corneas for integrin alpha4 and were prominent in corneas with grade I or higher haze. SMA-positive myofibroblasts tended to be present sub-adjacent to

Crosstalk between stromal cells and cancer cells in pancreatic cancer: New insights into stromal biology.

PubMed

Zhan, Han-Xiang; Zhou, Bin; Cheng, Yu-Gang; Xu, Jian-Wei; Wang, Lei; Zhang, Guang-Yong; Hu, San-Yuan

2017-04-28

Pancreatic cancer (PC) remains one of the most lethal malignancies worldwide. Increasing evidence has confirmed the pivotal role of stromal components in the regulation of carcinogenesis, invasion, metastasis, and therapeutic resistance in PC. Interaction between neoplastic cells and stromal cells builds a specific microenvironment, which further modulates the malignant properties of cancer cells. Instead of being a "passive bystander", stroma may play a role as a "partner in crime" in PC. However, the role of stromal components in PC is complex and requires further investigation. In this article, we review recent advances regarding the regulatory roles and mechanisms of stroma biology, especially the cellular components such as pancreatic stellate cells, macrophages, neutrophils, adipocytes, epithelial cells, pericytes, mast cells, and lymphocytes, in PC. Crosstalk between stromal cells and cancer cells is thoroughly investigated. We also review the prognostic value and molecular therapeutic targets of stroma in PC. This review may help us further understand the molecular mechanisms of stromal biology and its role in PC development and therapeutic resistance. Moreover, targeting stroma components may provide new therapeutic strategies for this stubborn disease. Copyright © 2017 Elsevier B.V. All rights reserved.

Papillary fibroblasts differentiate into reticular fibroblasts after prolonged in vitro culture.

PubMed

Janson, David; Saintigny, Gaëlle; Mahé, Christian; El Ghalbzouri, Abdoelwaheb

2013-01-01

The dermis can be divided into two morphologically different layers: the papillary and reticular dermis. Fibroblasts isolated from these layers behave differently when cultured in vitro. During skin ageing, the papillary dermis decreases in volume. Based on the functional differences in vitro, it is hypothesized that the loss of papillary fibroblasts contributes to skin ageing. In this study, we aimed to mimic certain aspects of skin ageing by using high-passage cultures of reticular and papillary fibroblasts and investigated the effect of these cells on skin morphogenesis in reconstructed human skin equivalents. Skin equivalents generated with reticular fibroblasts showed a reduced terminal differentiation and fewer proliferating basal keratinocytes. Aged in vitro papillary fibroblasts had increased expression of biomarkers specific to reticular fibroblasts. The phenotype and morphology of skin equivalents generated with high-passage papillary fibroblasts resembled that of reticular fibroblasts. This demonstrates that papillary fibroblasts can differentiate into reticular fibroblasts in vitro. Therefore, we hypothesize that papillary fibroblasts represent an undifferentiated phenotype, while reticular fibroblasts represent a more differentiated population. The differentiation process could be a new target for anti-skin-ageing strategies. © 2013 John Wiley & Sons A/S.

Genetics Home Reference: gastrointestinal stromal tumor

MedlinePlus

... Treatment of Gastrointestinal Stromal Tumours (Review of NICE Technology Appraisal Guidance 196) (National Institute for Health and ... Society: Treating Gastrointestinal Stromal Tumor (GIST) Cancer.Net: Gastrointestinal ...

Metabolic cooperation between cancer and non-cancerous stromal cells is pivotal in cancer progression.

PubMed

Lopes-Coelho, Filipa; Gouveia-Fernandes, Sofia; Serpa, Jacinta

2018-02-01

The way cancer cells adapt to microenvironment is crucial for the success of carcinogenesis, and metabolic fitness is essential for a cancer cell to survive and proliferate in a certain organ/tissue. The metabolic remodeling in a tumor niche is endured not only by cancer cells but also by non-cancerous cells that share the same microenvironment. For this reason, tumor cells and stromal cells constitute a complex network of signal and organic compound transfer that supports cellular viability and proliferation. The intensive dual-address cooperation of all components of a tumor sustains disease progression and metastasis. Herein, we will detail the role of cancer-associated fibroblasts, cancer-associated adipocytes, and inflammatory cells, mainly monocytes/macrophages (tumor-associated macrophages), in the remodeling and metabolic adaptation of tumors.

The use of bone marrow stromal cells (bone marrow-derived multipotent mesenchymal stromal cells) for alveolar bone tissue engineering: basic science to clinical translation.

PubMed

Kagami, Hideaki; Agata, Hideki; Inoue, Minoru; Asahina, Izumi; Tojo, Arinobu; Yamashita, Naohide; Imai, Kohzoh

2014-06-01

Bone tissue engineering is a promising field of regenerative medicine in which cultured cells, scaffolds, and osteogenic inductive signals are used to regenerate bone. Human bone marrow stromal cells (BMSCs) are the most commonly used cell source for bone tissue engineering. Although it is known that cell culture and induction protocols significantly affect the in vivo bone forming ability of BMSCs, the responsible factors of clinical outcome are poorly understood. The results from recent studies using human BMSCs have shown that factors such as passage number and length of osteogenic induction significantly affect ectopic bone formation, although such differences hardly affected the alkaline phosphatase activity or gene expression of osteogenic markers. Application of basic fibroblast growth factor helped to maintain the in vivo osteogenic ability of BMSCs. Importantly, responsiveness of those factors should be tested under clinical circumstances to improve the bone tissue engineering further. In this review, clinical application of bone tissue engineering was reviewed with putative underlying mechanisms.

CD34+ Testicular Stromal Cells Support Long-Term Expansion of Embryonic and Adult Stem and Progenitor Cells

PubMed Central

Kim, Jiyeon; Seandel, Marco; Falciatori, Ilaria; Wen, Duancheng; Rafii, Shahin

2010-01-01

Stem cells reside in specialized microenvironments created by supporting stromal cells that orchestrate self-renewal and lineage-specific differentiation. However, the precise identity of the cellular and molecular pathways that support self-renewal of stem cells is not known. For example, long-term culture of prototypical stem cells, such as adult spermatogonial stem and progenitor cells (SPCs), in vitro has been impeded by the lack of an optimal stromal cell line that initiates and sustains proliferation of these cells. Indeed, current methods, including the use of mouse embryonic fibroblasts (MEFs), have not been efficient and have generally led to inconsistent results. Here, we report the establishment of a novel CD34-positive cell line, referred to as JK1, derived from mouse testicular stromal cells that not only facilitated long-term SPC culture but also allowed faithful generation of SPCs and multipotent stem cells. SPCs generated on JK1 maintained key features of germ line stem cells, including expression of PLZF, DAZL, and GCNA. Furthermore, these feeders also promoted the long-term cultivation of other types of primitive cells including multi-potent adult spermatogonial-derived stem cells, pluripotent murine embryonic stem cells, and embryonic germ cells derived from primordial germ cells. Stem cells could be passaged serially and still maintained expression of characteristic markers such as OCT4 and NANOG in vitro, as well as the ability to generate all three germ layers in vivo. These results indicate that the JK1 cell line is capable of promoting long-term culture of primitive cells. As such, this cell line allows for identification of stromal-derived factors that support long-term proliferation of various types of stem cells and constitutes a convenient alternative to other types of feeder layers. PMID:18669907

Adipose Stromal Vascular Fraction Isolation: A Head-to-Head Comparison of 4 Cell Separation Systems #2.

PubMed

Aronowitz, Joel A; Lockhart, Ryan A; Hakakian, Cloe S; Birnbaum, Zoe E

2016-09-01

With stromal vascular fraction (SVF) cell and adipose-derived stem cell-based technologies translating into the clinical setting, numerous isolation systems have been developed for the point of care isolation of SVF cells from adipose tissue. A relative lack of performance data on these systems can make objective assessment difficult for prospective clinicians. This study compared the performance of 4 SVF cell isolation systems. Four isolation systems were compared: the MultiStation by PNC International, the LipoKit by MediKhan, the GID SVF-2 platform by GID Europe Ltd, and the StemSource 900/MB system by Cytori Therapeutics, Inc. Identical lipoaspirate samples for 5 separate donors were used. Stromal vascular fraction output was compared in terms of nucleated cell yield, viability, residual collagenase activity, sterility of the output, colony-forming unit-fibroblast frequency, frequency of CD31-/CD34+/CD45- cells, and operating statistics. Mean process time ranged from 65.4 to 120.8 minutes. Mean nucleated cell yield per milliliter of tissue processed ranged from 1.01 × 10 cells/mL to 6.24 × 10 cells/mL. Mean cellular viability ranged from 50.3% to 84.02%. Residual collagenase activity was negligible across all systems. Observed colony-forming unit-fibroblast frequency ranged from 0.495% to 1.704%. No significant difference was observed in frequency of CD31-/CD34+/CD45- cells. Results of the anaerobic/aerobic cultures were mixed. There was considerable variability between the outputs of each system. The system used by a clinician should be tailored to the individual needs of the practice. There is a range of cost options available. This study may help clinicians make more educated decisions when choosing an isolation system to meet their clinical needs.

Endometrial stromal tumors: the new WHO classification.

PubMed

Conklin, Christopher M J; Longacre, Teri A

2014-11-01

Endometrial stromal tumors are rare uterine mesenchymal neoplasms that have intrigued pathologists for years, not only because they commonly pose diagnostic dilemmas, but also because the classification and pathogenesis of these tumors has been widely debated. The current World Health Organization recognizes 4 categories of endometrial stromal tumor: endometrial stromal nodule (ESN), low-grade endometrial stromal sarcoma (LG-ESS), high-grade endometrial stromal sarcoma (HG-ESS), and undifferentiated uterine sarcoma (UUS). uterine sarcoma. These categories are defined by the presence of distinct translocations as well as tumor morphology and prognosis. Specifically, the JAZF1-SUZ12 (formerly JAZF1-JJAZ1) fusion identifies a large proportion of ESN and LG-ESSs, whereas the YWHAE-FAM22 translocation identifies HG-ESSs. The latter tumors appear to have a prognosis intermediate between LG-ESS and UUS, which exhibits no specific translocation pattern. This review (1) presents the clinicopathologic features of endometrial stromal tumors; (2) discusses their immunophenotype; and (3) highlights the recent advances in molecular genetics which explain their pathogenesis and lend support for a new classification system.

Whole transcriptome profiling of patient-derived xenograft models as a tool to identify both tumor and stromal specific biomarkers.

PubMed

Bradford, James R; Wappett, Mark; Beran, Garry; Logie, Armelle; Delpuech, Oona; Brown, Henry; Boros, Joanna; Camp, Nicola J; McEwen, Robert; Mazzola, Anne Marie; D'Cruz, Celina; Barry, Simon T

2016-04-12

The tumor microenvironment is emerging as a key regulator of cancer growth and progression, however the exact mechanisms of interaction with the tumor are poorly understood. Whilst the majority of genomic profiling efforts thus far have focused on the tumor, here we investigate RNA-Seq as a hypothesis-free tool to generate independent tumor and stromal biomarkers, and explore tumor-stroma interactions by exploiting the human-murine compartment specificity of patient-derived xenografts (PDX).Across a pan-cancer cohort of 79 PDX models, we determine that mouse stroma can be separated into distinct clusters, each corresponding to a specific stromal cell type. This implies heterogeneous recruitment of mouse stroma to the xenograft independent of tumor type. We then generate cross-species expression networks to recapitulate a known association between tumor epithelial cells and fibroblast activation, and propose a potentially novel relationship between two hypoxia-associated genes, human MIF and mouse Ddx6. Assessment of disease subtype also reveals MMP12 as a putative stromal marker of triple-negative breast cancer. Finally, we establish that our ability to dissect recruited stroma from trans-differentiated tumor cells is crucial to identifying stem-like poor-prognosis signatures in the tumor compartment.In conclusion, RNA-Seq is a powerful, cost-effective solution to global analysis of human tumor and mouse stroma simultaneously, providing new insights into mouse stromal heterogeneity and compartment-specific disease markers that are otherwise overlooked by alternative technologies. The study represents the first comprehensive analysis of its kind across multiple PDX models, and supports adoption of the approach in pre-clinical drug efficacy studies, and compartment-specific biomarker discovery.

The mandibular ridge oral mucosa model of stromal influences on the endothelial tip cells: an immunohistochemical and TEM study.

PubMed

Rusu, Mugurel Constantin; Didilescu, Andreea Cristiana; Stănescu, Ruxandra; Pop, Florinel; Mănoiu, Valentina Mariana; Jianu, Adelina Maria; Vâlcu, Marek

2013-02-01

This study aimed to evaluate by immunohistochemistry and transmission electron microscopy (TEM) the morphological features of the oral mucosa endothelial tip cells (ETCs) and to determine the immune and ultrastructural patterns of the stromal nonimmune cells which could influence healing processes. Immune labeling was performed on bioptic samples obtained from six edentulous patients undergoing surgery for dental implants placement; three normal samples were collected from patients prior to the extraction of the third mandibular molar. The antibodies were tested for CD34, CD117(c-kit), platelet derived growth factor receptor-alpha (PDGFR-α), Mast Cell Tryptase, CD44, vimentin, CD45, CD105, alpha-smooth muscle actin, FGF2, Ki67. In light microscopy, while stromal cells (StrCs) of the reparatory and normal oral mucosa, with a fibroblastic appearance, were found positive for a CD34/CD44/CD45/CD105/PDGFR-α/vimentin immune phenotype, the CD117/c-kit labeling led to a positive stromal reaction only in the reparatory mucosa. In TEM, non-immune StrCs presenting particular ultrastructural features were identified as circulating fibrocytes (CFCs). Within the lamina propria CFCs were in close contact with ETCs. Long processes of the ETCs were moniliform, and hook-like collaterals were arising from the dilated segments, suggestive for a different stage migration. Maintenance and healing of oral mucosa are so supported by extensive processes of angiogenesis, guided by ETCs that, in turn, are influenced by the CFCs that populate the stromal compartment both in normal and reparatory states. Therefore, CFCs could be targeted by specific therapies, with pro- or anti-angiogenic purposes. Copyright © 2012 Wiley Periodicals, Inc.

Stromal cell-based immunotherapy in transplantation.

PubMed

Charles, Ronald; Lu, Lina; Qian, Shiguang; Fung, John J

2011-12-01

Organs are composed of parenchymal cells that characterize organ function and nonparenchymal cells that are composed of cells in transit, as well as tissue connective tissue, also referred to as tissue stromal cells. It was originally thought that these tissue stromal cells provided only structural and functional support for parenchymal cells and were relatively inert. However, we have come to realize that tissue stromal cells, not restricted to in the thymus and lymphoid organs, also play an active role in modulating the immune system and its response to antigens. The recognition of these elements and the elucidation of their mechanisms of action have provided valuable insight into peripheral immune regulation. Extrapolation of these principles may allow us to utilize their potential for clinical application. In this article, we will summarize a number of tissue stromal elements/cell types that have been shown to induce hyporesponsiveness to transplants. We will also discuss the mechanisms by which these stromal cells create a tolerogenic environment, which in turn results in long-term allograft survival.

Effects of Prisma® Skin dermal regeneration device containing glycosaminoglycans on human keratinocytes and fibroblasts.

PubMed

Belvedere, Raffaella; Bizzarro, Valentina; Parente, Luca; Petrella, Francesco; Petrella, Antonello

2018-03-04

Prisma® Skin is a new pharmaceutical device developed by Mediolanum Farmaceutici S.p.a. It includes alginates, hyaluronic acid and mainly mesoglycan. The latter is a natural glycosaminoglycan preparation containing chondroitin sulfate, dermatan sulfate, heparan sulfate and heparin and it is used in the treatment of vascular disease. Glycosaminoglycans may contribute to the re-epithelialization in the skin wound healing, as components of the extracellular matrix. Here we describe, for the first time, the effects of Prisma® Skin in in vitro cultures of adult epidermal keratinocytes and dermal fibroblasts. Once confirmed the lack of cytotoxicity by mesoglycan and Prisma® Skin, we have shown the increase of S and G2 phases of fibroblasts cell cycle distribution. We further report the strong induction of cell migration rate and invasion capability on both cell lines, two key processes of wound repair. In support of these results, we found significant cytoskeletal reorganization, following the treatments with mesoglycan and Prisma® Skin, as confirmed by the formation of F-actin stress fibers. Additionally, together with a significant reduction of E-cadherin, keratinocytes showed an increase of CD44 expression and the translocation of ezrin to the plasma membrane, suggesting the involvement of CD44/ERM (ezrin-radixin-moesin) pathway in the induction of the analyzed processes. Furthermore, as showed by immunofluorescence assay, fibroblasts treated with mesoglycan and Prisma® Skin exhibited the increase of Fibroblast Activated Protein α and a remarkable change in shape and orientation, two common features of reactive stromal fibroblasts. In all experiments Prisma® Skin was slightly more potent than mesoglycan. In conclusion, based on these findings we suggest that Prisma® Skin may be able to accelerate the healing process in venous skin ulcers, principally enhancing re-epithelialization and granulation processes.

Hematopoietic progenitor cells grow on 3T3 fibroblast monolayers that overexpress growth arrest-specific gene-6 (GAS6)

PubMed Central

Dormady, Shane P.; Zhang, Xin-Min; Basch, Ross S.

2000-01-01

Pluripotential hematopoietic stem cells grow in close association with bone marrow stromal cells, which play a critical role in sustaining hematopoiesis in long-term bone marrow cultures. The mechanisms through which stromal cells act to support pluripotential hematopoietic stem cells are largely unknown. This study demonstrates that growth arrest-specific gene-6 (GAS6) plays an important role in this process. GAS6 is a ligand for the Axl (Ufo/Ark), Sky (Dtk/Tyro3/Rse/Brt/Tif), and Mer (Eyk) family of tyrosine kinase receptors and binds to these receptors via tandem G domains at its C terminus. After translation, GAS6 moves to the lumen of the endoplasmic reticulum, where it is extensively γ-carboxylated. The carboxylation process is vitamin K dependent, and current evidence suggests that GAS6 must be γ-carboxylated to bind and activate any of the cognate tyrosine kinase receptors. Here, we show that expression of GAS6 is highly correlated with the capacity of bone marrow stromal cells to support hematopoiesis in culture. Nonsupportive stromal cell lines express little to no GAS6, whereas supportive cell lines express high levels of GAS6. Transfection of the cDNA encoding GAS6 into 3T3 fibroblasts is sufficient to render this previously nonsupportive cell line capable of supporting long-term hematopoietic cultures. 3T3 cells, genetically engineered to stably express GAS6 (GAS6-3T3), produce a stromal layer that supports the generation of colony-forming units in culture (CFU-c) for up to 6 wk. Hematopoietic support by genetically engineered 3T3 is not vitamin K dependent, and soluble recombinant GAS6 does not substitute for coculturing the hematopoietic progenitors with genetically modified 3T3 cells. PMID:11050245

CD90/THY1 is over-expressed in prostate cancer-associated fibroblasts and could serve as a cancer biomarker

PubMed Central

True, Lawrence D; Zhang, Hui; Ye, Mingliang; Huang, Chung-Ying; Nelson, Peter S; von Haller, Priska D; Tjoelker, Larry W; Kim, Jong-Seo; Qian, Wei-Jun; Smith, Richard D; Ellis, William J; Liebeskind, Emily S; Liu, Alvin Y

2010-01-01

A by-product in the processing of prostate tissue for cell sorting by collagenase digestion is the media supernatant that remains after the cells are harvested. These supernatants contain proteins made by the cells within the tissue. Quantitative proteomic analysis of N-glycosylated proteins detected an increased amount of CD90/THY1 in cancer supernatants compared to non-cancer supernatants. Immunohistochemistry showed that in all carcinomas, regardless of Gleason grade, a layer of CD90-positive stromal fibroblastic cells, approximately 5-to-10 cells deep, was localized to tumor glands. In contrast, a no more than 1-cell wide girth of CD90-positive stromal cells was found around benign glands. The increased number of CD90-positive stromal cells in cancer correlated with overexpression of CD90 mRNA detected by gene expression analysis of stromal cells obtained by laser-capture microdissection. There is increasing evidence that cancer-associated stroma plays a role in both tumor progression and carcinogenesis. Most experiments to identify cancer biomarkers have focused on the cancer cells. CD90, being a marker for prostate cancer-associated stroma, might be a potential biomarker for this cancer. A non-invasive test could be provided by a urine test. Proteomic analysis of urine from patients with prostate cancer identified CD90; conversely, CD90 was not detected in the urine of post-prostatectomy patients. Furthermore, this urinary CD90 protein was a variant CD90 protein not known to be expressed by such cells as lymphocytes that express CD90. These CD90 results were obtained from ∼90 cases consisting of proteomic analysis of tissue and urine, immunohistochemistry, Western blot analysis of tissue media, flow cytometry of cells from digested tissue, and reverse transcriptase polymerase chain reaction analysis of isolated stromal cells. PMID:20562849

Accelerated Development of Supramolecular Corneal Stromal-Like Assemblies from Corneal Fibroblasts in the Presence of Macromolecular Crowders.

PubMed

Kumar, Pramod; Satyam, Abhigyan; Fan, Xingliang; Rochev, Yury; Rodriguez, Brian J; Gorelov, Alexander; Joshi, Lokesh; Raghunath, Michael; Pandit, Abhay; Zeugolis, Dimitrios I

2015-07-01

Tissue engineering by self-assembly uses the cells' secretome as a regeneration template and biological factory of trophic factors. Despite the several advantages that have been witnessed in preclinical and clinical settings, the major obstacle for wide acceptance of this technology remains the tardy extracellular matrix formation. In this study, we assessed the influence of macromolecular crowding (MMC)/excluding volume effect, a biophysical phenomenon that accelerates thermodynamic activities and biological processes by several orders of magnitude, in human corneal fibroblast (HCF) culture. Our data indicate that the addition of negatively charged galactose derivative (carrageenan) in HCF culture, even at 0.5% serum, increases by 12-fold tissue-specific matrix deposition, while maintaining physiological cell morphology and protein/gene expression. Gene analysis indicates that a glucose derivative (dextran sulfate) may drive corneal fibroblasts toward a myofibroblast lineage. Collectively, these results indicate that MMC may be suitable not only for clinical translation and commercialization of tissue engineering by self-assembly therapies, but also for the development of in vitro pathophysiology models.

Fibroblast-matrix interplay: Nintedanib and pirfenidone modulate the effect of IPF fibroblast-conditioned matrix on normal fibroblast phenotype.

PubMed

Epstein Shochet, Gali; Wollin, Lutz; Shitrit, David

2018-03-12

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis. Activated fibroblasts are the key effector cells in fibrosis, producing excessive amounts of collagen and extracellular matrix (ECM) proteins. Whether the ECM conditioned by IPF fibroblasts determines the phenotype of naïve fibroblasts is difficult to explore. IPF-derived primary fibroblasts were cultured on Matrigel and then cleared using ammonium hydroxide, creating an IPF-conditioned matrix (CM). Normal fibroblast CM served as control. Normal fibroblasts were cultured on both types of CM, and cell count, cell distribution and markers of myofibroblast differentiation; transforming growth factor beta (TGFβ) signalling; and ECM expression were assessed. The effects of the anti-fibrotic drugs nintedanib and pirfenidone at physiologically relevant concentrations were also explored. Normal fibroblasts cultured on IPF-CM arranged in large aggregates as a result of increased proliferation and migration. Moreover, increased levels of pSmad3, pSTAT3 (phospho signal transducer and activator of transcription 3), alpha smooth muscle actin (αSMA) and Collagen1a were found, suggesting a differentiation towards a myofibroblast-like phenotype. SB505124 (10 μmol/L) partially reversed these alterations, suggesting a TGFβ contribution. Furthermore, nintedanib at 100 nmol/L and, to a lesser extent, pirfenidone at 100 μmol/L prevented the IPF-CM-induced fibroblast phenotype alterations, suggesting an attenuation of the ECM-fibroblast interplay. IPF fibroblasts alter the ECM, thus creating a CM that further propagates an IPF-like phenotype in normal fibroblasts. This assay demonstrated differences in drug activities for approved IPF drugs at clinically relevant concentrations. Thus, the matrix-fibroblast phenotype interplay might be a relevant assay to explore drug candidates for IPF treatment. © 2018 Asian Pacific Society of Respirology.

Comparison of stromal hydration techniques for clear corneal cataract incisions: conventional hydration versus anterior stromal pocket hydration.

PubMed

Mifflin, Mark D; Kinard, Krista; Neuffer, Marcus C

2012-06-01

Anterior stromal pocket hydration was compared with conventional hydration for preventing wound leak after 2.8 mm uniplanar clear corneal incisions (CCIs) in patients having routine cataract surgery. Conventional hydration involves hydration of the lateral walls of the main incision with visible whitening of the stroma. The anterior stromal pocket hydration technique involves creation of an additional supraincisional stromal pocket overlying the main incision, which is then hydrated instead of the main incision. Sixty-six eyes of 48 patients were included in the data analysis with 33 assigned to each study group. The anterior stromal pocket hydration technique was significantly better than conventional hydration in preventing wound leak due to direct pressure on the posterior lip of the incision. Copyright © 2012 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

The prognostic value of stromal FK506-binding protein 1 and androgen receptor in prostate cancer outcome.

PubMed

Leach, Damien A; Trotta, Andrew P; Need, Eleanor F; Risbridger, Gail P; Taylor, Renea A; Buchanan, Grant

2017-02-01

Improving our ability to predict cancer progression and response to conservative or radical intent therapy is critical if we are to prevent under or over treatment of individual patients. Whereas the majority of solid tumors now have a range of molecular and/or immunological markers to help define prognosis and treatment options, prostate cancer still relies mainly on histological grading and clinical parameters. We have recently reported that androgen receptor (AR) expression in stroma inversely associates with prostate cancer-specific survival, and that stromal AR reduces metastasis. For this paper, we tested the hypothesis that the AR-regulated gene FKBP51 could be used as a marker of AR activity to better predict outcome. Using immunohistochemistry on a cohort of 64 patient-matched benign and malignant prostate tissues, we assessed patient outcome by FKBP51 and AR levels. Immunoblot and RT-qPCR were used to demonstrate androgen regulation of FKBP51 in primary and primary human prostatic fibroblasts and fibroblast cell-lines. As predicted by FKBP51 level, high AR activity in cancer stroma was associated with longer median survival (1,306 days) compared with high AR alone (699 days), whereas those with low AR and/or low FKBP51 did poorly (384 and 338 days, respectively). Survival could not be predicted on the basis cancer epithelial AR levels or activity, and was not associated with immunoreactivity in patient matched benign tissues. FKBP51 improves the ability of stromal AR to predict prostate cancer-specific mortality. By adding additional immunological assessment, similar to what is already in place in a number of other cancers, we could better serve patients with prostate cancer in prognosis and informed treatment choices. Prostate 77:185-195, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Pharmacological targeting of the PI3K/mTOR pathway alters the release of angioregulatory mediators both from primary human acute myeloid leukemia cells and their neighboring stromal cells.

PubMed

Reikvam, Håkon; Nepstad, Ina; Bruserud, Øystein; Hatfield, Kimberley Joanne

2013-06-01

Acute myeloid leukemia (AML) is a heterogeneous and aggressive malignancy with poor overall survival. Constitutive as well as cytokine-initiated activation of PI3K/Akt/mTOR signaling is a common feature of AML patients, and inhibition of this pathway is considered as a possible therapeutic strategy in AML. Human AML cells and different stromal cell populations were cultured under highly standardized in vitro conditions. We investigated the effects of mTOR inhibitors (rapamycin and temsirolimus) and PI3K inhibitors (GDC-0941 and 3-methyladenin (3-MA)) on cell proliferation and the constitutive release of angioregulatory mediators by AML and stromal cells. Primary human AML cells were heterogeneous, though most patients showed high CXCL8 levels and detectable release of CXCL10, Ang-1, HGF and MMP-9. Hierarchical clustering analysis showed that disruption of PI3K/Akt/mTOR pathways decreased AML cell release of CXCL8-11 for a large subset of patients, whereas the effects on other mediators were divergent. Various stromal cells (endothelial cells, fibroblasts, cells with osteoblastic phenotype) also showed constitutive release of angioregulatory mediators, and inhibitors of both the PI3K and mTOR pathway had anti-proliferative effects on stromal cells and resulted in decreased release of these angioregulatory mediators. PI3K and mTOR inhibitors can decrease constitutive cytokine release both by AML and stromal cells, suggesting potential direct and indirect antileukemic effects.

Pharmacological targeting of the PI3K/mTOR pathway alters the release of angioregulatory mediators both from primary human acute myeloid leukemia cells and their neighboring stromal cells

PubMed Central

Reikvam, Håkon; Nepstad, Ina; Bruserud, Øystein; Hatfield, Kimberley Joanne

2013-01-01

Acute myeloid leukemia (AML) is a heterogeneous and aggressive malignancy with poor overall survival. Constitutive as well as cytokine-initiated activation of PI3K/Akt/mTOR signaling is a common feature of AML patients, and inhibition of this pathway is considered as a possible therapeutic strategy in AML. Human AML cells and different stromal cell populations were cultured under highly standardized in vitro conditions. We investigated the effects of mTOR inhibitors (rapamycin and temsirolimus) and PI3K inhibitors (GDC-0941 and 3-methyladenin (3-MA)) on cell proliferation and the constitutive release of angioregulatory mediators by AML and stromal cells. Primary human AML cells were heterogeneous, though most patients showed high CXCL8 levels and detectable release of CXCL10, Ang-1, HGF and MMP-9. Hierarchical clustering analysis showed that disruption of PI3K/Akt/mTOR pathways decreased AML cell release of CXCL8-11 for a large subset of patients, whereas the effects on other mediators were divergent. Various stromal cells (endothelial cells, fibroblasts, cells with osteoblastic phenotype) also showed constitutive release of angioregulatory mediators, and inhibitors of both the PI3K and mTOR pathway had anti-proliferative effects on stromal cells and resulted in decreased release of these angioregulatory mediators. PI3K and mTOR inhibitors can decrease constitutive cytokine release both by AML and stromal cells, suggesting potential direct and indirect antileukemic effects. PMID:23919981

Stromal and Hematopoietic Progenitors from C57/BI/6N Murine Bone Marrow After 30-Day "BION-M1" Spaceflight.

PubMed

Markina, Elena; Andreeva, Elena; Andrianova, Irina; Sotnezova, Elena; Buravkova, Ludmila

2018-05-02

Elucidation of the spaceflight (SF) effects on the adult stem and progenitor cells is an important goal in space biology and medicine. A unique opportunity for this was provided by project "BION-M1". The purpose of this study was to evaluate the effects of 30-day SF on biosatellite, 7-day recovery (SFR), and subsequent ground control (GC) experiment on the mononuclear cells (MNCs) from C57/BI/6N murine tibia bone marrow. Also, hematopoietic and stromal precursor functions were characterized ex vivo. There was no significant difference in the total MNC number between experimental groups. After SF, immunophenotyping revealed an increase of large-sized CD45 + MNCs corresponded to committed hematopoietic progenitors. The total hematopoietic colony-forming unit (CFU) number decreased after SF and did not restore after 7 day of recovery due to predominant reduction of bi- and multipotent CFUs and primitive burst-forming units in favor of unipotent CFUs. Functional activity of stromal precursors in vitro was only slightly altered. SF cells displayed the enhanced expression of alkaline phosphatase. The data of the GC experiment demonstrated the preservation of the functional activity of progenitor cells from mice bone marrow. The activation of erythropoiesis in expense of burst-forming units of erythrocytes elevation was detected. After 7 days of recovery, the number of colony-forming units of fibroblast (CFUs-f) was similar to the vivarium control, while the proliferative activity of bone marrow stromal precursors decreased. The present study demonstrated that certain hematopoietic progenitors are susceptible to SF factors, while the stromal precursors displayed a certain degree of resistance. These data indicate mild and reversible alterations of bone marrow progenitors after SF.

Chromosome copy number variation in telomerized human bone marrow stromal cells; insights for monitoring safe ex-vivo expansion of adult stem cells.

PubMed

Burns, Jorge S; Harkness, Linda; Aldahmash, Abdullah; Gautier, Laurent; Kassem, Moustapha

2017-12-01

Adult human bone marrow stromal cells (hBMSC) cultured for cell therapy require evaluation of potency and stability for safe use. Chromosomal aberrations upsetting genomic integrity in such cells have been contrastingly described as "Limited" or "Significant". Previously reported stepwise acquisition of a spontaneous neoplastic phenotype during three-year continuous culture of telomerized cells (hBMSC-TERT20) didn't alter a diploid karyotype measured by spectral karyotype analysis (SKY). Such screening may not adequately monitor abnormal and potentially tumorigenic hBMSC in clinical scenarios. We here used array comparative genomic hybridization (aCGH) to more stringently compare non-tumorigenic parental hBMSC-TERT strains with their tumorigenic subcloned populations. Confirmation of a known chromosome 9p21 microdeletion at locus CDKN2A/B, showed it also impinged upon the adjacent MTAP gene. Compared to reference diploid human fibroblast genomic DNA, the non-tumorigenic hBMSC-TERT4 cells had a copy number variation (CNV) in at least 14 independent loci. The pre-tumorigenic hBMSC-TERT20 cell strain had further CNV including 1q44 gain enhancing SMYD3 expression and 11q13.1 loss downregulating MUS81 expression. Bioinformatic analysis of gene products reflecting 11p15.5 CNV gain in tumorigenic hBMSC-TERT20 cells highlighted networks implicated in tumorigenic progression involving cell cycle control and mis-match repair. We provide novel biomarkers for prospective risk assessment of expanded stem cell cultures. Copyright © 2017. Published by Elsevier B.V.

The differential effect of basic fibroblast growth factor and stromal cell‑derived factor‑1 pretreatment on bone morrow mesenchymal stem cells osteogenic differentiation potency.

PubMed

Wang, Ruolin; Liu, Wenhua; Du, Mi; Yang, Chengzhe; Li, Xuefen; Yang, Pishan

2018-03-01

In situ tissue engineering has become a novel strategy to repair periodontal/bone tissue defects. The choice of cytokines that promote the recruitment and proliferation, and potentiate and maintain the osteogenic differentiation ability of mesenchymal stem cells (MSCs) is the key point in this technique. Stromal cell‑derived factor‑1 (SDF‑1) and basic fibroblast growth factor (bFGF) have the ability to promote the recruitment, and proliferation of MSCs; however, the differential effect of SDF‑1 and bFGF pretreatment on MSC osteogenic differentiation potency remains to be explored. The present study comparatively observed osteogenic differentiation of bone morrow MSCs (BMMSCs) pretreated by bFGF or SDF‑1 in vitro. The gene and protein expression levels of alkaline phosphatase (ALP), runt related transcription factor 2 (Runx‑2) and bone sialoprotein (BSP) were detected using reverse transcription‑quantitative polymerase chain reaction and western blotting. The results showed that the expression of ALP mRNA on day 3, and BSP and Runx‑2 mRNA on day 7 in the bFGF pretreatment group was significantly higher than those in SDF‑1 pretreatment group. Expression levels of Runx‑2 mRNA, and ALP and Runx‑2 protein on day 3 in the SDF‑1 pretreatment group were higher than those in the bFGF pretreatment group. However, there was no significant difference in osteogenic differentiation ability on day 14 and 28 between the bFGF‑ or SDF‑1‑pretreatment groups and the control. In conclusion, bFGF and SDF‑1 pretreatment inhibits osteogenic differentiation of BMMSCs at the early stage, promotes it in the medium phase, and maintains it in the later stage during osteogenic induction, particularly at the mRNA level. Out of the two cytokines, bFGF appeared to have a greater effect on osteogenic differentiation.

Alterations of the bone marrow stromal microenvironment in adult patients with acute myeloid and lymphoblastic leukemias before and after allogeneic hematopoietic stem cell transplantation.

PubMed

Shipounova, Irina N; Petinati, Nataliya A; Bigildeev, Alexey E; Drize, Nina J; Sorokina, Tamara V; Kuzmina, Larisa A; Parovichnikova, Elena N; Savchenko, Valeri G

2017-02-01

Bone marrow (BM) derived adult multipotent mesenchymal stromal cells (MMSCs) and fibroblast colony-forming units (CFU-Fs) of 20 patients with acute myeloid leukemia (AML) and 15 patients with acute lymphoblastic leukemia (ALL) before and during 1 year after receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT) were studied. The growth characteristics of MMSCs of all patients before allo-HSCT were not altered; however, relative expression level (REL) of some genes in MMSCs, but not in CFU-Fs, from AML and ALL patients significantly changed. After allo-HSCT, CFU-F concentration and MMSC production were significantly decreased for 1 year; REL of several genes in MMSCs and CFU-F-derived colonies were also significantly downregulated. Thus, chemotherapy that was used for induction of remission did not impair the function of stromal precursors, but gene expression levels were altered. Allo-HSCT conditioning regimens significantly damaged MMSCs and CFU-Fs, and the effect lasted for at least 1 year.

Oncogenes and inflammation rewire host energy metabolism in the tumor microenvironment

PubMed Central

Martinez-Outschoorn, Ubaldo E; Curry, Joseph M; Ko, Ying-Hui; Lin, Zhao; Tuluc, Madalina; Cognetti, David; Birbe, Ruth C; Pribitkin, Edmund; Bombonati, Alessandro; Pestell, Richard G; Howell, Anthony; Sotgia, Federica; Lisanti, Michael P

2013-01-01

Here, we developed a model system to evaluate the metabolic effects of oncogene(s) on the host microenvironment. A matched set of “normal” and oncogenically transformed epithelial cell lines were co-cultured with human fibroblasts, to determine the “bystander” effects of oncogenes on stromal cells. ROS production and glucose uptake were measured by FACS analysis. In addition, expression of a panel of metabolic protein biomarkers (Caveolin-1, MCT1, and MCT4) was analyzed in parallel. Interestingly, oncogene activation in cancer cells was sufficient to induce the metabolic reprogramming of cancer-associated fibroblasts toward glycolysis, via oxidative stress. Evidence for “metabolic symbiosis” between oxidative cancer cells and glycolytic fibroblasts was provided by MCT1/4 immunostaining. As such, oncogenes drive the establishment of a stromal-epithelial “lactate-shuttle”, to fuel the anabolic growth of cancer cells. Similar results were obtained with two divergent oncogenes (RAS and NFκB), indicating that ROS production and inflammation metabolically converge on the tumor stroma, driving glycolysis and upregulation of MCT4. These findings make stromal MCT4 an attractive target for new drug discovery, as MCT4 is a shared endpoint for the metabolic effects of many oncogenic stimuli. Thus, diverse oncogenes stimulate a common metabolic response in the tumor stroma. Conversely, we also show that fibroblasts protect cancer cells against oncogenic stress and senescence by reducing ROS production in tumor cells. Ras-transformed cells were also able to metabolically reprogram normal adjacent epithelia, indicating that cancer cells can use either fibroblasts or epithelial cells as “partners” for metabolic symbiosis. The antioxidant N-acetyl-cysteine (NAC) selectively halted mitochondrial biogenesis in Ras-transformed cells, but not in normal epithelia. NAC also blocked stromal induction of MCT4, indicating that NAC effectively functions as an “MCT4

Keratinocyte growth factor expression in human gingival fibroblasts and stimulation of in vitro gene expression by retinoic acid.

PubMed

Mackenzie, I C; Gao, Z

2001-04-01

Keratinocyte growth factor (KGF) is a stromally derived growth factor of the fibroblast growth factor (FGF) family with paracrine effects targeted to influence the growth and differentiation of epithelia. Regional and temporal changes in KGF expression play important roles in the development and maintenance of epithelial structures and in epithelial wound healing. Differing patterns of expression of KGF by fibroblasts in the gingival region could therefore be related to the observed regional variation in the differentiation and behavior of gingival epithelia. The in vitro and in vivo patterns of expression of KGF mRNA in human gingival and periodontal fibroblasts were examined using reverse transcription polymerase chain reactions (RT-PCR) and in situ hybridization with digoxigenin-labeled riboprobes. The patterns observed for human gingiva were compared with those for human skin and for murine tissues. Gingival and periodontal fibroblasts showed expression of KGF transcripts in vitro, and the degree of expression was markedly influenced by the presence of retinoic acid, an agent known to influence patterns of epithelial differentiation. Sections of human and murine gingiva and skin showed regionally variable expression of transcripts with the cells expressing KGF in the subepithelial, rather than the deeper, connective tissues and periodontium. The results point to a role of KGF in the maintenance of normal growth and differentiation of gingival epithelia. A lack of KGF expression by periodontal fibroblasts in vivo is expected to hinder apical epithelial migration and thus stabilize the epithelial attachment. The effects of retinoic acid (RA) on KGF expression in vitro provide an indirect mechanism by which RA may regulate the growth and differentiation of gingival epithelia.

Columnar cell lesions and pseudoangiomatous hyperplasia like stroma: is there an epithelial-stromal interaction?

PubMed

Recavarren, Rosemary A; Chivukula, Mamatha; Carter, Gloria; Dabbs, David J

2009-10-10

The significance of association between cancer and its microenvironment has been increasingly recognized. It has been shown in animal models that interaction between neoplastic epithelial cells and adjacent stroma can modulate tumor behavior. Carcinoma associated stromal cells can transform normal epithelial cells into neoplastic cells. In breast, columnar cell lesions are non-obligate precursors of low grade ductal carcinoma in situ. Columnar cell lesions can be seen intimately associated with PASH-like-stroma, a lesion we termed as CCPLS. Our aim is to investigate epithelial-stromal interactions in CCPLS and compare them to PASH without columnar cell lesions in breast core needle biopsies. Normal terminal duct lobular unit (TDLU) epithelium was seen in association with columnar cell lesions as well as PASH. Eight (8) cases of each category were examined by a panel of immunostains: CD117 (C-kit), CD34, CD105, bFGF, AR, ER-beta, MIB-1. We observed a markedly decreased expression of c-kit in columnar cell lesions compared to TDLU-epithelium. CD105 showed a quantitative increase in activated vessels in CCPLS compared to PASH. A subset of CCPLS and PASH were androgen receptor positive. A strong nuclear positivity for ER-beta is observed in the epithelium and stroma of all CCPLS cases. We conclude that (1) activated blood vessels predominate in CCPLS; (2) A molecular alteration is signified by c-kit loss in columnar cell lesions; (3) ER-beta and androgen receptor positivity indicate CCPLS are hormonally responsive lesions. Our study suggests an intimate vascular and hormone dependent epithelial-stromal interaction exists in CCPLS lesions.

Ethanol exposure induces the cancer-associated fibroblast phenotype and lethal tumor metabolism

PubMed Central

Sanchez-Alvarez, Rosa; Martinez-Outschoorn, Ubaldo E.; Lin, Zhao; Lamb, Rebecca; Hulit, James; Howell, Anthony; Sotgia, Federica; Rubin, Emanuel; Lisanti, Michael P.

2013-01-01

Little is known about how alcohol consumption promotes the onset of human breast cancer(s). One hypothesis is that ethanol induces metabolic changes in the tumor microenvironment, which then enhances epithelial tumor growth. To experimentally test this hypothesis, we used a co-culture system consisting of human breast cancer cells (MCF7) and hTERT-immortalized fibroblasts. Here, we show that ethanol treatment (100 mM) promotes ROS production and oxidative stress in cancer-associated fibroblasts, which is sufficient to induce myofibroblastic differentiation. Oxidative stress in stromal fibroblasts also results in the onset of autophagy/mitophagy, driving the induction of ketone body production in the tumor microenvironment. Interestingly, ethanol has just the opposite effect in epithelial cancer cells, where it confers autophagy resistance, elevates mitochondrial biogenesis and induces key enzymes associated with ketone re-utilization (ACAT1/OXCT1). During co-culture, ethanol treatment also converts MCF7 cells from an ER(+) to an ER(-) status, which is thought to be associated with “stemness,” more aggressive behavior and a worse prognosis. Thus, ethanol treatment induces ketone production in cancer-associated fibroblasts and ketone re-utilization in epithelial cancer cells, fueling tumor cell growth via oxidative mitochondrial metabolism (OXPHOS). This “two-compartment” metabolic model is consistent with previous historical observations that ethanol is first converted to acetaldehyde (which induces oxidative stress) and then ultimately to acetyl-CoA (a high-energy mitochondrial fuel), or can be used to synthesize ketone bodies. As such, our results provide a novel mechanism by which alcohol consumption could metabolically convert “low-risk” breast cancer patients to “high-risk” status, explaining tumor recurrence or disease progression. Hence, our findings have clear implications for both breast cancer prevention and therapy. Remarkably, our results

PHD-2 Suppression in Mesenchymal Stromal Cells Enhances Wound Healing.

PubMed

Ko, Sae Hee; Nauta, Allison C; Morrison, Shane D; Hu, Michael S; Zimmermann, Andrew S; Chung, Michael T; Glotzbach, Jason P; Wong, Victor W; Walmsley, Graham G; Peter Lorenz, H; Chan, Denise A; Gurtner, Geoffrey C; Giaccia, Amato J; Longaker, Michael T

2018-01-01

Cell therapy with mesenchymal stromal cells is a promising strategy for tissue repair. Restoration of blood flow to ischemic tissues is a key step in wound repair, and mesenchymal stromal cells have been shown to be proangiogenic. Angiogenesis is critically regulated by the hypoxia-inducible factor (HIF) superfamily, consisting of transcription factors targeted for degradation by prolyl hydroxylase domain (PHD)-2. The aim of this study was to enhance the proangiogenic capability of mesenchymal stromal cells and to use these modified cells to promote wound healing. Mesenchymal stromal cells harvested from mouse bone marrow were transduced with short hairpin RNA (shRNA) against PHD-2; control cells were transduced with scrambled shRNA (shScramble) construct. Gene expression quantification, human umbilical vein endothelial cell tube formation assays, and wound healing assays were used to assess the effect of PHD knockdown mesenchymal stromal cells on wound healing dynamics. PHD-2 knockdown mesenchymal stromal cells overexpressed HIF-1α and multiple angiogenic factors compared to control (p < 0.05). Human umbilical vein endothelial cells treated with conditioned medium from PHD-2 knockdown mesenchymal stromal cells exhibited increased formation of capillary-like structures and enhanced migration compared with human umbilical vein endothelial cells treated with conditioned medium from shScramble-transduced mesenchymal stromal cells (p < 0.05). Wounds treated with PHD-2 knockdown mesenchymal stromal cells healed at a significantly accelerated rate compared with wounds treated with shScramble mesenchymal stromal cells (p < 0.05). Histologic studies revealed increased blood vessel density and increased cellularity in the wounds treated with PHD-2 knockdown mesenchymal stromal cells (p < 0.05). Silencing PHD-2 in mesenchymal stromal cells augments their proangiogenic potential in wound healing therapy. This effect appears to be mediated by overexpression of HIF family

Mesenteric lymph node stromal cell‐derived extracellular vesicles contribute to peripheral de novo induction of Foxp3+ regulatory T cells

PubMed Central

Pasztoi, Maria; Pezoldt, Joern; Beckstette, Michael; Lipps, Christoph; Wirth, Dagmar; Rohde, Manfred; Paloczi, Krisztina; Buzas, Edit Iren

2017-01-01

Abstract Intestinal regulatory T cells (Tregs) are fundamental in peripheral tolerance toward commensals and food‐borne antigens. Accordingly, gut‐draining mesenteric lymph nodes (mLNs) represent a site of efficient peripheral de novo Treg induction when compared to skin‐draining peripheral LNs (pLNs), and we had recently shown that LN stromal cells substantially contribute to this process. Here, we aimed to unravel the underlying molecular mechanisms and generated immortalized fibroblastic reticular cell lines (iFRCs) from mLNs and pLNs, allowing unlimited investigation of this rare stromal cell subset. In line with our previous findings, mLN‐iFRCs showed a higher Treg‐inducing capacity when compared to pLN‐iFRCs. RNA‐seq analysis focusing on secreted molecules revealed a more tolerogenic phenotype of mLN‐ as compared to pLN‐iFRCs. Remarkably, mLN‐iFRCs produced substantial numbers of microvesicles (MVs) that carried elevated levels of TGF‐β when compared to pLN‐iFRC‐derived MVs, and these novel players of intercellular communication were shown to be responsible for the tolerogenic properties of mLN‐iFRCs. Thus, stromal cells originating from mLNs contribute to peripheral tolerance by fostering de novo Treg induction using TGF‐β‐carrying MVs. This finding provides novel insights into the subcellular/molecular mechanisms of de novo Treg induction and might serve as promising tool for future therapeutic applications to treat inflammatory disorders. PMID:28833065

Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer. | Office of Cancer Genomics

Cancer.gov

Pancreatic stellate cells (PSCs) differentiate into cancer-associated fibroblasts (CAFs) that produce desmoplastic stroma, thereby modulating disease progression and therapeutic response in pancreatic ductal adenocarcinoma (PDA). However, it is unknown whether CAFs uniformly carry out these tasks or if subtypes of CAFs with distinct phenotypes in PDA exist. We identified a CAF subpopulation with elevated expression of α-smooth muscle actin (αSMA) located immediately adjacent to neoplastic cells in mouse and human PDA tissue.

Colorectal cancer cell-derived exosomes containing miR-10b regulate fibroblast cells via the PI3K/Akt pathway.

PubMed

Dai, Guangyao; Yao, Xiaoguang; Zhang, Yubin; Gu, Jianbin; Geng, Yunfeng; Xue, Fei; Zhang, Jingcheng

2018-04-01

Cancer-associated fibroblasts (CAFs) contribute to the proliferation of colorectal cancer(CRC) cells. However, the mechanism by which CAFs develop in the tumor microenvironment remains unknown. Exosomes may be involved in activating CAFs. Using a miRNA expression profiling array, we determined the miRNA expression profile of secretory exosomes in CRC cells and then identified potential miRNAs with significant differential expression compared to normal cells via enrichment analysis. Predicted targets of candidate miRNAs were then assessed via bioinformatics analysis. Realtime qPCR, western blot, and cell cycle analyses were performed to evaluate the role of candidate exosomal miRNAs. Luciferase reporter assays were applied to confirm whether candidate exosomal miRNAs control target pathway expression. A CRC xenograft mouse model was constructed to evaluate tumor growth in vivo. Exosomes from CRC cells contained significantly higher levels of miR-10b than did exosomes from normal colorectal epithelial cells. Moreover, exosomes containing miR-10b were transferred to fibroblasts. Bioinformatics analysis identified PIK3CA, as a potential target of miR-10b. Luciferase reporter assays confirmed that miR-10b directly inhibited PIK3CA expression. Co-culturing fibroblasts with exosomes containing miR-10b significantly suppressed PIK3CA expression and decreased PI3K/Akt/mTOR pathway activity. Finally, exosomes containing miR-10b reduced fibroblast proliferation but promoted expression of TGF-β and SM α-actin, suggesting that exosomal miR-10b may activate fibroblasts to become CAFs that express myofibroblast markers. These activated fibroblasts were able to promote CRC growth in vitro and in vivo. CRC-derived exosomes actively promote disease progression by modulating surrounding stromal cells, which subsequently acquire features of CAFs. Copyright © 2018 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.

Enabling screening in 3D microenvironments: probing matrix and stromal effects on the morphology and proliferation of T47D breast carcinoma cells.

PubMed

Montanez-Sauri, Sara I; Sung, Kyung Eun; Berthier, Erwin; Beebe, David J

2013-03-01

During breast carcinoma progression, the three-dimensional (3D) microenvironment is continuously remodeled, and changes in the composition of the extracellular matrix (ECM) occur. High throughput screening platforms have been used to decipher the complexity of the microenvironment and to identify ECM components responsible for cancer progression. However, traditional screening platforms are typically limited to two-dimensional (2D) cultures, and often exclude the influence of ECM and stromal components. In this work, a system that integrates 3-dimensional cell culture techniques with an automated microfluidic platform was used to create a new ECM screening platform that cultures cells in more physiologically relevant 3D in vitro microenvironments containing stromal cells and different ECM molecules. This new ECM screening platform was used to culture T47D breast carcinoma cells in mono- and co-culture with human mammary fibroblasts (HMF) with seven combinations of three different ECM proteins (collagen, fibronectin, laminin). Differences in the morphology of T47D clusters, and the proliferation of T47D cells were found in ECM compositions rich in fibronectin or laminin. In addition, an MMP enzyme activity inhibition screening showed the capabilities of the platform for small molecule screening. The platform presented in this work enables screening for the effects of matrix and stromal compositions and show promises for providing new insights in the identification of key ECM components involved in breast cancer.

Extragastrointestinal Stromal Tumor during Pregnacy.

PubMed

Gözükara, Ilay; Dilek, T U Kutlu; Durukan, Hüseyin; Düsmez Apa, Duygu; Kabil Kucur, Suna; Dilek, Saffet

2012-01-01

Extragastrointestinal stromal tumors (EGISTs) are mesenchymal neoplasms without connection to the gastrointestinal tract. Gastrointestinal stromal tumors (GISTs) and EGIST are similar according to their clinicopathologic and histomorphologic features. Both of them most often express immunoreactivity for CD-117, a c-kit proto-oncogene protein. The coexistence of GIST and pregnancy is very rare, with only two cases reported in the literature. In this paper, we presented the first EGIST case during pregnancy in the literature.

Matrix metalloproteinase (MMP)-9 in cancer-associated fibroblasts (CAFs) is suppressed by omega-3 polyunsaturated fatty acids in vitro and in vivo.

PubMed

Taguchi, Ayumi; Kawana, Kei; Tomio, Kensuke; Yamashita, Aki; Isobe, Yosuke; Nagasaka, Kazunori; Koga, Kaori; Inoue, Tomoko; Nishida, Haruka; Kojima, Satoko; Adachi, Katsuyuki; Matsumoto, Yoko; Arimoto, Takahide; Wada-Hiraike, Osamu; Oda, Katsutoshi; Kang, Jing X; Arai, Hiroyuki; Arita, Makoto; Osuga, Yutaka; Fujii, Tomoyuki

2014-01-01

Cancer associated fibroblasts (CAFs) are responsible for tumor growth, angiogenesis, invasion, and metastasis. Matrix metalloproteinase (MMP)-9 secreted from cancer stroma populated by CAFs is a prerequisite for cancer angiogenesis and metastasis. Omega-3 polyunsaturated fatty acids (omega-3 PUFA) have been reported to have anti-tumor effects on diverse types of malignancies. Fat-1 mice, which can convert omega-6 to omega-3 PUFA independent of diet, are useful to investigate the functions of endogenous omega-3 PUFA. To examine the effect of omega-3 PUFA on tumorigenesis, TC-1 cells, a murine epithelial cell line immortalized by human papillomavirus (HPV) oncogenes, were injected subcutaneously into fat-1 or wild type mice. Tumor growth and angiogenesis of the TC-1 tumor were significantly suppressed in fat-1 compared to wild type mice. cDNA microarray of the tumors derived from fat-1 and wild type mice revealed that MMP-9 is downregulated in fat-1 mice. Immunohistochemical study demonstrated immunoreactivity for MMP-9 in the tumor stromal fibroblasts was diffusely positive in wild type whereas focal in fat-1 mice. MMP-9 was expressed in primary cultured fibroblasts isolated from fat-1 and wild type mice but was not expressed in TC-1 cells. Co-culture of fibroblasts with TC-1 cells enhanced the expression and the proteinase activity of MMP-9, although the protease activity of MMP-9 in fat-1-derived fibroblasts was lower than that in wild type fibroblasts. Our data suggests that omega-3 PUFAs suppress MMP-9 induction and tumor angiogenesis. These findings may provide insight into mechanisms by which omega-3 PUFAs exert anti-tumor effects by modulating tumor microenvironment.

Nuclear alternate estrogen receptor GPR30 mediates 17beta-estradiol-induced gene expression and migration in breast cancer-associated fibroblasts.

PubMed

Madeo, Antonio; Maggiolini, Marcello

2010-07-15

Fibroblasts are the principal cellular component of connective tissue and are associated with cancer cells at all stages of tumor progression. Structural and functional contributions of fibroblasts to the growth, survival, and invasive capacity of cancer cells are beginning to emerge. In breast carcinoma, approximately 80% of stromal fibroblasts termed cancer-associated fibroblasts (CAF) are thought to manifest an activated phenotype that promotes cancer cell proliferation tumor growth at metastatic sites similar to the primary tumor. In this report, we show that CAFs respond to physiologic concentrations of 17beta-estradiol (E2) by rapidly inducing extracellular signal-regulated kinase phosphorylation and immediate early gene expression, including c-fos and connective tissue growth factor, and cyclin D1. Notably, the E2 response is mediated by the alternate estrogen receptor GPR30, which interfaces with the epidermal growth factor receptor (EGFR) signaling pathway. In particular, E2 stimulates a physical interaction between GPR30 and phosphorylated EGFR, recruiting them to the cyclin D1 gene promoter. Nuclear localization induced by E2 was confirmed by cellular immunofluorescence methods. GPR30 was required for CAF proliferation and migration induced by E2. Our results provide important new mechanistic insights into how CAFs are stimulated by estrogen through a GPR30-mediated nuclear signaling pathway. More generally, they define estrogenic GPR30 signaling as a functionally important component of the tumor microenvironment. (c)2010 AACR.

Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblasts synergizes with anti-PD-L1 immunotherapy in pancreatic cancer.

PubMed

Feig, Christine; Jones, James O; Kraman, Matthew; Wells, Richard J B; Deonarine, Andrew; Chan, Derek S; Connell, Claire M; Roberts, Edward W; Zhao, Qi; Caballero, Otavia L; Teichmann, Sarah A; Janowitz, Tobias; Jodrell, Duncan I; Tuveson, David A; Fearon, Douglas T

2013-12-10

An autochthonous model of pancreatic ductal adenocarcinoma (PDA) permitted the analysis of why immunotherapy is ineffective in this human disease. Despite finding that PDA-bearing mice had cancer cell-specific CD8(+) T cells, the mice, like human patients with PDA, did not respond to two immunological checkpoint antagonists that promote the function of T cells: anti-cytotoxic T-lymphocyte-associated protein 4 (α-CTLA-4) and α-programmed cell death 1 ligand 1 (α-PD-L1). Immune control of PDA growth was achieved, however, by depleting carcinoma-associated fibroblasts (CAFs) that express fibroblast activation protein (FAP). The depletion of the FAP(+) stromal cell also uncovered the antitumor effects of α-CTLA-4 and α-PD-L1, indicating that its immune suppressive activity accounts for the failure of these T-cell checkpoint antagonists. Three findings suggested that chemokine (C-X-C motif) ligand 12 (CXCL12) explained the overriding immunosuppression by the FAP(+) cell: T cells were absent from regions of the tumor containing cancer cells, cancer cells were coated with the chemokine, CXCL12, and the FAP(+) CAF was the principal source of CXCL12 in the tumor. Administering AMD3100, a CXCL12 receptor chemokine (C-X-C motif) receptor 4 inhibitor, induced rapid T-cell accumulation among cancer cells and acted synergistically with α-PD-L1 to greatly diminish cancer cells, which were identified by their loss of heterozygosity of Trp53 gene. The residual tumor was composed only of premalignant epithelial cells and inflammatory cells. Thus, a single protein, CXCL12, from a single stromal cell type, the FAP(+) CAF, may direct tumor immune evasion in a model of human PDA.

Hedgehog signaling contributes to basic fibroblast growth factor-regulated fibroblast migration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu, Zhong Xin; Sun, Cong Cong; Wenzhou People's Hospital, Wenzhou, Zhejiang

Fibroblast migration is a central process in skin wound healing, which requires the coordination of several types of growth factors. bFGF, a well-known fibroblast growth factor (FGF), is able to accelerate fibroblast migration; however, the underlying mechanism of bFGF regulation fibroblast migration remains unclear. Through the RNA-seq analysis, we had identified that the hedgehog (Hh) canonical pathway genes including Smoothened (Smo) and Gli1, were regulated by bFGF. Further analysis revealed that activation of the Hh pathway via up-regulation of Smo promoted fibroblast migration, invasion, and skin wound healing, but which significantly reduced by GANT61, a selective antagonist of Gli1/Gli2. Westernmore » blot analyses and siRNA transfection assays demonstrated that Smo acted upstream of phosphoinositide 3-kinase (PI3K)-c-Jun N-terminal kinase (JNK)-β-catenin to promote cell migration. Moreover, RNA-seq and qRT-PCR analyses revealed that Hh pathway genes including Smo and Gli1 were under control of β-catenin, suggesting that β-catenin turn feedback activates Hh signaling. Taken together, our analyses identified a new bFGF-regulating mechanism by which Hh signaling regulates human fibroblast migration, and the data presented here opens a new avenue for the wound healing therapy. - Highlights: • bFGF regulates Hedgehog (Hh) signaling in fibroblasts. • The Smo and Gli two master regulators of Hh signaling positively regulate fibroblast migration. • Smo facilitates β-catenin nuclear translocation via activation PI3K/JNK/GSK3β. • β-catenin positively regulates fibroblast cell migration and the expression of Hh signaling genes including Smo and Gli.« less

Extragastrointestinal Stromal Tumor during Pregnacy

PubMed Central

Gözükara, Ilay; Dilek, T. U. Kutlu; Durukan, Hüseyin; Düsmez Apa, Duygu; Kabil Kucur, Suna; Dilek, Saffet

2012-01-01

Extragastrointestinal stromal tumors (EGISTs) are mesenchymal neoplasms without connection to the gastrointestinal tract. Gastrointestinal stromal tumors (GISTs) and EGIST are similar according to their clinicopathologic and histomorphologic features. Both of them most often express immunoreactivity for CD-117, a c-kit proto-oncogene protein. The coexistence of GIST and pregnancy is very rare, with only two cases reported in the literature. In this paper, we presented the first EGIST case during pregnancy in the literature. PMID:23119199

Single cell dual adherent-suspension co-culture micro-environment for studying tumor-stromal interactions with functionally selected cancer stem-like cells.

PubMed

Chen, Yu-Chih; Zhang, Zhixiong; Fouladdel, Shamileh; Deol, Yadwinder; Ingram, Patrick N; McDermott, Sean P; Azizi, Ebrahim; Wicha, Max S; Yoon, Euisik

2016-08-07

Considerable evidence suggests that cancer stem-like cells (CSCs) are critical in tumor pathogenesis, but their rarity and transience has led to much controversy about their exact nature. Although CSCs can be functionally identified using dish-based tumorsphere assays, it is difficult to handle and monitor single cells in dish-based approaches; single cell-based microfluidic approaches offer better control and reliable single cell derived sphere formation. However, like normal stem cells, CSCs are heavily regulated by their microenvironment, requiring tumor-stromal interactions for tumorigenic and proliferative behaviors. To enable single cell derived tumorsphere formation within a stromal microenvironment, we present a dual adherent/suspension co-culture device, which combines a suspension environment for single-cell tumorsphere assays and an adherent environment for co-culturing stromal cells in close proximity by selectively patterning polyHEMA in indented microwells. By minimizing dead volume and improving cell capture efficiency, the presented platform allows for the use of small numbers of cells (<100 cells). As a proof of concept, we co-cultured single T47D (breast cancer) cells and primary cancer associated fibroblasts (CAF) on-chip for 14 days to monitor sphere formation and growth. Compared to mono-culture, co-cultured T47D have higher tumorigenic potential (sphere formation rate) and proliferation rates (larger sphere size). Furthermore, 96-multiplexed single-cell transcriptome analyses were performed to compare the gene expression of co-cultured and mono-cultured T47D cells. Phenotypic changes observed in co-culture correlated with expression changes in genes associated with proliferation, apoptotic suppression, tumorigenicity and even epithelial-to-mesechymal transition. Combining the presented platform with single cell transcriptome analysis, we successfully identified functional CSCs and investigated the phenotypic and transcriptome effects induced

Effects of conditioned medium from LL-37 treated adipose stem cells on human fibroblast migration.

PubMed

Yang, Eun-Jung; Bang, Sa-Ik

2017-07-01

Adipose stem cell-conditioned medium may promote human dermal fibroblast (HDF) proliferation and migration by activating paracrine peptides during the re-epithelization phase of wound healing. Human antimicrobial peptide LL-37 is upregulated in the skin epithelium as part of the normal response to injury. The effects of conditioned medium (CM) from LL-37 treated adipose stem cells (ASCs) on cutaneous wound healing, including the mediation of fibroblast migration, remain to be elucidated, therefore the aim of the present study was to determine how ASCs would react to an LL-37-rich microenvironment and if CM from LL-37 treated ASCs may influence the migration of HDFs. The present study conducted migration assays with HDFs treated with CM from LL-37 treated ASCs. Expression of CXC chemokine receptor 4 (CXCR4), which controls the recruitment of HDFs, was analyzed at the mRNA and protein levels. To further characterize the stimulatory effects of LL-37 on ASCs, the expression of stromal cell-derived factor-1α (SDF-1α), a CXC chemokine, was investigated. CM from LL-37-treated ASCs induced migration of HDFs in a time- and dose-dependent manner, with a maximum difference in migration observed 24 h following stimulation with LL-37 at a concentration of 10 µg/ml. The HDF migration and the expression of CXCR4 in fibroblasts was markedly increased upon treatment with CM from LL-37-treated ASCs compared with CM from untreated ASCs. SDF-1α expression was markedly increased in CM from LL-37 treated ASCs. It was additionally observed that SDF-1α blockade significantly reduced HDF migration. These findings suggest the feasibility of CM from LL-37-treated ASCs as a potential therapeutic for human dermal fibroblast migration.

IL-6 secreted by cancer-associated fibroblasts promotes epithelial-mesenchymal transition and metastasis of gastric cancer via JAK2/STAT3 signaling pathway

PubMed Central

Li, Jie; Yu, Zhenjia; Wang, Xiaofeng; Li, Jiaanfang; Li, Chen; Yan, Min; Zhu, Zhenggang; Liu, Bingya; Su, Liping

2017-01-01

Cancer-associated fibroblasts (CAFs), as the activated fibroblasts in tumor stroma, are important modifiers of tumor progression. However, the molecular mechanisms underlying the tumor-promoting properties of CAFs in gastric cancer remain unclear. Here, we show that CAFs isolated from gastric cancer produce significant amounts of interleukin-6 (IL-6). CAFs enhances the migration and EMT of gastric cancer cells through the secretion of IL-6 that activates Janus kinase 2/signal transducers and activators of transcription (JAK2/STAT3) pathway in gastric cancer cells, while deprivation of IL-6 using a neutralizing antibody or inhibition of JAK/STAT3 pathway with specific inhibitor AG490 markedly attenuates these phenotypes in gastric cancer cells induced by CAFs. Moreover, silencing IL-6 expression in CAFs or inhibiting JAK2/STAT3 pathway in gastric cancer cells impairs tumor peritoneal metastasis induced by CAFs in vivo. Taken together, these results suggest that CAFs in the tumor microenvironment promote the progression of gastric cancer through IL-6/JAK2/STAT3 signaling, and IL-6 targeted therapy could be a complementary approach against gastric cancer by exerting their action on stromal fibroblasts. PMID:28186964

Nitric oxide regulates tumor cell cross-talk with stromal cells in the tumor microenvironment of the liver.

PubMed

Decker, Ningling Kang; Abdelmoneim, Soha S; Yaqoob, Usman; Hendrickson, Helen; Hormes, Joe; Bentley, Mike; Pitot, Henry; Urrutia, Raul; Gores, Greg J; Shah, Vijay H

2008-10-01

Tumor progression is regulated through paracrine interactions between tumor cells and stromal cells in the microenvironment, including endothelial cells and myofibroblasts. Nitric oxide (NO) is a key molecule in the regulation of tumor-microenvironment interactions, although its precise role is incompletely defined. By using complementary in vitro and in vivo approaches, we studied the effect of endothelial NO synthase (eNOS)-derived NO on liver tumor growth and metastasis in relation to adjacent stromal myofibroblasts and matrix because liver tumors maintain a rich, vascular stromal network enriched with phenotypically heterogeneous myofibroblasts. Mice with an eNOS deficiency developed liver tumors more frequently in response to carcinogens compared with control animals. In a surgical model of pancreatic cancer liver metastasis, eNOS overexpression in the tumor microenvironment attenuated both the number and size of tumor implants. NO promoted anoikis of tumor cells in vitro and limited their invasive capacity. Because tumor cell anoikis and invasion are both regulated by myofibroblast-derived matrix, we explored the effect of NO on tumor cell protease expression. Both microarray and Western blot analysis revealed eNOS-dependent down-regulation of the matrix protease cathepsin B within tumor cells, and silencing of cathepsin B attenuated tumor cell invasive capacity in a similar manner to that observed with eNOS overexpression. Thus, a NO gradient within the tumor microenvironment influences tumor progression through orchestrated molecular interactions between tumor cells and stroma.

Hemangioblastomas: histogenesis of the stromal cell studied by immunocytochemistry.

PubMed

Jurco, S; Nadji, M; Harvey, D G; Parker, J C; Font, R L; Morales, A R

1982-01-01

Twenty-one cases of hemangioblastoma from the cerebellum, spinal cord and retina were studied using the unlabeled antibody peroxidase-antiperoxidase technique with antibodies directed against glial fibrillary acidic protein (GFAP) and factor VIII related antigen (VIIIR:Ag). In 19 of 21 cases studied with anti-GFAP, astrocytes were identified peripherally, and in 13 cases they were found centrally within the tumor. In no instance did stromal cells react positively for GFAP. Sixteen cases with anti-VIIIR:Ag antibody were examined, and in all cases many stromal cells showed positive staining. It is concluded that the stromal cells were of endothelial origin. The occasional stromal cells that other investigators have identified as reacting positively for GFAP may represent stromal cells capable of ingesting extracellular GFAP derived from reactive astrocytes within the tumor, or they may be lipidized astrocytes.

Cisplatin-induced mesenchymal stromal cells-mediated mechanism contributing to decreased antitumor effect in breast cancer cells.

PubMed

Skolekova, Svetlana; Matuskova, Miroslava; Bohac, Martin; Toro, Lenka; Durinikova, Erika; Tyciakova, Silvia; Demkova, Lucia; Gursky, Jan; Kucerova, Lucia

2016-01-12

Cells of the tumor microenvironment are recognized as important determinants of the tumor biology. The adjacent non-malignant cells can regulate drug responses of the cancer cells by secreted paracrine factors and direct interactions with tumor cells. Human mesenchymal stromal cells (MSC) actively contribute to tumor microenvironment. Here we focused on their response to chemotherapy as during the treatment these cells become affected. We have shown that the secretory phenotype and behavior of mesenchymal stromal cells influenced by cisplatin differs from the naïve MSC. MSC were more resistant to the concentrations of cisplatin, which was cytotoxic for tumor cells. They did not undergo apoptosis, but a part of MSC population underwent senescence. However, MSC pretreatment with cisplatin led to changes in phosphorylation profiles of many kinases and also increased secretion of IL-6 and IL-8 cytokines. These changes in cytokine and phosphorylation profile of MSC led to increased chemoresistance and stemness of breast cancer cells. Taken together here we suggest that the exposure of the chemoresistant cells in the tumor microenvironment leads to substantial alterations and might lead to promotion of acquired microenvironment-mediated chemoresistance and stemness.

Pirfenidone and nintedanib modulate properties of fibroblasts and myofibroblasts in idiopathic pulmonary fibrosis.

PubMed

Lehtonen, Siri T; Veijola, Anniina; Karvonen, Henna; Lappi-Blanco, Elisa; Sormunen, Raija; Korpela, Saara; Zagai, Ulrika; Sköld, Magnus C; Kaarteenaho, Riitta

2016-02-04

Idiopathic pulmonary fibrosis (IPF) is an incurable lung disease with a poor prognosis. Fibroblasts and myofibroblasts are the key cells in the fibrotic process. Recently two drugs, pirfenidone and nintedanib, were approved for clinical use as they are able to slow down the disease progression. The mechanisms by which these two drugs act in in vitro cell systems are not known. The aim of this study was therefore to examine the effects of pirfenidone and nintedanib on fibroblasts and myofibroblasts structure and function established from patients with or without IPF. Stromal cells were collected and cultured from control lung (n = 4) or IPF (n = 7). The cells were treated with pirfenidone and/or nintedanib and the effect of treatment was evaluated by measuring cell proliferation, alpha smooth muscle actin (α-SMA) and fibronectin expression by Western analysis and/or immunoelectron microscopy, ultrastructural properties by transmission electron microscopy and functional properties by collagen gel contraction and invasion assays. Both pirfenidone and nintedanib reduced in vitro proliferation of fibroblastic cells in a dose dependent manner. The number of cells from control lung was reduced to 47 % (p = 0.04) and of IPF cells to 42 % (p = 0.04) by 1 mM pirfenidone and correspondingly to 67 % (p = 0.04) and 68 % (p = 0.04), by 1 μM nintedanib. If both drugs were used together, a further reduced proliferation was observed. Both pirfenidone and nintedanib were able to reduce the amount of α-SMA and the myofibroblastic appearance although the level of reduction was cell line dependent. In functional assays, the effect of both drugs was also variable. We conclude that the ultrastructure and function of fibroblasts and myofibroblasts are affected by pirfenidone and nintedanib. Combination of the drugs reduced cell proliferation more than either of them individually. Human lung derived cell culture systems represent a potential platform for

Direct Reprogramming of Murine Fibroblasts to Hematopoietic Progenitor Cells

PubMed Central

Batta, Kiran; Florkowska, Magdalena; Kouskoff, Valerie; Lacaud, Georges

2014-01-01

Summary Recent reports have shown that somatic cells, under appropriate culture conditions, could be directly reprogrammed to cardiac, hepatic, or neuronal phenotype by lineage-specific transcription factors. In this study, we demonstrate that both embryonic and adult somatic fibroblasts can be efficiently reprogrammed to clonal multilineage hematopoietic progenitors by the ectopic expression of the transcription factors ERG, GATA2, LMO2, RUNX1c, and SCL. These reprogrammed cells were stably expanded on stromal cells and possessed short-term reconstitution ability in vivo. Loss of p53 function facilitated reprogramming to blood, and p53−/− reprogrammed cells efficiently generated erythroid, megakaryocytic, myeloid, and lymphoid lineages. Genome-wide analyses revealed that generation of hematopoietic progenitors was preceded by the appearance of hemogenic endothelial cells expressing endothelial and hematopoietic genes. Altogether, our findings suggest that direct reprogramming could represent a valid alternative approach to the differentiation of embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) for disease modeling and autologous blood cell therapies. PMID:25466247

Clinical-grade production of human mesenchymal stromal cells: occurrence of aneuploidy without transformation.

PubMed

Tarte, Karin; Gaillard, Julien; Lataillade, Jean-Jacques; Fouillard, Loic; Becker, Martine; Mossafa, Hossein; Tchirkov, Andrei; Rouard, Hélène; Henry, Catherine; Splingard, Marie; Dulong, Joelle; Monnier, Delphine; Gourmelon, Patrick; Gorin, Norbert-Claude; Sensebé, Luc

2010-02-25

Clinical-grade human mesenchymal stromal cells (MSCs) have been expanded in vitro for tissue engineering or immunoregulatory purposes without standardized culture conditions or release criteria. Although human MSCs show poor susceptibility for oncogenic transformation, 2 recent studies described their capacity to accumulate chromosomal instability and to give rise to carcinoma in immunocompromised mice after long-term culture. We thus investigated the immunologic and genetic features of MSCs expanded with fetal calf serum and fibroblast growth factor or with platelet lysate in 4 cell-therapy facilities during 2 multicenter clinical trials. Cultured MSCs showed a moderate expression of human leukocyte antigen-DR without alteration of their low immunogenicity or their immunomodulatory capacity. Moreover, some transient and donor-dependent recurring aneuploidy was detected in vitro, independently of the culture process. However, MSCs with or without chromosomal alterations showed progressive growth arrest and entered senescence without evidence of transformation either in vitro or in vivo.

CASTIN: a system for comprehensive analysis of cancer-stromal interactome.

PubMed

Komura, Daisuke; Isagawa, Takayuki; Kishi, Kazuki; Suzuki, Ryohei; Sato, Reiko; Tanaka, Mariko; Katoh, Hiroto; Yamamoto, Shogo; Tatsuno, Kenji; Fukayama, Masashi; Aburatani, Hiroyuki; Ishikawa, Shumpei

2016-11-09

Cancer microenvironment plays a vital role in cancer development and progression, and cancer-stromal interactions have been recognized as important targets for cancer therapy. However, identifying relevant and druggable cancer-stromal interactions is challenging due to the lack of quantitative methods to analyze whole cancer-stromal interactome. We present CASTIN (CAncer-STromal INteractome analysis), a novel framework for the evaluation of cancer-stromal interactome from RNA-Seq data using cancer xenograft models. For each ligand-receptor interaction which is derived from curated protein-protein interaction database, CASTIN summarizes gene expression profiles of cancer and stroma into three evaluation indices. These indices provide quantitative evaluation and comprehensive visualization of interactome, and thus enable to identify critical cancer-microenvironment interactions, which would be potential drug targets. We applied CASTIN to the dataset of pancreas ductal adenocarcinoma, and successfully characterized the individual cancer in terms of cancer-stromal relationships, and identified both well-known and less-characterized druggable interactions. CASTIN provides comprehensive view of cancer-stromal interactome and is useful to identify critical interactions which may serve as potential drug targets in cancer-microenvironment. CASTIN is available at: http://github.com/tmd-gpat/CASTIN .

Seminal plasma induces global transcriptomic changes associated with cell migration, proliferation and viability in endometrial epithelial cells and stromal fibroblasts.

PubMed

Chen, Joseph C; Johnson, Brittni A; Erikson, David W; Piltonen, Terhi T; Barragan, Fatima; Chu, Simon; Kohgadai, Nargis; Irwin, Juan C; Greene, Warner C; Giudice, Linda C; Roan, Nadia R

2014-06-01

How does seminal plasma (SP) affect the transcriptome of human primary endometrial epithelial cells (eEC) and stromal fibroblasts (eSF)? Exposure of eEC and eSF to SP in vitro increases expression of genes and secreted proteins associated with cellular migration, proliferation, viability and inhibition of cell death. Studies in both humans and animals suggest that SP can access and induce physiological changes in the upper female reproductive tract (FRT), which may participate in promoting reproductive success. This is a cross sectional study involving control samples versus treatment. SP (pooled from twenty donors) was first tested for dose- and time-dependent cytotoxic effects on eEC and eSF (n = 4). As exposure of eEC or eSF to 1% SP for 6 h proved to be non-toxic, a second set of eEC/eSF samples (n = 4) was treated under these conditions for transcriptome, protein and functional analysis. With a third set of samples (n = 3), we further compared the transcriptional response of the cells to SP versus fresh semen. eEC and eSF were isolated from endometrial biopsies from women of reproductive age undergoing benign gynecologic procedures and maintained in vitro. RNA was isolated and processed for microarray studies to analyze global transcriptomic changes. Secreted factors in conditioned media from SP-treated cells were analyzed by Luminex and for the ability to stimulate migration of CD14+ monocytes and CD4+ T cells. Pathway identifications were determined using the Z-scoring system in Ingenuity Pathways Analysis (Z scores ≥|1.5|). SP induced transcriptomic changes (P < 0.05) associated with promoting leukocyte and endothelial cell recruitment, and proliferation of eEC and eSF. Cell viability pathways were induced, while those associated with cell death were suppressed (P < 0.05). SP and fresh semen induced similar sets of pathways, suggesting that SP can model the signaling effects of semen in the endometrium. SP also induced secretion of pro-inflammatory and

Changes in compartments of hemospoietic and stromal marrow progenitor cells after continuous low dose gamma-irradiation

NASA Astrophysics Data System (ADS)

Domaratskaya, E.; Starostin, V.

The low dose continuous gamma-irradiation chosen corresponded with that affected the organisms onboard a spacecraft (Mitrikas, Tsetlin, 2000). F1 (CBAxC57Bl/6) male and female mice were used at 3 4 months of age. Experimental mice were- irradiated during 10 days to a total dose of 15 mGy (Co60 gamma-sources, mean dose rate of 1.5-2.0 mGy/day). Another group of intact mice served as control. Younger and advanced hemopoietic progenitors measured at day 11 (i.e. CFU -S-11) and day 7 (i.e. CFU-S-7), respectively, after transplantation of test donor cells were assayed by the method of Till and McCulloch (1961). Stromal changes were evaluated by estimation of in vitro fibroblastic colony-forming units (CFU -F ) content and by the ability of ectopically grafted (under renal capsule) stroma to regenerate the new bone marrow organ. CFU-S-11 number increased of 40% as compared with control and almost 2-fold higher than that of CFU-S-7. The CFU-F content increased almost of 3-fold. Size of ectopic marrow transplants was estimated at day 70 following grafting by counting myelokariocyte and CFU -S number that repopulated the newly formed bone marrow organ. It was found more than 2-fold increase of myelokariocytes in transplants produced by marrow stroma of irradiated donors. CFU -S contents in transplants increased strikingly in comparison to control level. CFU-S-7 and CFU-S-11 increased of 7.5- and of 3.7-fold, respectively, i.e. the rate of advanced CFU - S predominated. It should be noted a good correlation between number of stromal progenitor cells (CFU-F) and ectopic transplant sizes evaluated as myelokaryocyte counts when irradiated donors used. In the same time, if sizes of transplants was measured as CFU-S-7 and CFU - S-11 numbers, their increases were more pronounced. Therefore, continuous low dose gamma- irradiation augments significantly both hemopoietic and stromal progenitor cell number in bone marrow. Additionally, the ratio of distinct CFU -S subpopulations

Involvement of stromal p53 in tumor-stroma interactions

PubMed Central

Bar, Jair; Moskovits, Neta; Oren, Moshe

2009-01-01

p53 is a major tumor-suppressor gene, inactivated by mutations in about half of all human cancer cases, and probably incapacitated by other means in most other cases. Most research regarding the role of p53 in cancer has focused on its ability to elicit apoptosis or growth arrest of cells that are prone to become malignant owing to DNA damage or oncogene activation, i.e. cell-autonomous activities of p53. However, p53 activation within a cell can also exert a variety of effects upon neighboring cells, through secreted factors and paracrine and endocrine mechanisms. Of note, p53 within cancer stromal cells can inhibit tumor growth and malignant progression. Cancer cells that evolve under this inhibitory influence acquire mechanisms to silence stromal p53, either by direct inhibition of p53 within stromal cells, or through pressure for selection of stromal cells with compromised p53 function. Hence, activation of stromal p53 by chemotherapy or radiotherapy might be part of the mechanisms by which these treatments cause cancer regression. However, in certain circumstances, activation of stromal p53 by cytotoxic anti-cancer agents might actually promote treatment resistance, probably through stromal p53-mediated growth arrest of the cancer cells or through protection of the tumor vasculature. Better understanding of the underlying molecular mechanisms is thus required. Hopefully, this will allow their manipulation towards better inhibition of cancer initiation, progression and metastasis. PMID:19914385

Intraperitoneal but not intravenous cryopreserved mesenchymal stromal cells home to the inflamed colon and ameliorate experimental colitis.

PubMed

Castelo-Branco, Morgana T L; Soares, Igor D P; Lopes, Daiana V; Buongusto, Fernanda; Martinusso, Cesonia A; do Rosario, Alyson; Souza, Sergio A L; Gutfilen, Bianca; Fonseca, Lea Mirian B; Elia, Celeste; Madi, Kalil; Schanaider, Alberto; Rossi, Maria Isabel D; Souza, Heitor S P

2012-01-01

Mesenchymal stromal cells (MSCs) were shown to have immunomodulatory activity and have been applied for treating immune-mediated disorders. We compared the homing and therapeutic action of cryopreserved subcutaneous adipose tissue (AT-MSCs) and bone marrow-derived mesenchymal stromal cells (BM-MSCs) in rats with trinitrobenzene sulfonic acid (TNBS)-induced colitis. After colonoscopic detection of inflammation AT-MSCs or BM-MSCs were injected intraperitoneally. Colonoscopic and histologic scores were obtained. Density of collagen fibres and apoptotic rates were evaluated. Cytokine levels were measured in supernatants of colon explants. For cell migration studies MSCs and skin fibroblasts were labelled with Tc-99m or CM-DiI and injected intraperitonealy or intravenously. Intraperitoneal injection of AT-MSCs or BM-MSCs reduced the endoscopic and histopathologic severity of colitis, the collagen deposition, and the epithelial apoptosis. Levels of TNF-α and interleukin-1β decreased, while VEGF and TGF-β did not change following cell-therapy. Scintigraphy showed that MSCs migrated towards the inflamed colon and the uptake increased from 0.5 to 24 h. Tc-99m-MSCs injected intravenously distributed into various organs, but not the colon. Cm-DiI-positive MSCs were detected throughout the colon wall 72 h after inoculation, predominantly in the submucosa and muscular layer of inflamed areas. Intraperitoneally injected cryopreserved MSCs home to and engraft into the inflamed colon and ameliorate TNBS-colitis.

Sequential growth factor application in bone marrow stromal cell ligament engineering.

PubMed

Moreau, Jodie E; Chen, Jingsong; Horan, Rebecca L; Kaplan, David L; Altman, Gregory H

2005-01-01

In vitro bone marrow stromal cell (BMSC) growth may be enhanced through culture medium supplementation, mimicking the biochemical environment in which cells optimally proliferate and differentiate. We hypothesize that the sequential administration of growth factors to first proliferate and then differentiate BMSCs cultured on silk fiber matrices will support the enhanced development of ligament tissue in vitro. Confluent second passage (P2) BMSCs obtained from purified bone marrow aspirates were seeded on RGD-modified silk matrices. Seeded matrices were divided into three groups for 5 days of static culture, with medium supplement of basic fibroblast growth factor (B) (1 ng/mL), epidermal growth factor (E; 1 ng/mL), or growth factor-free control (C). After day 5, medium supplementation was changed to transforming growth factor-beta1 (T; 5 ng/mL) or C for an additional 9 days of culture. Real-time RT-PCR, SEM, MTT, histology, and ELISA for collagen type I of all sample groups were performed. Results indicated that BT supported the greatest cell ingrowth after 14 days of culture in addition to the greatest cumulative collagen type I expression measured by ELISA. Sequential growth factor application promoted significant increases in collagen type I transcript expression from day 5 of culture to day 14, for five of six groups tested. All T-supplemented samples surpassed their respective control samples in both cell ingrowth and collagen deposition. All samples supported spindle-shaped, fibroblast cell morphology, aligning with the direction of silk fibers. These findings indicate significant in vitro ligament development after only 14 days of culture when using a sequential growth factor approach.

Cancer cells metabolically "fertilize" the tumor microenvironment with hydrogen peroxide, driving the Warburg effect: implications for PET imaging of human tumors.

PubMed

Martinez-Outschoorn, Ubaldo E; Lin, Zhao; Trimmer, Casey; Flomenberg, Neal; Wang, Chenguang; Pavlides, Stephanos; Pestell, Richard G; Howell, Anthony; Sotgia, Federica; Lisanti, Michael P

2011-08-01

Previously, we proposed that cancer cells behave as metabolic parasites, as they use targeted oxidative stress as a "weapon" to extract recycled nutrients from adjacent stromal cells. Oxidative stress in cancer-associated fibroblasts triggers autophagy and  mitophagy, resulting in compartmentalized cellular catabolism, loss of mitochondrial function, and the onset of aerobic glycolysis, in the tumor stroma. As such, cancer-associated fibroblasts produce high-energy nutrients (such as lactate and ketones) that fuel mitochondrial biogenesis, and oxidative metabolism in cancer cells. We have termed this new energy-transfer mechanism the "reverse Warburg effect." To further test the validity of this hypothesis, here we used an in vitro MCF7-fibroblast co-culture system, and quantitatively measured a variety of metabolic parameters by FACS analysis (analogous to laser-capture micro-dissection).  Mitochondrial activity, glucose uptake, and ROS production were measured with highly-sensitive fluorescent probes (MitoTracker, NBD-2-deoxy-glucose, and DCF-DA). Interestingly, using this approach, we directly show that cancer cells initially secrete hydrogen peroxide that then triggers oxidative stress in neighboring fibroblasts. Thus, oxidative stress is contagious (spreads like a virus) and is propagated laterally and vectorially from cancer cells to adjacent fibroblasts. Experimentally, we show that oxidative stress in cancer-associated fibroblasts quantitatively reduces mitochondrial activity, and increases glucose uptake, as the fibroblasts become more dependent on aerobic glycolysis.  Conversely, co-cultured cancer cells show significant increases in mitochondrial activity, and corresponding reductions in both glucose uptake and GLUT1 expression. Pre-treatment of co-cultures with extracellular catalase (an anti-oxidant enzyme that detoxifies hydrogen peroxide) blocks the onset of oxidative stress, and potently induces the death of cancer cells, likely via starvation

Gastrointestinal Stromal Tumors Treatment (PDQ®)—Patient Version

Cancer.gov

Gastrointestinal stromal tumors (GIST) are usually found on the stomach or small intestine, but they can be found anywhere in or near the GI tract. Find out about risk factors, symptoms, tests to diagnose, prognosis, staging, and treatment for gastrointestinal stromal tumors.

Ethanol exposure induces the cancer-associated fibroblast phenotype and lethal tumor metabolism: implications for breast cancer prevention.

PubMed

Sanchez-Alvarez, Rosa; Martinez-Outschoorn, Ubaldo E; Lin, Zhao; Lamb, Rebecca; Hulit, James; Howell, Anthony; Sotgia, Federica; Rubin, Emanuel; Lisanti, Michael P

2013-01-15

Little is known about how alcohol consumption promotes the onset of human breast cancer(s). One hypothesis is that ethanol induces metabolic changes in the tumor microenvironment, which then enhances epithelial tumor growth. To experimentally test this hypothesis, we used a co-culture system consisting of human breast cancer cells (MCF7) and hTERT-immortalized fibroblasts. Here, we show that ethanol treatment (100 mM) promotes ROS production and oxidative stress in cancer-associated fibroblasts, which is sufficient to induce myofibroblastic differentiation. Oxidative stress in stromal fibroblasts also results in the onset of autophagy/mitophagy, driving the induction of ketone body production in the tumor microenvironment. Interestingly, ethanol has just the opposite effect in epithelial cancer cells, where it confers autophagy resistance, elevates mitochondrial biogenesis and induces key enzymes associated with ketone re-utilization (ACAT1/OXCT1). During co-culture, ethanol treatment also converts MCF7 cells from an ER(+) to an ER(-) status, which is thought to be associated with "stemness," more aggressive behavior and a worse prognosis. Thus, ethanol treatment induces ketone production in cancer-associated fibroblasts and ketone re-utilization in epithelial cancer cells, fueling tumor cell growth via oxidative mitochondrial metabolism (OXPHOS). This "two-compartment" metabolic model is consistent with previous historical observations that ethanol is first converted to acetaldehyde (which induces oxidative stress) and then ultimately to acetyl-CoA (a high-energy mitochondrial fuel), or can be used to synthesize ketone bodies. As such, our results provide a novel mechanism by which alcohol consumption could metabolically convert "low-risk" breast cancer patients to "high-risk" status, explaining tumor recurrence or disease progression. Hence, our findings have clear implications for both breast cancer prevention and therapy. Remarkably, our results also show that

Management of hemorrhage in gastrointestinal stromal tumors: a review

PubMed Central

Liu, Qi; Kong, Fanmin; Zhou, Jianping; Dong, Ming; Dong, Qi

2018-01-01

Gastrointestinal stromal tumors (GISTs) are relatively common mesenchymal tumors. They originate from the wall of hollow viscera and may be found in any part of the digestive tract. The prognosis of patients with stromal tumors depends on various risk factors, including size, location, presence of mitotic figures, and tumor rupture. Emergency surgery is often required for stromal tumors with hemorrhage. The current literature suggests that stromal tumor hemorrhage indicates poor prognosis. Although the optimal treatment options for hemorrhagic GISTs are based on surgical experience, there remains controversy with regard to optimum postoperative management as well as the classification of malignant potential. This article reviews the biological characteristics, diagnostic features, prognostic factors, treatment, and postoperative management of GISTs with hemorrhage. PMID:29695930

Expression of Pleiotrophin in the Prostate is Androgen Regulated and it Functions as an Autocrine Regulator of Mesenchyme and Cancer Associated Fibroblasts and as a Paracrine Regulator of Epithelia

PubMed Central

Orr, Brigid; Vanpoucke, Griet; Grace, O Cathal; Smith, Lee; Anderson, Richard A; Riddick, Antony CP; Franco, Omar E; Hayward, Simon W; Thomson, Axel A

2011-01-01

BACKGROUND Androgens and paracrine signaling from mesenchyme/stroma regulate development and disease of the prostate, and gene profiling studies of inductive prostate mesenchyme have identified candidate molecules such as pleiotrophin (Ptn). METHODS Ptn transcripts and protein were localized by in situ and immunohistochemistry and Ptn mRNA was quantitated by Northern blot and qRT-PCR. Ptn function was examined by addition of hPTN protein to rat ventral prostate organ cultures, primary human fetal prostate fibroblasts, prostate cancer associated fibroblasts, and BPH1 epithelia. RESULTS During development, Ptn transcripts and protein were expressed in ventral mesenchymal pad (VMP) and prostatic mesenchyme. Ptn was localized to mesenchyme surrounding ductal epithelial tips undergoing branching morphogenesis, and was located on the surface of epithelia. hPTN protein stimulated branching morphogenesis and stromal and epithelial proliferation, when added to rat VP cultures, and also stimulated growth of fetal human prostate fibroblasts, prostate cancer associated fibroblasts, and BPH1 epithelia. PTN mRNA was enriched in patient-matched normal prostate fibroblasts versus prostate cancer associated fibroblasts. PTN also showed male enriched expression in fetal human male urethra versus female, and between wt male and ARKO male mice. Transcripts for PTN were upregulated by testosterone in fetal human prostate fibroblasts and organ cultures of female rat VMP. Ptn protein was increased by testosterone in organ cultures of female rat VMP and in rat male urethra compared to female. CONCLUSIONS Our data suggest that in the prostate Ptn functions as a regulator of both mesenchymal and epithelial proliferation, and that androgens regulate Ptn levels. Prostate 71:305–317, 2011. © 2010 Wiley-Liss, Inc. PMID:20812209

Autoimmune Th17 Cells Induced Synovial Stromal and Innate Lymphoid Cell Secretion of the Cytokine GM-CSF to Initiate and Augment Autoimmune Arthritis.

PubMed

Hirota, Keiji; Hashimoto, Motomu; Ito, Yoshinaga; Matsuura, Mayumi; Ito, Hiromu; Tanaka, Masao; Watanabe, Hitomi; Kondoh, Gen; Tanaka, Atsushi; Yasuda, Keiko; Kopf, Manfred; Potocnik, Alexandre J; Stockinger, Brigitta; Sakaguchi, Noriko; Sakaguchi, Shimon

2018-06-19

Despite the importance of Th17 cells in autoimmune diseases, it remains unclear how they control other inflammatory cells in autoimmune tissue damage. Using a model of spontaneous autoimmune arthritis, we showed that arthritogenic Th17 cells stimulated fibroblast-like synoviocytes via interleukin-17 (IL-17) to secrete the cytokine GM-CSF and also expanded synovial-resident innate lymphoid cells (ILCs) in inflamed joints. Activated synovial ILCs, which expressed CD25, IL-33Ra, and TLR9, produced abundant GM-CSF upon stimulation by IL-2, IL-33, or CpG DNA. Loss of GM-CSF production by either ILCs or radio-resistant stromal cells prevented Th17 cell-mediated arthritis. GM-CSF production by Th17 cells augmented chronic inflammation but was dispensable for the initiation of arthritis. We showed that GM-CSF-producing ILCs were present in inflamed joints of rheumatoid arthritis patients. Thus, a cellular cascade of autoimmune Th17 cells, ILCs, and stromal cells, via IL-17 and GM-CSF, mediates chronic joint inflammation and can be a target for therapeutic intervention. Copyright © 2018 Elsevier Inc. All rights reserved.

Stromal p16 Overexpression in Adult Granulosa Cell Tumors of the Ovary.

PubMed

Na, Kiyong; Sung, Ji-Youn; Kim, Hyun-Soo

2017-05-01

Adult granulosa cell tumor of the ovary is usually diagnosed at an early stage. However, most patients with advanced or recurrent disease will die of the disease due to limited treatment options. Data on the stromal p16 expression of ovarian adult granulosa cell tumors are limited. The aim of this study was to analyze the immunohistochemical p16 expression in the peritumoral stroma of primary and recurrent adult granulosa cell tumors and investigate whether there were significant differences in stromal p16 expression among nonpathological ovaries, benign sex cord-stromal tumors, and adult granulosa cell tumors. This study included 13 and 11 cases of primary and recurrent adult granulosa cell tumors, respectively. Non-pathological ovaries and benign sex cord-stromal tumors showed negative or weak positive expression, whereas most of the adult granulosa cell tumors showed diffuse and moderate-to-strong immunostaining. Primary adult granulosa cell tumors had significantly higher stromal p16 expression levels than nonpathological ovaries and benign sex cord-stromal tumors (p<0.001). Moreover, recurrent adult granulosa cell tumors showed significantly elevated levels of stromal p16 expression compared to primary adult granulosa cell tumors (p=0.032). In contrast, the difference in stromal p16 expression between non-pathological ovaries and benign sex cord-stromal tumors was not statistically significant (p=0.522). Our observations suggest that stromal p16 expression may be involved in the development and progression of ovarian adult granulosa cell tumors. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Underestimation of malignancy in biopsy-proven cases of stromal fibrosis

PubMed Central

Lad, S; Seely, J M; Schweitzer, M E

2014-01-01

Objective: To determine the rate of underestimation of malignancy in patients with biopsy-proven stromal fibrosis. Methods: Following institutional review board approval, we retrospectively reviewed the charts of patients with biopsy-proven stromal fibrosis who underwent percutaneous breast biopsy in the 5-year period between 1 January 2005 and 31 December 2009. The medical records and the histopathology in patients who underwent repeat biopsy and/or surgical excision at the site of stromal fibrosis within 2 years were reviewed. Interval stability for up to 2 years was documented in patients who did not undergo additional biopsy or surgical excision. An upgrade was defined as any patient with biopsy-proven stromal fibrosis or fibroadenoma with evidence of malignancy at the site of biopsy within 2 years. Results: 365 cases of stromal fibrosis were identified, of which 25 (7%) were upgraded to in situ or invasive malignancy on repeat biopsy or surgical excision. 7 were upgraded to ductal carcinoma in situ and 18 were upgraded to invasive cancer. Of the upgraded cases, 8 out of 24 (32%) were considered concordant with a benign diagnosis. The false-negative rate, that is, cases of stromal fibrosis concordant with benignity, but with subsequent upgrade, comprised 2% of all cases. Conclusion: In biopsy-proven cases of stromal fibrosis, there is a 7% upgrade to malignancy. We recommend that all instances of stromal fibrosis with radiology–pathology discordance undergo repeat biopsy or surgical excision. Cases that demonstrate radiology–pathology concordance can be safely categorized as a Breast Imaging Reporting and Data System 3 (BI-RADS® 3) lesion with a 6-month follow-up, owing to a false-negative rate for missed cancer of 2%. Advances in knowledge: We now recommend that concordant cases of stromal fibrosis be categorized as BI-RADS 3 with a short-term follow-up, as this results in a missed cancer rate of 2%. PMID:24846442

The gene expression program of prostate fibroblast senescence modulates neoplastic epithelial cell proliferation through paracrine mechanisms.

PubMed

Bavik, Claes; Coleman, Ilsa; Dean, James P; Knudsen, Beatrice; Plymate, Steven; Nelson, Peter S

2006-01-15

The greatest risk factor for developing carcinoma of the prostate is advanced age. Potential molecular and physiologic contributors to the frequency of cancer occurrence in older individuals include the accumulation of somatic mutations through defects in genome maintenance, epigenetic gene silencing, oxidative stress, loss of immune surveillance, telomere dysfunction, chronic inflammation, and alterations in tissue microenvironment. In this context, the process of prostate carcinogenesis can be influenced through interactions between intrinsic cellular alterations and the extrinsic microenvironment and macroenvironment, both of which change substantially as a consequence of aging. In this study, we sought to characterize the molecular alterations that occur during the process of prostate fibroblast senescence to identify factors in the aged tissue microenvironment capable of promoting the proliferation and potentially the neoplastic progression of prostate epithelium. We evaluated three mechanisms leading to cell senescence: oxidative stress, DNA damage, and replicative exhaustion. We identified a consistent program of gene expression that includes a subset of paracrine factors capable of influencing adjacent prostate epithelial growth. Both direct coculture and conditioned medium from senescent prostate fibroblasts stimulated epithelial cell proliferation, 3-fold and 2-fold, respectively. The paracrine-acting proteins fibroblast growth factor 7, hepatocyte growth factor, and amphiregulin (AREG) were elevated in the extracellular environment of senescent prostate fibroblasts. Exogenous AREG alone stimulated prostate epithelial cell growth, and neutralizing antibodies and small interfering RNA targeting AREG attenuated, but did not completely abrogate the growth-promoting effects of senescent fibroblast conditioned medium. These results support the concept that aging-related changes in the prostate microenvironment may contribute to the progression of prostate

PD-1 ligand expression by human colonic myofibroblasts/fibroblasts regulates CD4+ T-cell activity.

PubMed

Pinchuk, Irina V; Saada, Jamal I; Beswick, Ellen J; Boya, Gushyalatha; Qiu, Sumin M; Mifflin, Randy C; Raju, Gottumukkala S; Reyes, Victor E; Powell, Don W

2008-10-01

A prominent role for inhibitory molecules PD-L1 and PD-L2 in peripheral tolerance has been proposed. However, the phenotype and function of PD-L-expressing cells in human gut remains unclear. Recent studies suggest that colonic myofibroblasts (CMFs) and fibroblasts are important in the switch from acute inflammation to adaptive immunity. In the normal human colon, CMFs represent a distinct population of major histocompatibility complex class II(+) cells involved in the regulation of mucosal CD4(+) T-cell responses. PD-L1 and PD-L2 expression on human CMFs was determined using Western blot, fluorescence-activated cell sorter analysis and confocal microscopy. Lymphoproliferation assays and cytokine enzyme-linked immunosorbent assays were used to evaluate the role of B7 costimulators expressed by CMFs with regard to the regulation of preactivated T-helper cell responses. We demonstrate here the expression of PD-L1/2 molecules by normal human CMF and fibroblasts in situ and in culture. Both molecules support suppressive functions of CMFs in the regulation of activated CD4(+) T-helper cell proliferative responses; blocking this interaction reverses the suppressive effect of CMFs on T-cell proliferation and leads to increased production of the major T-cell growth factor, interleukin (IL)-2. PD-L1/2-mediated CMF suppressive functions are mainly due to the inhibition of IL-2 production, because supplementation of the coculture media with exogenous IL-2 led to partial recovery of activated T-cell proliferation. Our data suggest that stromal myofibroblasts and fibroblasts may limit T-helper cell proliferative activity in the gut and, thus, might play a prominent role in mucosal intestinal tolerance.

[Risk factors for malignant evolution of gastrointestinal stromal tumors].

PubMed

Andrei, S; Andrei, Adriana; Tonea, A; Andronesi, D; Becheanu, G; Dumbravă, Mona; Pechianu, C; Herlea, V; Popescu, I

2007-01-01

Gastrointestinal stromal tumors are the most frequent non-epithelial digestive tumors, being classified in the group of primitive mesenchymal tumors of the digestive tract. These tumors have a non predictable evolution and where stratified regarding the risk for malignant behavior in 4 categories: very low risk, low risk, intermediate risk and high risk. We performed a retrospective non randomised study including the patients with gastrointestinal stromal tumors treated in the Department of General Surgery and Liver Transplantation of Fundeni Clinical Institute in the period January 2002 - June 2007, to define the epidemiological, clinico-paraclinical, histological and especially evolutive features of the gastrointestinal stromal tumors from this group, with a special regard to the risk factors for their malignant behavior. The most important risk factors in gastrointestinal stromal tumors are the tumor size and the mitotic index, based on them being realised the classification of Fletcher in the 4 risk categories mentioned above. In our group all the local advanced or metastatic gastrointestinal stromal tumors, regardless of their location, were classified in the group of high risk for the malignant behavior. The gastric location and the epithelioid type were positive prognostic factors, and the complete resection of the tumor, an other important positive prognostic feature, was possible in about 80% of the cases, probably because the gastrointestinal stromal tumors in our study were diagnosed in less advanced evolutive situations, only about one third being metastatic and about 14% being locally advanced at the time of diagnose. The association with other neoplasias was in our cases insignificant, only 5% of the patients presenting concomitant malignant digestive tumors and 7.6% intraabdominal benign tumors. Gastrointestinal stromal tumors remain a challenge for the medical staff, regarding their diagnose and therapeutical management, the stratification of the

Cellular Dysfunction in the Diabetic Fibroblast

PubMed Central

Lerman, Oren Z.; Galiano, Robert D.; Armour, Mary; Levine, Jamie P.; Gurtner, Geoffrey C.

2003-01-01

Although it is known that systemic diseases such as diabetes result in impaired wound healing, the mechanism for this impairment is not understood. Because fibroblasts are essential for wound repair, we compared the in vitro behavior of fibroblasts cultured from diabetic, leptin receptor-deficient (db/db) mice with wild-type fibroblasts from mice of the same genetic background in processes important during tissue repair. Adult diabetic mouse fibroblast migration exhibited a 75% reduction in migration compared to normal fibroblasts (P < 0.001) and was not significantly stimulated by hypoxia (1% O2), whereas wild-type fibroblast migration was up-regulated nearly twofold in hypoxic conditions (P < 0.05). Diabetic fibroblasts produced twice the amount of pro-matrix metalloproteinase-9 as normal fibroblasts, as measured by both gelatin zymography and enzyme-linked immunosorbent assay (P < 0.05). Adult diabetic fibroblasts exhibited a sevenfold impairment in vascular endothelial growth factor (VEGF) production (4.5 ± 1.3 pg/ml versus 34.8 ± 3.3 pg/ml, P < 0.001) compared to wild-type fibroblasts. Moreover, wild-type fibroblast production of VEGF increased threefold in response to hypoxia, whereas diabetic fibroblast production of VEGF was not up-regulated in hypoxic conditions (P < 0.001). To address the question whether these differences resulted from chronic hyperglycemia or absence of the leptin receptor, fibroblasts were harvested from newborn db/db mice before the onset of diabetes (4 to 5 weeks old). These fibroblasts showed no impairments in VEGF production under basal or hypoxic conditions, confirming that the results from db/db fibroblasts in mature mice resulted from the diabetic state and were not because of alterations in the leptin-leptin receptor axis. Markers of cellular viability including proliferation and senescence were not significantly different between diabetic and wild-type fibroblasts. We conclude that, in vitro, diabetic fibroblasts show

Stromal-Based Signatures for the Classification of Gastric Cancer.

PubMed

Uhlik, Mark T; Liu, Jiangang; Falcon, Beverly L; Iyer, Seema; Stewart, Julie; Celikkaya, Hilal; O'Mahony, Marguerita; Sevinsky, Christopher; Lowes, Christina; Douglass, Larry; Jeffries, Cynthia; Bodenmiller, Diane; Chintharlapalli, Sudhakar; Fischl, Anthony; Gerald, Damien; Xue, Qi; Lee, Jee-Yun; Santamaria-Pang, Alberto; Al-Kofahi, Yousef; Sui, Yunxia; Desai, Keyur; Doman, Thompson; Aggarwal, Amit; Carter, Julia H; Pytowski, Bronislaw; Jaminet, Shou-Ching; Ginty, Fiona; Nasir, Aejaz; Nagy, Janice A; Dvorak, Harold F; Benjamin, Laura E

2016-05-01

Treatment of metastatic gastric cancer typically involves chemotherapy and monoclonal antibodies targeting HER2 (ERBB2) and VEGFR2 (KDR). However, reliable methods to identify patients who would benefit most from a combination of treatment modalities targeting the tumor stroma, including new immunotherapy approaches, are still lacking. Therefore, we integrated a mouse model of stromal activation and gastric cancer genomic information to identify gene expression signatures that may inform treatment strategies. We generated a mouse model in which VEGF-A is expressed via adenovirus, enabling a stromal response marked by immune infiltration and angiogenesis at the injection site, and identified distinct stromal gene expression signatures. With these data, we designed multiplexed IHC assays that were applied to human primary gastric tumors and classified each tumor to a dominant stromal phenotype representative of the vascular and immune diversity found in gastric cancer. We also refined the stromal gene signatures and explored their relation to the dominant patient phenotypes identified by recent large-scale studies of gastric cancer genomics (The Cancer Genome Atlas and Asian Cancer Research Group), revealing four distinct stromal phenotypes. Collectively, these findings suggest that a genomics-based systems approach focused on the tumor stroma can be used to discover putative predictive biomarkers of treatment response, especially to antiangiogenesis agents and immunotherapy, thus offering an opportunity to improve patient stratification. Cancer Res; 76(9); 2573-86. ©2016 AACR. ©2016 American Association for Cancer Research.

Fibroblast heterogeneity: implications for human disease.

PubMed

Lynch, Magnus D; Watt, Fiona M

2018-01-02

Fibroblasts synthesize the extracellular matrix of connective tissue and play an essential role in maintaining the structural integrity of most tissues. Researchers have long suspected that fibroblasts exhibit functional specialization according to their organ of origin, body site, and spatial location. In recent years, a number of approaches have revealed the existence of fibroblast subtypes in mice. Here, we discuss fibroblast heterogeneity with a focus on the mammalian dermis, which has proven an accessible and tractable system for the dissection of these relationships. We begin by considering differences in fibroblast identity according to anatomical site of origin. Subsequently, we discuss new results relating to the existence of multiple fibroblast subtypes within the mouse dermis. We consider the developmental origin of fibroblasts and how this influences heterogeneity and lineage restriction. We discuss the mechanisms by which fibroblast heterogeneity arises, including intrinsic specification by transcriptional regulatory networks and epigenetic factors in combination with extrinsic effects of the spatial context within tissue. Finally, we discuss how fibroblast heterogeneity may provide insights into pathological states including wound healing, fibrotic diseases, and aging. Our evolving understanding suggests that ex vivo expansion or in vivo inhibition of specific fibroblast subtypes may have important therapeutic applications.

Free bone graft reconstruction of irradiated facial tissue: Experimental effects of basic fibroblast growth factor stimulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eppley, B.L.; Connolly, D.T.; Winkelmann, T.

1991-07-01

A study was undertaken to evaluate the potential utility of basic fibroblast growth factor in the induction of angiogenesis and osseous healing in bone previously exposed to high doses of irradiation. Thirty New Zealand rabbits were evaluated by introducing basic fibroblast growth factor into irradiated mandibular resection sites either prior to or simultaneous with reconstruction by corticocancellous autografts harvested from the ilium. The fate of the free bone grafts was then evaluated at 90 days postoperatively by microangiographic, histologic, and fluorochrome bone-labeling techniques. Sequestration, necrosis, and failure to heal to recipient osseous margins was observed both clinically and histologically inmore » all nontreated irradiated graft sites as well as those receiving simultaneous angiogenic stimulation at the time of graft placement. No fluorescent activity was seen in these graft groups. In the recipient sites pretreated with basic fibroblast growth factor prior to placement of the graft, healing and reestablishment of mandibular contour occurred in nearly 50 percent of the animals. Active bone formation was evident at cortical margins adjacent to the recipient sites but was absent in the more central cancellous regions of the grafts.« less

Stromal liver kinase B1 [STK11] signaling loss induces oviductal adenomas and endometrial cancer by activating mammalian Target of Rapamycin Complex 1.

PubMed

Tanwar, Pradeep S; Kaneko-Tarui, Tomoko; Zhang, Lihua; Tanaka, Yoshihiro; Crum, Christopher P; Teixeira, Jose M

2012-01-01

Germline mutations of the Liver Kinase b1 (LKB1/STK11) tumor suppressor gene have been linked to Peutz-Jeghers Syndrome (PJS), an autosomal-dominant, cancer-prone disorder in which patients develop neoplasms in several organs, including the oviduct, ovary, and cervix. We have conditionally deleted Lkb1 in Müllerian duct mesenchyme-derived cells of the female reproductive tract and observed expansion of the stromal compartment and hyperplasia and/or neoplasia of adjacent epithelial cells throughout the reproductive tract with paratubal cysts and adenomyomas in oviducts and, eventually, endometrial cancer. Examination of the proliferation marker phospho-histone H3 and mammalian Target Of Rapamycin Complex 1 (mTORC1) pathway members revealed increased proliferation and mTORC1 activation in stromal cells of both the oviduct and uterus. Treatment with rapamycin, an inhibitor of mTORC1 activity, decreased tumor burden in adult Lkb1 mutant mice. Deletion of the genes for Tuberous Sclerosis 1 (Tsc1) or Tsc2, regulators of mTORC1 that are downstream of LKB1 signaling, in the oviductal and uterine stroma phenocopies some of the defects observed in Lkb1 mutant mice, confirming that dysregulated mTORC1 activation in the Lkb1-deleted stroma contributes to the phenotype. Loss of PTEN, an upstream regulator of mTORC1 signaling, along with Lkb1 deletion significantly increased tumor burden in uteri and induced tumorigenesis in the cervix and vagina. These studies show that LKB1/TSC1/TSC2/mTORC1 signaling in mesenchymal cells is important for the maintenance of epithelial integrity and suppression of carcinogenesis in adjacent epithelial cells. Because similar changes in the stromal population are also observed in human oviductal/ovarian adenoma and endometrial adenocarcinoma patients, we predict that dysregulated mTORC1 activity by upstream mechanisms similar to those described in these model systems contributes to the pathogenesis of these human diseases.

Stromal Liver Kinase B1 [STK11] Signaling Loss Induces Oviductal Adenomas and Endometrial Cancer by Activating Mammalian Target of Rapamycin Complex 1

PubMed Central

Zhang, LiHua; Tanaka, Yoshihiro; Crum, Christopher P.; Teixeira, Jose M.

2012-01-01

Germline mutations of the Liver Kinase b1 (LKB1/STK11) tumor suppressor gene have been linked to Peutz-Jeghers Syndrome (PJS), an autosomal-dominant, cancer-prone disorder in which patients develop neoplasms in several organs, including the oviduct, ovary, and cervix. We have conditionally deleted Lkb1 in Müllerian duct mesenchyme-derived cells of the female reproductive tract and observed expansion of the stromal compartment and hyperplasia and/or neoplasia of adjacent epithelial cells throughout the reproductive tract with paratubal cysts and adenomyomas in oviducts and, eventually, endometrial cancer. Examination of the proliferation marker phospho-histone H3 and mammalian Target Of Rapamycin Complex 1 (mTORC1) pathway members revealed increased proliferation and mTORC1 activation in stromal cells of both the oviduct and uterus. Treatment with rapamycin, an inhibitor of mTORC1 activity, decreased tumor burden in adult Lkb1 mutant mice. Deletion of the genes for Tuberous Sclerosis 1 (Tsc1) or Tsc2, regulators of mTORC1 that are downstream of LKB1 signaling, in the oviductal and uterine stroma phenocopies some of the defects observed in Lkb1 mutant mice, confirming that dysregulated mTORC1 activation in the Lkb1-deleted stroma contributes to the phenotype. Loss of PTEN, an upstream regulator of mTORC1 signaling, along with Lkb1 deletion significantly increased tumor burden in uteri and induced tumorigenesis in the cervix and vagina. These studies show that LKB1/TSC1/TSC2/mTORC1 signaling in mesenchymal cells is important for the maintenance of epithelial integrity and suppression of carcinogenesis in adjacent epithelial cells. Because similar changes in the stromal population are also observed in human oviductal/ovarian adenoma and endometrial adenocarcinoma patients, we predict that dysregulated mTORC1 activity by upstream mechanisms similar to those described in these model systems contributes to the pathogenesis of these human diseases. PMID:22916036

Cytoplasmic GPER translocation in cancer-associated fibroblasts mediates cAMP/PKA/CREB/glycolytic axis to confer tumor cells with multidrug resistance.

PubMed

Yu, T; Yang, G; Hou, Y; Tang, X; Wu, C; Wu, X-A; Guo, L; Zhu, Q; Luo, H; Du, Y-E; Wen, S; Xu, L; Yin, J; Tu, G; Liu, M

2017-04-01

Multiple drug resistance is a challenging issue in the clinic. There is growing evidence that the G-protein-coupled estrogen receptor (GPER) is a novel mediator in the development of multidrug resistance in both estrogen receptor (ER)-positive and -negative breast cancers, and that cancer-associated fibroblasts (CAFs) in the tumor microenvironment may be a new agent that promotes drug resistance in tumor cells. However, the role of cytoplasmic GPER of CAFs on tumor therapy remains unclear. Here we first show that the breast tumor cell-activated PI3K/AKT (phosphoinositide 3-kinase/AKT) signaling pathway induces the cytoplasmic GPER translocation of CAFs in a CRM1-dependent pattern, and leads to the activation of a novel estrogen/GPER/cAMP/PKA/CREB signaling axis that triggers the aerobic glycolysis switch in CAFs. The glycolytic CAFs feed the extra pyruvate and lactate to tumor cells for augmentation of mitochondrial activity, and this energy metabolically coupled in a 'host-parasite relationship' between catabolic CAFs and anabolic cancer cells confers the tumor cells with multiple drug resistance to several conventional clinical treatments including endocrine therapy (tamoxifen), Her-2-targeted therapy (herceptin) and chemotherapy (epirubicin). Moreover, the clinical data from 18 F-fluorodeoxyglucose positron emission tomography/computed tomography further present a strong association between the GPER/cAMP/PKA/CREB pathway of stromal fibroblasts with tumor metabolic activity and clinical treatment, suggesting that targeting cytoplasmic GPER in CAFs may rescue the drug sensitivity in patients with breast cancer. Thus, our data define novel insights into the stromal GPER-mediated multiple drug resistance from the point of reprogramming of tumor energy metabolism and provide the rationale for CAFs as a promising target for clinical therapy.

Comparison of stromal corneal nerves between normal and keratoconus patients using confocal microscopy.

PubMed

Ramírez Fernández, M; Hernández Quintela, E; Naranjo Tackman, R

2014-08-01

To evaluate the differences in stromal corneal nerves between normal patients and keratoconus patients. A total of 140 eyes of 70 normal patients (group A) and 122 eyes of 87 keratoconus patients (group B) were examined with the confocal microscope, with a central scan of the total corneal thickness being taken. The morphology and thickness of the corneal stromal nerves were evaluated by using the Navis v. 3.5.0. software. Nerve thickness was obtained from the mean between the widest and the narrowest portions of each stromal nerve. Corneal stromal nerves were observed as irregular linear hyper-reflective structures with wide and narrow portions in all cases. Mean corneal stromal nerves thickness in group A was 5.7±1.7 (range from 3.3 to 10.4 μ), mean corneal stromal nerves thickness in group B was 7.2±1.9 (range from 3.5 to 12.0 μ). There was a statistical significant difference (P<.05) in stromal corneal nerves thickness between group A and group B. Stromal corneal nerves morphology was similar in both groups, but stromal nerves were thicker in keratoconus patients. Copyright © 2013 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

Differences between the endocytosis of horseradish peroxidase and its conjugate with wheat germ agglutinin by cultured fibroblasts.

PubMed

Stieber, A; Gonatas, J O; Gonatas, N K

1984-04-01

A covalent conjugate of wheat germ agglutinin (WGA) with horseradish peroxidase (HRP) was used for a morphologic study of its adsorptive endocytosis by cultured human fibroblasts. Initial binding at 4 degrees C of the conjugate was observed over the entire plasma membrane, including "coated" and smooth pits. Endocytosis of HRP and the WGA-HRP conjugate was observed in lysosomes, but only the conjugate was seen in a cisterna of the Golgi apparatus (GERL), and in adjacent coated vesicles.

MiR-16 regulates the pro-tumorigenic potential of lung fibroblasts through the inhibition of HGF production in an FGFR-1- and MEK1-dependent manner.

PubMed

Andriani, Francesca; Majorini, Maria Teresa; Mano, Miguel; Landoni, Elena; Miceli, Rosalba; Facchinetti, Federica; Mensah, Mavis; Fontanella, Enrico; Dugo, Matteo; Giacca, Mauro; Pastorino, Ugo; Sozzi, Gabriella; Delia, Domenico; Roz, Luca; Lecis, Daniele

2018-03-20

Fibroblasts are crucial mediators of tumor-stroma cross-talk through synthesis and remodeling of the extracellular matrix and production of multiple soluble factors. Nonetheless, little is still known about specific determinants of fibroblast pro-tumorigenic activity in lung cancer. Here, we aimed at understanding the role of miRNAs, which are often altered in stromal cells, in reprogramming fibroblasts towards a tumor-supporting phenotype. We employed a co-culture-based high-throughput screening to identify specific miRNAs modulating the pro-tumorigenic potential of lung fibroblasts. Multiplex assays and ELISA were instrumental to study the effect of miRNAs on the secretome of both primary and immortalized lung fibroblasts from lung cancer patients and to evaluate plasmatic levels of HGF in heavy smokers. Direct mRNA targeting by miRNAs was investigated through dual-luciferase reporter assay and western blot. Finally, the pro-tumorigenic activity of fibroblasts and their conditioned media was tested by employing in vitro migration experiments and mouse xenografts. We identified miR-16 as a master regulator of fibroblast secretome and showed that its upregulation reduces HGF secretion by fibroblasts, impairing their capacity to promote cancer cell migration. This effect is due to a pleiotropic activity of miR-16 which prevents HGF expression through direct inhibition of FGFR-1 signaling and targeting of HGF mRNA. Mechanistically, miR-16 targets FGFR-1 downstream mediator MEK1, thus reducing ERK1/2 activation. Consistently, chemical or genetic inhibition of FGFR-1 mimics miR-16 activity and prevents HGF production. Of note, we report that primary fibroblast cell lines derived from lungs of heavy smokers express reduced miR-16 levels compared to those from lungs not exposed to smoke and that HGF concentration in heavy smokers' plasma correlates with levels of tobacco exposure. Finally, in vivo experiments confirmed that restoration of miR-16 expression in fibroblasts

Fibroblast activation protein is dispensable in the anti-influenza immune response in mice

PubMed Central

Chowdhury, Sumaiya; Polak, Natasa

2017-01-01

Fibroblast activation protein alpha (FAP) is a unique dual peptidase of the S9B serine protease family, being capable of both dipeptidyl peptidase and endopeptidase activities. FAP is expressed at low level in healthy adult organs including the pancreas, cervix, uterus, submaxillary gland and the skin, and highly upregulated in embryogenesis, chronic inflammation and tissue remodelling. It is also expressed by cancer-associated stromal fibroblasts in more than 90% of epithelial tumours. FAP has enzymatic and non-enzymatic functions in the growth, immunosuppression, invasion and cell signalling of tumour cells. FAP deficient mice are fertile and viable with no gross abnormality, but little data exist on the role of FAP in the immune system. FAP is upregulated in association with microbial stimulation and chronic inflammation, but its function in infection remains unknown. We showed that major populations of immune cells including CD4+ and CD8+ T cells, B cells, dendritic cells and neutrophils are generated and maintained normally in FAP knockout mice. Upon intranasal challenge with influenza virus, FAP mRNA was increased in the lungs and lung-draining lymph nodes. Nonetheless, FAP deficient mice showed similar pathologic kinetics to wildtype controls, and were capable of supporting normal anti-influenza T and B cell responses. There was no evidence of compensatory upregulation of other DPP4 family members in influenza-infected FAP-deficient mice. FAP appears to be dispensable in anti-influenza adaptive immunity. PMID:28158223

Co-Culturing of Multipotent Mesenchymal Stromal Cells with Autological and Allogenic Lymphocytes.

PubMed

Kapranov, N M; Davydova, Yu O; Gal'tseva, I V; Petinati, N A; Bakshinskaitė, M V; Drize, N I; Kuz'mina, L A; Parovichnikova, E N; Savchenko, V G

2018-03-01

We studied the effect of autologous and allogeneic lymphocytes on multipotent mesenchymal stromal cells in co-culture. It is shown that changes in multipotent mesenchymal stromal cells and in lymphocytes did not depend on the source of lymphocytes. Contact with lymphocytes triggers expression of HLA-DR molecules on multipotent mesenchymal stromal cells and these cells lose their immune privilege. In multipotent mesenchymal stromal cells, the relative level of expression of factors involved in immunomodulation (IDO1, PTGES, and IL-6) and expression of adhesion molecule ICAM1 increased, while expression of genes involved in the differentiation of multipotent mesenchymal stromal cells remained unchanged. Priming of multipotent mesenchymal stromal cells with IFN did not affect these changes. In turn, lymphocytes underwent activation, expression of HLA-DR increased, subpopulation composition of lymphocytes changed towards the increase in the content of naïve T cells. These findings are important for cell therapy.

In vitro effects of direct current electric fields on adipose-derived stromal cells.

PubMed

Hammerick, Kyle E; Longaker, Michael T; Prinz, Fritz B

2010-06-18

Endogenous electric fields play an important role in embryogenesis, regeneration, and wound repair and previous studies have shown that many populations of cells, leukocytes, fibroblasts, epithelial cells, and endothelial cells, exhibit directed migration in response to electric fields. As regenerative therapies continue to explore ways to control mesenchymal progenitor cells to recreate desirable tissues, it is increasingly necessary to characterize the vast nature of biological responses imposed by physical phenomena. Murine adipose-derived stromal cells (mASCs) migrated toward the cathode in direct current (DC) fields of physiologic strength and show a dose dependence of migration rate to stronger fields. Electric fields also caused mASCs to orient perpendicularly to the field vector and elicited a transient increase in cytosolic calcium. Additionally, their galvanotactic response appears to share classic chemotactic signaling pathways that are involved in the migration of other cell types. Galvanotaxis is one predominant result of electric fields on mASCs and it may be exploited to engineer adult stem cell concentrations and locations within implanted grafts or toward sites of wound repair. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Pro-tumorigenic roles of fibroblast activation protein in cancer: back to the basics.

PubMed

Puré, Ellen; Blomberg, Rachel

2018-05-03

Fibroblast activation protein (FAP) is a cell-surface serine protease that acts on various hormones and extracellular matrix components. FAP is highly upregulated in a wide variety of cancers, and is often used as a marker for pro-tumorigenic stroma. It has also been proposed as a molecular target of cancer therapies, and, especially in recent years, a great deal of research has gone into design and testing of diverse FAP-targeted treatments. Yet despite this growing field of research, our knowledge of FAP's basic biology and functional roles in various cancers has lagged behind its use as a tumor-stromal marker. In this review, we summarize and analyze recent advances in understanding the functions of FAP in cancer, most notably its prognostic value in various tumor types, cellular effects on various cell types, and potential as a therapeutic target. We highlight outstanding questions in the field, the answers to which could shape preclinical and clinical studies of FAP.

Targeting Stromal Recruitment by Prostate Cancer Cells

DTIC Science & Technology

2006-03-01

Ensinger, C., Tumer , Z., Tommerup, N. et al.: Hedgehog signaling in small-cell lung cancer : frequent in vivo but a rare event in vitro. Lung Cancer , 52...W81XWH-04-1-0157 TITLE: Targeting Stromal Recruitment by Prostate Cancer Cells PRINCIPAL INVESTIGATOR: Jingxian Zhang, Ph.D...DATES COVERED (From - To) 15 Feb 2004 – 14 Feb 2006 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Targeting Stromal Recruitment by Prostate Cancer

A subset of myofibroblastic cancer-associated fibroblasts regulate collagen fiber elongation, which is prognostic in multiple cancers.

PubMed

Hanley, Christopher J; Noble, Fergus; Ward, Matthew; Bullock, Marc; Drifka, Cole; Mellone, Massimiliano; Manousopoulou, Antigoni; Johnston, Harvey E; Hayden, Annette; Thirdborough, Steve; Liu, Yuming; Smith, David M; Mellows, Toby; Kao, W John; Garbis, Spiros D; Mirnezami, Alex; Underwood, Tim J; Eliceiri, Kevin W; Thomas, Gareth J

2016-02-02

Collagen structure has been shown to influence tumor cell invasion, metastasis and clinical outcome in breast cancer. However, it remains unclear how it affects other solid cancers. Here we utilized multi-photon laser scanning microscopy and Second Harmonic Generation to identify alterations to collagen fiber structure within the tumor stroma of head & neck, esophageal and colorectal cancers. Image segmentation algorithms were then applied to quantitatively characterize these morphological changes, showing that elongated collagen fibers significantly correlated with poor clinical outcome (Log Rank p < 0.05). We used TGF-β treatment to model fibroblast conversion to smooth muscle actin SMA-positive cancer associated fibroblasts (CAFs) and found that these cells induce the formation of elongated collagen fibers in vivo. However, proteomic/transcriptomic analysis of SMA-positive CAFs cultured ex-vivo showed significant heterogeneity in the expression of genes with collagen fibril organizing gene ontology. Notably, stratifying patients according to stromal SMA-positivity and collagen fiber elongation was found to provide a highly significant correlation with poor survival in all 3 cancer types (Log Rank p ≤ 0.003). In summary, we show that increased collagen fiber length correlates with poor patient survival in multiple tumor types and that only a sub-set of SMA-positive CAFs can mediate the formation of this collagen structure.

The Interaction between Human Papillomaviruses and the Stromal Microenvironment

PubMed Central

Woodby, Brittany; Scott, Matthew; Bodily, Jason

2017-01-01

Human papillomaviruses (HPV) are small, double-stranded DNA viruses that replicate in stratified squamous epithelia and cause a variety of malignancies. Current efforts in HPV biology are focused on understanding the virus-host interactions that enable HPV to persist for years or decades in the tissue. The importance of interactions between tumor cells and the stromal microenvironment has become increasingly apparent in recent years, but how stromal interactions impact the normal, benign life cycle of HPVs or progression of lesions to cancer is less understood. Furthermore, how productively replicating HPV impacts cells in the stromal environment is also unclear. Here we bring together some of the relevant literature on keratinocyte-stromal interactions and their impacts on HPV biology. We discuss how HPV oncogenes in infected cells manipulate other cells in their environment, and, conversely, how neighboring cells may impact the efficiency or course of HPV infection. PMID:27865458

Seminal plasma induces global transcriptomic changes associated with cell migration, proliferation and viability in endometrial epithelial cells and stromal fibroblasts

PubMed Central

Chen, Joseph C.; Johnson, Brittni A.; Erikson, David W.; Piltonen, Terhi T.; Barragan, Fatima; Chu, Simon; Kohgadai, Nargis; Irwin, Juan C.; Greene, Warner C.; Giudice, Linda C.; Roan, Nadia R.

2014-01-01

STUDY QUESTION How does seminal plasma (SP) affect the transcriptome of human primary endometrial epithelial cells (eEC) and stromal fibroblasts (eSF)? SUMMARY ANSWER Exposure of eEC and eSF to SP in vitro increases expression of genes and secreted proteins associated with cellular migration, proliferation, viability and inhibition of cell death. WHAT IS KNOWN ALREADY Studies in both humans and animals suggest that SP can access and induce physiological changes in the upper female reproductive tract (FRT), which may participate in promoting reproductive success. STUDY DESIGN, SIZE, DURATION This is a cross sectional study involving control samples versus treatment. SP (pooled from twenty donors) was first tested for dose- and time-dependent cytotoxic effects on eEC and eSF (n = 4). As exposure of eEC or eSF to 1% SP for 6 h proved to be non-toxic, a second set of eEC/eSF samples (n = 4) was treated under these conditions for transcriptome, protein and functional analysis. With a third set of samples (n = 3), we further compared the transcriptional response of the cells to SP versus fresh semen. PARTICIPANTS/MATERIALS, SETTING, METHODS eEC and eSF were isolated from endometrial biopsies from women of reproductive age undergoing benign gynecologic procedures and maintained in vitro. RNA was isolated and processed for microarray studies to analyze global transcriptomic changes. Secreted factors in conditioned media from SP-treated cells were analyzed by Luminex and for the ability to stimulate migration of CD14+ monocytes and CD4+ T cells. MAIN RESULTS AND THE ROLE OF CHANCE Pathway identifications were determined using the Z-scoring system in Ingenuity Pathways Analysis (Z scores ≥|1.5|). SP induced transcriptomic changes (P < 0.05) associated with promoting leukocyte and endothelial cell recruitment, and proliferation of eEC and eSF. Cell viability pathways were induced, while those associated with cell death were suppressed (P < 0.05). SP and fresh semen induced

[Clinical analysis of 31 patients with gastric stromal tumors].

PubMed

Li, Junxia; Liu, Ping; Wang, Huahong; Yu, Jing; Xie, Pengyan; Liu, Xinguang

2002-11-01

To investigate the clinical manifestations, diagnosis and treatment of gastric stromal tumors. 31 patients with gastric stromal tumors treated from 1993, 1 - 2001, 9 were analyzed retrospectively. All cases were diagnosed by pathological and immunohistochemistry examinations. According to Levin's standard combining with Hurliman's and Goldbum's methods, the patients were classified. There are no significant difference between male and female patients. 50 - 60 years old patients have high incidence. The distribution of gastric tromal tumors is fundus > body > antrum. Diagnosis of this condition is sometimes difficult and treatment is often delayed because patients usually present with nonspecific abdominal symptoms. The main manifestations of gastric stromal tumors are upper gastrointestinal hemorrhage 61.3% (19/31), 7 patients with acute hemorrhage and 12 with chronic hemorrhage. Most of them were malignant. Abdominal malaises and/or distention 32.3% (10/31) and abdominal pain 22.6% (7/31). Gastroscopy, ultrasound gastroscopy, computed tomography, B type ultrasound and upper gastrointestinal X-ray series are helpful to diagnosis. But the final diagnosis is decided by pathological and immunohistochemistry examinations. Gastric stromal tumors exhibit consistent immunohistochemical expressions of CD(117) and/or CD(34). The operative treatment is thought of the first choice. Effect of the chemotherapy isn't satisfied. There is no standard chemotherapy for gastric stromal tumors. Gastric stromal tumor is a kind of separated submucosal tumor which is different from leiomyoma, leiomyosarcoma and neurogenic tumors. Pathological and immunohistochemistry inspectations are very important to make clear diagnosis. Early diagnosis and rational treatment are the keys to improve the prognosis.

Dendritic Cells Control Fibroblastic Reticular Network Tension and Lymph Node Expansion

PubMed Central

Acton, Sophie E.; Farrugia, Aaron J.; Astarita, Jillian L.; Mourão-Sá, Diego; Jenkins, Robert P.; Nye, Emma; Hooper, Steven; van Blijswijk, Janneke; Rogers, Neil C.; Snelgrove, Kathryn J.; Rosewell, Ian; Moita, Luis F.; Stamp, Gordon; Turley, Shannon J.; Sahai, Erik; Sousa, Caetano Reis e

2014-01-01

Following immunogenic challenge, infiltrating and dividing lymphocytes significantly increase lymph node (LN) cellularity leading to organ expansion1,2. Here we report that the physical elasticity of LNs is maintained in part by podoplanin (PDPN) signalling in stromal fibroblastic reticular cells (FRCs) and its modulation by CLEC-2 expressed on dendritic cells (DCs). We show that PDPN induces actomyosin contractility in FRCs via activation of RhoA/C and downstream Rho-kinase. Engagement by CLEC-2 causes PDPN clustering and rapidly uncouples PDPN from RhoA/C activation, relaxing the actomyosin cytoskeleton and permitting FRC stretching. Notably, administration of CLEC-2 protein to immunised mice augments LN expansion. In contrast, the latter is significantly constrained in mice selectively lacking CLEC-2 expression in DCs. Thus, the same DCs that initiate immunity by presenting antigens to T lymphocytes3 also initiate remodeling of LNs by delivering CLEC-2 to FRCs. CLEC-2 modulation of PDPN signalling permits FRC network stretching and allows for the rapid LN expansion driven by lymphocyte influx and proliferation that is the critical hallmark of adaptive immunity. PMID:25341788

Mesenchymal Stromal Cells for Antineoplastic Drug Loading and Delivery.

PubMed

Petrella, Francesco; Rimoldi, Isabella; Rizzo, Stefania; Spaggiari, Lorenzo

2017-11-23

Mesenchymal stromal cells are a population of undifferentiated multipotent adult cells possessing extensive self-renewal properties and the potential to differentiate into a variety of mesenchymal lineage cells. They express broad anti-inflammatory and immunomodulatory activity on the immune system and after transplantation can interact with the surrounding microenvironment, promoting tissue healing and regeneration. For this reason, mesenchymal stromal cells have been widely used in regenerative medicine, both in preclinical and clinical settings. Another clinical application of mesenchymal stromal cells is the targeted delivery of chemotherapeutic agents to neoplastic cells, maximizing the cytotoxic activity against cancer cells and minimizing collateral damage to non-neoplastic tissues. Mesenchymal stem cells are home to the stroma of several primary and metastatic neoplasms and hence can be used as vectors for targeted delivery of antineoplastic drugs to the tumour microenvironment, thereby reducing systemic toxicity and maximizing antitumour effects. Paclitaxel and gemcitabine are the chemotherapeutic drugs best loaded by mesenchymal stromal cells and delivered to neoplastic cells, whereas other agents, like pemetrexed, are not internalized by mesenchymal stromal cells and therefore are not suitable for advanced antineoplastic therapy. This review focuses on the state of the art of advanced antineoplastic cell therapy and its future perspectives, emphasizing in vitro and in vivo preclinical results and future clinical applications.

Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival.

PubMed

Özdemir, Berna C; Pentcheva-Hoang, Tsvetelina; Carstens, Julienne L; Zheng, Xiaofeng; Wu, Chia-Chin; Simpson, Tyler R; Laklai, Hanane; Sugimoto, Hikaru; Kahlert, Christoph; Novitskiy, Sergey V; De Jesus-Acosta, Ana; Sharma, Padmanee; Heidari, Pedram; Mahmood, Umar; Chin, Lynda; Moses, Harold L; Weaver, Valerie M; Maitra, Anirban; Allison, James P; LeBleu, Valerie S; Kalluri, Raghu

2014-06-16

Pancreatic ductal adenocarcinoma (PDAC) is associated with marked fibrosis and stromal myofibroblasts, but their functional contribution remains unknown. Transgenic mice with the ability to delete αSMA(+) myofibroblasts in pancreatic cancer were generated. Depletion starting at either noninvasive precursor (pancreatic intraepithelial neoplasia) or the PDAC stage led to invasive, undifferentiated tumors with enhanced hypoxia, epithelial-to-mesenchymal transition, and cancer stem cells, with diminished animal survival. In PDAC patients, fewer myofibroblasts in their tumors also correlated with reduced survival. Suppressed immune surveillance with increased CD4(+)Foxp3(+) Tregs was observed in myofibroblast-depleted mouse tumors. Although myofibroblast-depleted tumors did not respond to gemcitabine, anti-CTLA4 immunotherapy reversed disease acceleration and prolonged animal survival. This study underscores the need for caution in targeting carcinoma-associated fibroblasts in PDAC. Copyright © 2014 Elsevier Inc. All rights reserved.

Defining the role of mesenchymal stromal cells on the regulation of matrix metalloproteinases in skeletal muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sassoli, Chiara; Nosi, Daniele; Tani, Alessia

Recent studies indicate that mesenchymal stromal cell (MSC) transplantation improves healing of injured and diseased skeletal muscle, although the mechanisms of benefit are poorly understood. In the present study, we investigated whether MSCs and/or their trophic factors were able to regulate matrix metalloproteinase (MMP) expression and activity in different cells of the muscle tissue. MSCs in co-culture with C2C12 cells or their conditioned medium (MSC-CM) up-regulated MMP-2 and MMP-9 expression and function in the myoblastic cells; these effects were concomitant with the down-regulation of the tissue inhibitor of metalloproteinases (TIMP)-1 and -2 and with increased cell motility. In the singlemore » muscle fiber experiments, MSC-CM administration increased MMP-2/9 expression in Pax-7{sup +} satellite cells and stimulated their mobilization, differentiation and fusion. The anti-fibrotic properties of MSC-CM involved also the regulation of MMPs by skeletal fibroblasts and the inhibition of their differentiation into myofibroblasts. The treatment with SB-3CT, a potent MMP inhibitor, prevented in these cells, the decrease of α-smooth actin and type-I collagen expression induced by MSC-CM, suggesting that MSC-CM could attenuate the fibrogenic response through mechanisms mediated by MMPs. Our results indicate that growth factors and cytokines released by these cells may modulate the fibrotic response and improve the endogenous mechanisms of muscle repair/regeneration. - Highlights: • MSC-CM contains paracrine factors that up-regulate MMP expression and function in different skeletal muscle cells. • MSC-CM promotes myoblast and satellite cell migration, proliferation and differentiation. • MSC-CM negatively interferes with fibroblast-myoblast transition in primary skeletal fibroblasts. • Paracrine factors from MSCs modulate the fibrotic response and improve the endogenous mechanisms of muscle regeneration.« less

Stromal cells in breast cancer as a potential therapeutic target

PubMed Central

Dykes, Samantha S.; Hughes, Veronica S.; Wiggins, Jennifer M.; Fasanya, Henrietta O.; Tanaka, Mai; Siemann, Dietmar

2018-01-01

Breast cancer in the United States is the second most commonly diagnosed cancer in women. About 1 in 8 women will develop invasive breast cancer over the course of her lifetime and breast cancer remains the second leading cause of cancer-related death. In pursuit of novel therapeutic strategies, researchers have examined the tumor microenvironment as a potential anti-cancer target. In addition to neoplastic cells, the tumor microenvironment is composed of several critical normal cell types, including fibroblasts, vascular and lymph endothelial cells, osteoclasts, adipocytes, and immune cells. These cells have important roles in healthy tissue stasis, which frequently are altered in tumors. Indeed, tumor-associated stromal cells often contribute to tumorigenesis, tumor progression, and metastasis. Consequently, these host cells may serve as a possible target in anti-tumor and anti-metastatic therapeutic strategies. Targeting the tumor associated host cells offers the benefit that such cells do not mutate and develop resistance in response to treatment, a major cause of failure in cancer therapeutics targeting neoplastic cells. This review discusses the role of host cells in the tumor microenvironment during tumorigenesis, progression, and metastasis, and provides an overview of recent developments in targeting these cell populations to enhance cancer therapy efficacy.

The hallmarks of fibroblast ageing.

PubMed

Tigges, Julia; Krutmann, Jean; Fritsche, Ellen; Haendeler, Judith; Schaal, Heiner; Fischer, Jens W; Kalfalah, Faiza; Reinke, Hans; Reifenberger, Guido; Stühler, Kai; Ventura, Natascia; Gundermann, Sabrina; Boukamp, Petra; Boege, Fritz

2014-06-01

Ageing is influenced by the intrinsic disposition delineating what is maximally possible and extrinsic factors determining how that frame is individually exploited. Intrinsic and extrinsic ageing processes act on the dermis, a post-mitotic skin compartment mainly consisting of extracellular matrix and fibroblasts. Dermal fibroblasts are long-lived cells constantly undergoing damage accumulation and (mal-)adaptation, thus constituting a powerful indicator system for human ageing. Here, we use the systematic of ubiquitous hallmarks of ageing (Lopez-Otin et al., 2013, Cell 153) to categorise the available knowledge regarding dermal fibroblast ageing. We discriminate processes inducible in culture from phenomena apparent in skin biopsies or primary cells from old donors, coming to the following conclusions: (i) Fibroblasts aged in culture exhibit most of the established, ubiquitous hallmarks of ageing. (ii) Not all of these hallmarks have been detected or investigated in fibroblasts aged in situ (in the skin). (iii) Dermal fibroblasts aged in vitro and in vivo exhibit additional features currently not considered ubiquitous hallmarks of ageing. (iv) The ageing process of dermal fibroblasts in their physiological tissue environment has only been partially elucidated, although these cells have been a preferred model of cell ageing in vitro for decades. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Automated wholeslide analysis of multiplex-brightfield IHC images for cancer cells and carcinoma-associated fibroblasts

NASA Astrophysics Data System (ADS)

Lorsakul, Auranuch; Andersson, Emilia; Vega Harring, Suzana; Sade, Hadassah; Grimm, Oliver; Bredno, Joerg

2017-03-01

Multiplex-brightfield immunohistochemistry (IHC) staining and quantitative measurement of multiple biomarkers can support therapeutic targeting of carcinoma-associated fibroblasts (CAF). This paper presents an automated digitalpathology solution to simultaneously analyze multiple biomarker expressions within a single tissue section stained with an IHC duplex assay. Our method was verified against ground truth provided by expert pathologists. In the first stage, the automated method quantified epithelial-carcinoma cells expressing cytokeratin (CK) using robust nucleus detection and supervised cell-by-cell classification algorithms with a combination of nucleus and contextual features. Using fibroblast activation protein (FAP) as biomarker for CAFs, the algorithm was trained, based on ground truth obtained from pathologists, to automatically identify tumor-associated stroma using a supervised-generation rule. The algorithm reported distance to nearest neighbor in the populations of tumor cells and activated-stromal fibroblasts as a wholeslide measure of spatial relationships. A total of 45 slides from six indications (breast, pancreatic, colorectal, lung, ovarian, and head-and-neck cancers) were included for training and verification. CK-positive cells detected by the algorithm were verified by a pathologist with good agreement (R2=0.98) to ground-truth count. For the area occupied by FAP-positive cells, the inter-observer agreement between two sets of ground-truth measurements was R2=0.93 whereas the algorithm reproduced the pathologists' areas with R2=0.96. The proposed methodology enables automated image analysis to measure spatial relationships of cells stained in an IHC-multiplex assay. Our proof-of-concept results show an automated algorithm can be trained to reproduce the expert assessment and provide quantitative readouts that potentially support a cutoff determination in hypothesis testing related to CAF-targeting-therapy decisions.

Identification of inhibitory scFv antibodies targeting fibroblast activation protein utilizing phage display functional screens

PubMed Central

Zhang, Jiping; Valianou, Matthildi; Simmons, Heidi; Robinson, Matthew K.; Lee, Hyung-Ok; Mullins, Stefanie R.; Marasco, Wayne A.; Adams, Gregory P.; Weiner, Louis M.; Cheng, Jonathan D.

2013-01-01

Fibroblast activation protein (FAP) is a serine protease selectively expressed on tumor stromal fibroblasts in epithelial carcinomas and is important in cancer growth, adhesion, and metastases. As FAP enzymatic activity is a potent therapeutic target, we aimed to identify inhibitory antibodies. Using a competitive inhibition strategy, we used phage display techniques to identify 53 single-chain variable fragments (scFvs) after three rounds of panning against FAP. These scFvs were expressed and characterized for binding to FAP by surface plasmon resonance and flow cytometry. Functional assessment of these antibodies yielded an inhibitory scFv antibody, named E3, which could attenuate 35% of FAP cleavage of the fluorescent substrate Ala-Pro-7-amido-4-trifluoromethylcoumarin compared with nonfunctional scFv control. Furthermore, a mutant E3 scFv was identified by yeast affinity maturation. It had higher affinity (4-fold) and enhanced inhibitory effect on FAP enzyme activity (3-fold) than E3. The application of both inhibitory anti-FAP scFvs significantly affected the formation of 3-dimensional FAP-positive cell matrix, as demonstrated by reducing the fibronectin fiber orientation from 41.18% (negative antibody control) to 34.06% (E3) and 36.15% (mutant E3), respectively. Thus, we have identified and affinity-maturated the first scFv antibody capable of inhibiting FAP function. This scFv antibody has the potential to disrupt the role of FAP in tumor invasion and metastasis.—Zhang, J., Valianou, M., Simmons, H., Robinson, M. K., Lee, H.-O., Mullins, S. R., Marasco, W. A., Adams, G. P., Weiner, L. M., Cheng, J. D. Identification of inhibitory ScFv antibodies targeting fibroblast activation protein utilizing phage display functional screens. PMID:23104982

miRNA Signature and Dicer Requirement during Human Endometrial Stromal Decidualization In Vitro

PubMed Central

Estella, Carlos; Herrer, Isabel; Moreno-Moya, Juan Manuel; Quiñonero, Alicia; Martínez, Sebastián; Pellicer, Antonio; Simón, Carlos

2012-01-01

Decidualization is a morphological and biochemical transformation of endometrial stromal fibroblast into differentiated decidual cells, which is critical for embryo implantation and pregnancy establishment. The complex regulatory networks have been elucidated at both the transcriptome and the proteome levels, however very little is known about the post-transcriptional regulation of this process. miRNAs regulate multiple physiological pathways and their de-regulation is associated with human disorders including gynaecological conditions such as endometriosis and preeclampsia. In this study we profile the miRNAs expression throughout human endometrial stromal (hESCs) decidualization and analyze the requirement of the miRNA biogenesis enzyme Dicer during this process. A total of 26 miRNAs were upregulated and 17 miRNAs downregulated in decidualized hESCs compared to non-decidualized hESCs. Three miRNAs families, miR-181, miR-183 and miR-200, are down-regulated during the decidualization process. Using miRNAs target prediction algorithms we have identified the potential targets and pathways regulated by these miRNAs. The knockdown of Dicer has a minor effect on hESCs during in vitro decidualization. We have analyzed a battery of decidualization markers such as cell morphology, Prolactin, IGFBP-1, MPIF-1 and TIMP-3 secretion as well as HOXA10, COX2, SP1, C/EBPß and FOXO1 expression in decidualized hESCs with decreased Dicer function. We found decreased levels of HOXA10 and altered intracellular organization of actin filaments in Dicer knockdown decidualized hESCs compared to control. Our results provide the miRNA signature of hESC during the decidualization process in vitro. We also provide the first functional characterization of Dicer during human endometrial decidualization although surprisingly we found that Dicer plays a minor role regulating this process suggesting that alternative biogenesis miRNAs pathways must be involved in human endometrial decidualization

Sex cord-gonadal stromal tumor of the rete testis.

PubMed

Sajadi, Kamran P; Dalton, Rory R; Brown, James A

2009-01-01

A 34-year-old tetraplegic patient with suppurative epididymitis was found on follow-up examination and ultrasonography to have a testicular mass. The radical orchiectomy specimen contained an undifferentiated spindled sex cord-stromal tumor arising in the rete testis. Testicular sex cord-stromal tumors are far less common than germ cell neoplasms and are usually benign. The close relationship between sex cords and ductules of the rete testis during development provides the opportunity for these uncommon tumors to arise anatomically within the rete tesis. This undifferentiated sex cord-stromal tumor, occurring in a previously unreported location, is an example of an unusual lesion mimicking an intratesticular malignant neoplasm.

Expression and role of the cell surface protease seprase/fibroblast activation protein-α (FAP-α) in astroglial tumors.

PubMed

Mentlein, Rolf; Hattermann, Kirsten; Hemion, Charles; Jungbluth, Achim A; Held-Feindt, Janka

2011-03-01

Seprase or fibroblast activation protein-α (FAP-α) is a cell-surface serine protease that was previously described nearly exclusively on reactive and tumor stromal fibroblasts and thought to be involved in tissue remodeling. We investigated the expression and significance of FAP-α in astrocytomas/glioblastomas. As shown by quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, FAP-α was elevated in whole glioblastoma tissues and in particular in most glioma cells in situ and in vitro. In glioma stem-like cells (gliospheres), FAP-α was detected at low levels; however, FAP-α was considerably induced upon differentiation with 10% fetal calf serum. To explore its functional role, FAP-α was silenced by siRNA transfection. In Boyden chamber assays, FAP-α silenced cells migrated similar as control cells through non-coated or Matrigel (basal lamina)-coated porous membranes, but significantly slower through membranes coated with gelatin or brevican, a major component of brain extracellular matrix. Furthermore, FAP-α-silenced glioma cells migrated through murine brain slices much slower under the conditions tested than differentially fluorescent-labeled control cells. Thus, FAP-α is highly expressed on the surface of glioma cells and contributes to diffuse glioma invasion through extracellular matrix components.

Stromal p16 expression is significantly increased in endometrial carcinoma

PubMed Central

Yoon, Nara; Kim, Ji-Ye; Kim, Hyun-Soo

2017-01-01

p16 is a negative regulator of cell proliferation and is considered a tumor suppressor protein. Alterations in p16 protein expression are associated with tumor development and progression. However, the p16 expression status in the peritumoral stroma has not been investigated in the endometrium. Therefore, we evaluated stromal p16 expression in different types of endometrial lesions using immunohistochemistry. Differences in the p16 expression status according to the degree of malignancy and histological type were analyzed. This study included 62, 26, and 36 cases of benign, precancerous, and malignant endometrial lesions, respectively. Most benign lesions showed negative or weak expression, whereas precancerous lesions showed a variable degree of staining proportion and intensity. Atypical hyperplasia/endometrial intraepithelial neoplasia (AH/EIN) and serous endometrial intraepithelial carcinoma (SEIC) had significantly higher stromal p16 expression levels than benign lesions. Endometrioid carcinoma (EC), serous carcinoma (SC), and carcinosarcoma showed significantly elevated stromal p16 expression levels compared with benign and precancerous lesions. In addition, there were significant differences in stromal p16 expression between AH/EIN and SEIC and between EC and SC. In contrast, differences in stromal p16 expression among nonpathological endometrium, atrophic endometrium, endometrial polyp, and hyperplasia without atypia were not statistically significant. Our observations suggest that stromal p16 expression is involved in the development and progression of endometrial carcinoma, and raise the possibility that p16 overexpression in the peritumoral stroma is associated with aggressive oncogenic behavior of endometrial SC. PMID:27902476

GPER-mediated proliferation and estradiol production in breast cancer-associated fibroblasts.

PubMed

Luo, Haojun; Yang, Guanglun; Yu, Tenghua; Luo, Shujuan; Wu, Chengyi; Sun, Yan; Liu, Manran; Tu, Gang

2014-04-01

Cancer-associated fibroblasts (CAFs) are crucial co-mediators of breast cancer progression. Estrogen is the predominant driving force in the cyclic regulation of the mammary extracellular matrix, thus potentially affecting the tumor-associated stroma. Recently, a third estrogen receptor, estrogen (G-protein-coupled) receptor (GPER), has been reported to be expressed in breast CAFs. In this study, GPER was detected by immunohistochemical analysis in stromal fibroblasts of 41.8% (59/141) of the primary breast cancer samples. GPER expression in CAFs isolated from primary breast cancer tissues was confirmed by immunostaining and RT-PCR analyses. Tamoxifen (TAM) in addition to 17β-estradiol (E₂) and the GPER agonist G1 activated GPER, resulting in transient increases in cell index, intracellular calcium, and ERK1/2 phosphorylation. Furthermore, TAM, E₂, and G1 promoted CAF proliferation and cell-cycle progression, both of which were blocked by GPER interference, the selective GPER antagonist G15, the epidermal growth factor receptor (EGFR) inhibitor AG1478, and the ERK1/2 inhibitor U0126. Importantly, TAM as well as G1 increased E₂ production in breast CAFs via GPER/EGFR/ERK signaling when the substrate of E₂, testosterone, was added to the medium. GPER-induced aromatase upregulation was probably responsible for this phenomenon, as TAM- and G1-induced CYP19A1 gene expression was reduced by GPER knockdown and G15, AG1478, and U0126 administration. Accordingly, GPER-mediated CAF-dependent estrogenic effects on the tumor-associated stroma are conceivable, and CAF is likely to contribute to breast cancer progression, especially TAM resistance, via a positive feedback loop involving GPER/EGFR/ERK signaling and E₂ production.

Fibroblast growth factor receptors in breast cancer.

PubMed

Wang, Shuwei; Ding, Zhongyang

2017-05-01

Fibroblast growth factor receptors are growth factor receptor tyrosine kinases, exerting their roles in embryogenesis, tissue homeostasis, and development of breast cancer. Recent genetic studies have identified some subtypes of fibroblast growth factor receptors as strong genetic loci associated with breast cancer. In this article, we review the recent epidemiological findings and experiment results of fibroblast growth factor receptors in breast cancer. First, we summarized the structure and physiological function of fibroblast growth factor receptors in humans. Then, we discussed the common genetic variations in fibroblast growth factor receptors that affect breast cancer risk. In addition, we also introduced the potential roles of each fibroblast growth factor receptors isoform in breast cancer. Finally, we explored the potential therapeutics targeting fibroblast growth factor receptors for breast cancer. Based on the biological mechanisms of fibroblast growth factor receptors leading to the pathogenesis in breast cancer, targeting fibroblast growth factor receptors may provide new opportunities for breast cancer therapeutic strategies.

Reciprocal changes in gene expression profiles of cocultured breast epithelial cells and primary fibroblasts.

PubMed

Rozenchan, Patricia Bortman; Carraro, Dirce Maria; Brentani, Helena; de Carvalho Mota, Louise Danielle; Bastos, Elen Pereira; e Ferreira, Elisa Napolitano; Torres, Cesar H; Katayama, Maria Lúcia Hirata; Roela, Rosimeire Aparecida; Lyra, Eduardo C; Soares, Fernando Augusto; Folgueira, Maria Aparecida Azevedo Koike; Góes, João Carlos Guedes Sampaio; Brentani, Maria Mitzi

2009-12-15

The importance of epithelial-stroma interaction in normal breast development and tumor progression has been recognized. To identify genes that were regulated by these reciprocal interactions, we cocultured a nonmalignant (MCF10A) and a breast cancer derived (MDA-MB231) basal cell lines, with fibroblasts isolated from breast benign-disease adjacent tissues (NAF) or with carcinoma-associated fibroblasts (CAF), in a transwell system. Gene expression profiles of each coculture pair were compared with the correspondent monocultures, using a customized microarray. Contrariwise to large alterations in epithelial cells genomic profiles, fibroblasts were less affected. In MDA-MB231 highly represented genes downregulated by CAF derived factors coded for proteins important for the specificity of vectorial transport between ER and golgi, possibly affecting cell polarity whereas the response of MCF10A comprised an induction of genes coding for stress responsive proteins, representing a prosurvival effect. While NAF downregulated genes encoding proteins associated to glycolipid and fatty acid biosynthesis in MDA-MB231, potentially affecting membrane biogenesis, in MCF10A, genes critical for growth control and adhesion were altered. NAFs responded to coculture with MDA-MB231 by a decrease in the expression of genes induced by TGFbeta1 and associated to motility. However, there was little change in NAFs gene expression profile influenced by MCF10A. CAFs responded to the presence of both epithelial cells inducing genes implicated in cell proliferation. Our data indicate that interactions between breast fibroblasts and basal epithelial cells resulted in alterations in the genomic profiles of both cell types which may help to clarify some aspects of this heterotypic signaling. Copyright (c) 2009 UICC.

Fibroblast growth factor signaling in myofibroblasts differs from lipofibroblasts during alveolar septation in mice

PubMed Central

McCoy, Diann M.

2015-01-01

Pulmonary alveolar fibroblasts produce extracellular matrix in a temporally and spatially regulated pattern to yield a durable yet pliable gas-exchange surface. Proliferation ensures a sufficient complement of cells, but they must differentiate into functionally distinct subtypes: contractile myofibroblasts (MF), which generate elastin and regulate air-flow at the alveolar ducts, and, in mice and rats, lipofibroblasts (LF), which store neutral lipids. PDGF-A is required but acts in conjunction with other differentiation factors arising from adjacent epithelia or within fibroblasts. We hypothesized that FGF receptor (FGFR) expression and function vary for MF and LF and contributes to their divergent differentiation. Whereas approximately half of the FGFR3 was extracellular in MF, FGFR2 and FGFR4 were primarily intracellular. Intracellular FGFR3 localized to the multivesicular body, and its abundance may be modified by Sprouty and interaction with heat shock protein-90. FGF18 mRNA is more abundant in MF, whereas FGF10 mRNA predominated in LF, which also express FGFR1 IIIb, a receptor for FGF10. FGF18 diminished fibroblast proliferation and was chemotactic for cultured fibroblasts. Although PDGF receptor-α (PDGFR-α) primarily signals through phosphoinositide 3-kinase and Akt, p42/p44 MAP kinase (Erk1/2), a major signaling pathway for FGFRs, influenced the abundance of cell-surface PDGFR-α. Observing different FGFR and ligand profiles in MF and LF is consistent with their divergent differentiation although both subpopulations express PDGFR-α. These studies also emphasize the importance of particular cellular locations of FGFR3 and PDGFR-α, which may modify their effects during alveolar development or repair. PMID:26138642

Targeting stromal glutamine synthetase in tumors disrupts tumor microenvironment-regulated cancer cell growth

USDA-ARS?s Scientific Manuscript database

Reactive stromal cells are an integral part of tumor microenvironment (TME) and interact with cancer cells to regulate their growth. Although targeting stromal cells could be a viable therapy to regulate the communication between TME and cancer cells, identification of stromal targets that make canc...

Endometrial Cancer-Associated FGF18 Expression Is Reduced by Bazedoxifene in Human Endometrial Stromal Cells In Vitro and in Murine Endometrium

PubMed Central

Flannery, Clare A.; Fleming, Andrew G.; Choe, Gina H.; Naqvi, Hanyia; Zhang, Margaret; Sharma, Anu

2016-01-01

Endometrial cancer develops during exposure to estrogen unopposed by progesterone. Traditional formulations for menopausal hormone therapy include a progestin in women with a uterus. However, progestin exposure increases breast cancer risk in postmenopausal women. Alternatives to progestin include bazedoxifene (BZA), a selective estrogen receptor modulator, which prevents estrogen induced endometrial hyperplasia in clinical trials. Molecular mechanisms responsible for BZA's antiproliferative effect are not fully elucidated. We profiled endometrial adenocarcinoma, hyperplasia, and normal proliferative endometrium for differential expression in genes known to be regulated by estrogens or progesterone. Fibroblast growth factor (FGF)18, a paracrine growth factor promoting epithelial proliferation, was significantly increased in adenocarcinoma. Progesterone represses FGF18 by inducing heart and neural crest derivatives expressed transcript 2 (HAND2) in stromal cells. Notably, we confirmed lower HAND2 mRNA in adenocarcinoma, along with higher FGF tyrosine kinase receptor 2 and E74-like factor 5, collectively promoting FGF18 activity. We hypothesized BZA reduces epithelial proliferation by inhibiting FGF18 synthesis in stromal cells. To determine whether BZA regulates FGF18, we treated primary stromal cells with BZA or vehicle. In vitro, BZA reduced FGF18, but did not affect, HAND2. CD1 female mice received either BZA, conjugated estrogen (CE), or combined BZA/CE for 8 weeks. CE-treated mice had nearly 3-fold higher FGF18 expression. In contrast, BZA-treated mice, alone or with CE, had similar FGF18 as controls. Unexpectedly, BZA, alone or with CE, reduced HAND2 more than 80%, differing from progesterone regulation. Reduction of FGF18 is a potential mechanism by which BZA reduces endometrial proliferation and hyperplasia induced by estrogens. However, BZA works independently of HAND2, revealing a novel mechanism for progestin-free hormone therapy in postmenopausal women

Gastrointestinal stromal tumors.

PubMed

Sekac, J; Labas, P; Skultety, J; Prochotsky, A; Durdik, J; Hrbaty, B

2008-01-01

In the last 3 years 9 patients with gastrointestinal stromal tumors (GIST) underwent surgery at our department. All cases were with very atypical process. From these patients 3 interesting cases are described in more details. A 75-years-old woman with gastroscopically verified endoluminal tumour in the proximal third of stomach, 6x7 cm, 76-years-old man with a large endoluminal tumour in D2-D3 part of the duodenum, 4x4 cm, and 62-years-old man with verified extraluminal tumour by CT examination in the middle part of stomach. In all cases, gastrointestinal stromal tumour was histologically confirmed. Work is well photo-documented pre-surgically with endoscopic and CT-findings and during surgery: individual steps during the removal of these tumours. In assessment of the size and number of mitoses, tumours belonged to a group with highly malignant potential. Patients are regularly checked in 3-months intervals and also examination by positron emission tomography was performed--it seems to have the best demonstrability of possible relapse. All three patients live and are subjectively and objectively without significant problems (Tab. 5, Fig. 5, Ref. 7). Full Text (Free, PDF) www.bmj.sk.

Androgen Receptor Expression in Epithelial and Stromal Cells of Prostatic Carcinoma and Benign Prostatic Hyperplasia.

PubMed

Filipovski, Vanja; Kubelka-Sabit, Katerina; Jasar, Dzengis; Janevska, Vesna

2017-08-15

Prostatic carcinoma (PCa) derives from prostatic epithelial cells. However stromal microenvironment, associated with malignant epithelium, also plays a role in prostatic carcinogenesis. Alterations in prostatic stromal cells contribute to the loss of growth control in epithelial cells that lead to progression of PCa. To analyse the differences between Androgen Receptor (AR) expression in both epithelial and stromal cells in PCa and the surrounding benign prostatic hyperplasia (BPH) and to compare the results with tumour grade. Samples from 70 cases of radical prostatectomy specimens were used. The expression and intensity of the signal for AR was analysed in the epithelial and stromal cells of PCa and BPH, and the data was quantified using histological score (H-score). AR showed significantly lower expression in both epithelial and stromal cells of PCa compared to BPH. In PCa a significant positive correlation of AR expression was found between stromal and epithelial cells of PCa. AR expression showed a correlation between the stromal cells of PCa and tumour grade. AR expression is reduced in epithelial and stromal cells of PCa. Expression of AR in stromal cells of PCa significantly correlates with tumour grade.

Transcriptome-derived stromal and immune scores infer clinical outcomes of patients with cancer.

PubMed

Liu, Wei; Ye, Hua; Liu, Ying-Fu; Xu, Chao-Qun; Zhong, Yue-Xian; Tian, Tian; Ma, Shi-Wei; Tao, Huan; Li, Ling; Xue, Li-Chun; He, Hua-Qin

2018-04-01

The stromal and immune cells that form the tumor microenvironment serve a key role in the aggressiveness of tumors. Current tumor-centric interpretations of cancer transcriptome data ignore the roles of stromal and immune cells. The aim of the present study was to investigate the clinical utility of stromal and immune cells in tissue-based transcriptome data. The 'Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data' (ESTIMATE) algorithm was used to probe diverse cancer datasets and the fraction of stromal and immune cells in tumor tissues was scored. The association between the ESTIMATE scores and patient survival data was asessed; it was indicated that the two scores have implications for patient survival, metastasis and recurrence. Analysis of a colorectal cancer progression dataset revealed that decreased levels immune cells could serve an important role in cancer progression. The results of the present study indicated that trasncriptome-derived stromal and immune scores may be a useful indicator of cancer prognosis.

Parenchymal and Stromal Cells Contribute to Pro-Inflammatory Myocardial Environment at Early Stages of Diabetes: Protective Role of Resveratrol.

PubMed

Savi, Monia; Bocchi, Leonardo; Sala, Roberto; Frati, Caterina; Lagrasta, Costanza; Madeddu, Denise; Falco, Angela; Pollino, Serena; Bresciani, Letizia; Miragoli, Michele; Zaniboni, Massimiliano; Quaini, Federico; Del Rio, Daniele; Stilli, Donatella

2016-11-16

Background: Little information is currently available concerning the relative contribution of cardiac parenchymal and stromal cells in the activation of the pro-inflammatory signal cascade, at the initial stages of diabetes. Similarly, the effects of early resveratrol (RSV) treatment on the negative impact of diabetes on the different myocardial cell compartments remain to be defined. Methods: In vitro challenge of neonatal cardiomyocytes and fibroblasts to high glucose and in vivo/ex vivo experiments on a rat model of Streptozotocin-induced diabetes were used to specifically address these issues. Results: In vitro data indicated that, besides cardiomyocytes, neonatal fibroblasts contribute to generating initial changes in the myocardial environment, in terms of pro-inflammatory cytokine expression. These findings were mostly confirmed at the myocardial tissue level in diabetic rats, after three weeks of hyperglycemia. Specifically, monocyte chemoattractant protein-1 and Fractalkine were up-regulated and initial abnormalities in cardiomyocyte contractility occurred. At later stages of diabetes, a selective enhancement of pro-inflammatory macrophage M1 phenotype and a parallel reduction of anti-inflammatory macrophage M2 phenotype were associated with a marked disorganization of cardiomyocyte ultrastructural properties. RSV treatment inhibited pro-inflammatory cytokine production, leading to a recovery of cardiomyocyte contractile efficiency and a reduced inflammatory cell recruitment. Conclusion: Early RSV administration could inhibit the pro-inflammatory diabetic milieu sustained by different cardiac cell types.

Photobiomodulation of freshly isolated human adipose tissue-derived stromal vascular fraction cells by pulsed light-emitting diodes for direct clinical application.

PubMed

Priglinger, E; Maier, J; Chaudary, S; Lindner, C; Wurzer, C; Rieger, S; Redl, H; Wolbank, S; Dungel, P

2018-06-01

A highly interesting source for adult stem cells is adipose tissue, from which the stromal vascular fraction (SVF)-a heterogeneous cell population including the adipose-derived stromal/stem cells-can be obtained. To enhance the regenerative potential of freshly isolated SVF cells, low-level light therapy (LLLT) was used. The effects of pulsed blue (475 nm), green (516 nm), and red (635 nm) light from light-emitting diodes applied on freshly isolated SVF were analysed regarding cell phenotype, cell number, viability, adenosine triphosphate content, cytotoxicity, and proliferation but also osteogenic, adipogenic, and proangiogenic differentiation potential. The colony-forming unit fibroblast assay revealed a significantly increased colony size after LLLT with red light compared with untreated cells, whereas the frequency of colony-forming cells was not affected. LLLT with green and red light resulted in a stronger capacity to form vascular tubes by SVF when cultured within 3D fibrin matrices compared with untreated cells, which was corroborated by increased number and length of the single tubes and a significantly higher concentration of vascular endothelial growth factor. Our study showed beneficial effects after LLLT on the vascularization potential and proliferation capacity of SVF cells. Therefore, LLLT using pulsed light-emitting diode light might represent a new approach for activation of freshly isolated SVF cells for direct clinical application. Copyright © 2018 John Wiley & Sons, Ltd.

Cancer/stroma interplay via cyclooxygenase-2 and indoleamine 2,3-dioxygenase promotes breast cancer progression.

PubMed

Chen, Jing-Yi; Li, Chien-Feng; Kuo, Cheng-Chin; Tsai, Kelvin K; Hou, Ming-Feng; Hung, Wen-Chun

2014-07-25

Expression of indoleamine 2,3-dioxygenase (IDO) in primary breast cancer increases tumor growth and metastasis. However, the clinical significance of stromal IDO and the regulation of stromal IDO are unclear. Metabolomics and enzyme-linked immunosorbent assay (ELISA) were used to study the effect of cyclooxygenase-2 (COX-2)-overexpressing breast cancer cells on IDO expression in co-cultured human breast fibroblasts. Biochemical inhibitors and short-hairpin RNA (shRNA) were used to clarify how prostaglandin E2 (PGE2) upregulates IDO expression. Associations of stromal IDO with clinicopathologic parameters were tested in tumor specimens. An orthotopic animal model was used to examine the effect of COX-2 and IDO inhibitors on tumor growth. Kynurenine, the metabolite generated by IDO, increases in the supernatant of fibroblasts co-cultured with COX-2-overexpressing breast cancer cells. PGE2 released by cancer cells upregulates IDO expression in fibroblasts through an EP4/signal transducer and activator of transcription 3 (STAT3)-dependent pathway. Conversely, fibroblast-secreted kynurenine promotes the formation of the E-cadherin/Aryl hydrocarbon receptor (AhR)/S-phase kinase-associated protein 2 (Skp2) complex, resulting in degradation of E-cadherin to increase breast cancer invasiveness. The enhancement of motility of breast cancer cells induced by co-culture with fibroblasts is suppressed by the IDO inhibitor 1-methyl-tryptophan. Pathological analysis demonstrates that upregulation of stromal IDO is a poor prognosis factor and is associated with of COX-2 overexpression. Co-expression of cancer COX-2 and stromal IDO predicts a worse disease-free and metastasis-free survival. Finally, COX-2 and IDO inhibitors inhibit tumor growth in vivo. Integration of metabolomics and molecular and pathological approaches reveals the interplay between cancer and stroma via COX-2, and IDO promotes tumor progression and predicts poor patient survival.

High secreted protein acidic and rich in cysteine expression in peritumoral fibroblasts predicts better prognosis in patients with resectable gastric cancer

PubMed Central

Nakajima, Masao; Yoshino, Shigefumi; Kanekiyo, Shinsuke; Maeda, Noriko; Sakamoto, Kazuhiko; Tsunedomi, Ryoichi; Suzuki, Nobuaki; Takeda, Shigeru; Yamamoto, Shigeru; Hazama, Shoichi; Hoshii, Yoshinobu; Oga, Atsunori; Itoh, Hiroshi; Ueno, Tomio; Nagano, Hiroaki

2018-01-01

Secreted protein acidic and rich in cysteine (SPARC) is an extracellular matrix glycoprotein that may serve an important role in epithelial-mesenchymal transition. Recent studies have demonstrated that SPARC status is a prognostic indicator in various cancer types; however, its value remains unclear in gastric cancer (GC). In the present study, the localization and prognostic impact of SPARC expression were evaluated in patients with GC. Immunohistochemical analysis of SPARC expression was performed in 117 surgically resected GC specimens, and the localization of SPARC positive cells, as well as the rassociation between SPARC expression and clinicopathological characteristics were evaluated. High SPARC expression was observed in 47 cases; the glycoprotein was localized in the peritumoral fibroblasts, but was rarely observed in the cytoplasm of cancer cells. Heterogeneity of SPARC expression was observed in 52 cases. High stromal SPARC expression was identified to be an independent predictor of more favorable prognosis (overall survival and recurrence free survival) in all patients (P<0.001). On subgroup analysis, this association remained significant in patients who received adjuvant chemotherapy, but not in patients who did not (P<0.001). Stromal SPARC expression predicts better prognosis in GC patients who underwent curative resection; this appears to be associated with improved response to chemotherapy. PMID:29403557

Pericellular Versican Regulates the Fibroblast-Myofibroblast Transition

PubMed Central

Hattori, Noriko; Carrino, David A.; Lauer, Mark E.; Vasanji, Amit; Wylie, James D.; Nelson, Courtney M.; Apte, Suneel S.

2011-01-01

The cell and its glycosaminoglycan-rich pericellular matrix (PCM) comprise a functional unit. Because modification of PCM influences cell behavior, we investigated molecular mechanisms that regulate PCM volume and composition. In fibroblasts and other cells, aggregates of hyaluronan and versican are found in the PCM. Dermal fibroblasts from Adamts5−/− mice, which lack a versican-degrading protease, ADAMTS5, had reduced versican proteolysis, increased PCM, altered cell shape, enhanced α-smooth muscle actin (SMA) expression and increased contractility within three-dimensional collagen gels. The myofibroblast-like phenotype was associated with activation of TGFβ signaling. We tested the hypothesis that fibroblast-myofibroblast transition in Adamts5−/− cells resulted from versican accumulation in PCM. First, we noted that versican overexpression in human dermal fibroblasts led to increased SMA expression, enhanced contractility, and increased Smad2 phosphorylation. In contrast, dermal fibroblasts from Vcan haploinsufficient (Vcanhdf/+) mice had reduced contractility relative to wild type fibroblasts. Using a genetic approach to directly test if myofibroblast transition in Adamts5−/− cells resulted from increased PCM versican content, we generated Adamts5−/−;Vcanhdf/+ mice and isolated their dermal fibroblasts for comparison with dermal fibroblasts from Adamts5−/− mice. In Adamts5−/− fibroblasts, Vcan haploinsufficiency or exogenous ADAMTS5 restored normal fibroblast contractility. These findings demonstrate that altering PCM versican content through proteolytic activity of ADAMTS5 profoundly influenced the dermal fibroblast phenotype and may regulate a phenotypic continuum between the fibroblast and its alter ego, the myofibroblast. We propose that a physiological function of ADAMTS5 in dermal fibroblasts is to maintain optimal versican content and PCM volume by continually trimming versican in hyaluronan-versican aggregates. PMID:21828051

Neuropeptide Y, B-type natriuretic peptide, substance P and peptide YY are novel substrates of fibroblast activation protein-α.

PubMed

Keane, Fiona M; Nadvi, Naveed A; Yao, Tsun-Wen; Gorrell, Mark D

2011-04-01

Fibroblast activation protein-α (FAP) is a cell surface-expressed and soluble enzyme of the prolyl oligopeptidase family, which includes dipeptidyl peptidase 4 (DPP4). FAP is not generally expressed in normal adult tissues, but is found at high levels in activated myofibroblasts and hepatic stellate cells in fibrosis and in stromal fibroblasts of epithelial tumours. FAP possesses a rare catalytic activity, hydrolysis of the post-proline bond two or more residues from the N-terminus of target substrates. α(2)-antiplasmin is an important physiological substrate of FAP endopeptidase activity. This study reports the first natural substrates of FAP dipeptidyl peptidase activity. Neuropeptide Y, B-type natriuretic peptide, substance P and peptide YY were the most efficiently hydrolysed substrates and the first hormone substrates of FAP to be identified. In addition, FAP slowly hydrolysed other hormone peptides, such as the incretins glucagon-like peptide-1 and glucose-dependent insulinotropic peptide, which are efficient DPP4 substrates. FAP showed negligible or no hydrolysis of eight chemokines that are readily hydrolysed by DPP4. This novel identification of FAP substrates furthers our understanding of this unique protease by indicating potential roles in cardiac function and neurobiology. © 2011 The Authors Journal compilation © 2011 FEBS.

Bone marrow stromal-B cell interactions in polycyclic aromatic hydrocarbon-induced pro/pre-B cell apoptosis.

PubMed

Allan, Lenka L; Mann, Koren K; Matulka, Raymond A; Ryu, Heui-Young; Schlezinger, Jennifer J; Sherr, David H

2003-12-01

Environmental polycyclic aromatic hydrocarbons (PAH) and related halogenated hydrocarbons are immunotoxic in a variety of systems. In a model system of B lymphopoiesis, PAH exposure rapidly induces apoptosis in CD43- pre-B and CD43+ pro/pre-B cells. Apoptosis induction by 7,12-dimethylbenzo[a]anthracene (DMBA) is dependent upon AhR+ bone marrow stromal cells and likely involves DMBA metabolism within the stromal cell. However, it is not known if PAH-treated stromal cells release free metabolites or soluble factors that may directly induce B cell death or if the effector death signal is delivered by stromal cell-B cell contact. Here, we demonstrate that supernatants from DMBA-treated bone marrow stromal cells contain an activity capable of inducing apoptosis in pro/pre-B cells cocultured with stromal cells. This activity (1) is not produced when stromal cells are cotreated with DMBA and alpha-naphthoflavone (alpha-NF), an aryl hydrocarbon receptor (AhR) and cytochrome P-450 inhibitor, (2) is > or = 50 kDa, (3) is trypsin and heat sensitive, and (4) is dependent on AhR+ stromal cells, which in turn deliver the effector death signal to pro/pre-B cells. The results (1) argue against a role for a soluble, stromal cell-derived cytokine as the effector of PAH-induced pro/pre-B cell death, (2) exclude the possibility of a free metabolite acting directly on AhR- pro/pre-B cell targets, and (3) suggest the elaboration by stromal cells of a relatively stable, DMBA metabolite-protein complex capable of acting on other stromal cells at some distance. Collectively, these studies suggest that, while stromal cell products, e.g., metabolite-protein complexes, may affect the function of distant stromal cells, the effector death signal delivered by stromal cells to bone marrow B cells is mediated by cell-cell contact.

Basic Fibroblast Growth Factor Influences Epidermal Homeostasis of Living Skin Equivalents through Affecting Fibroblast Phenotypes and Functions.

PubMed

Yang, Lujun; Zhang, Dangui; Wu, Hongjuan; Xie, Sitian; Zhang, Mingjun; Zhang, Bingna; Tang, Shijie

2018-05-30

To elucidate the possible mechanisms of how basic fibroblast growth factor (bFGF) influences epidermal homeostasis in a living skin equivalent (LSE) model. Several wound healing-related growth factors were analyzed at protein and mRNA levels for dermal fibroblasts of induced alpha-smooth muscle actin (α-SMA)-positive or α-SMA-negative phenotypes. During culturing an LSE model by seeding normal human keratinocytes on a fibroblast-populated type I collagen gel, bFGF or neutralizing antibody for keratinocyte growth factor (KGF) was added to investigate its effects on fibroblast phenotypes and, subsequently, epidermal homeostasis by histology and immunohistochemistry. The α-SMA-positive phenotype of fibroblasts induced by transforming growth factor beta-1 (TGF-β1) markedly suppressed the expression of KGF and hepatocyte growth factor (HGF), and slightly upregulated vascular endothelial growth factor (VEGF) and TGF-β1 at mRNA and protein levels, compared with α-SMA-negative fibroblasts treated with bFGF. α-SMA expression of fibroblasts at the epidermal-mesenchymal junction of the LSEs was suppressed by the addition of bFGF, and a better-differentiated epidermis was presented. The abrogation of KGF from fibroblasts by the addition of the KGF neutralizing antibody disenabled the LSE culturing system to develop an epidermis. bFGF, through affecting the phenotypes and functions of fibroblasts, especially KGF expression, influenced epidermal homeostasis in an LSE model. © 2018 S. Karger AG, Basel.

Mineralization by mesenchymal stromal cells is variously modulated depending on commercial platelet lysate preparations.

PubMed

Boraldi, Federica; Burns, Jorge S; Bartolomeo, Angelica; Dominici, Massimo; Quaglino, Daniela

2018-03-01

Numerous cellular models have been developed to investigate calcification for regenerative medicine applications and for the identification of therapeutic targets in various complications associated with age-related diseases. However, results have often been contradictory due to specific culture conditions, cell type ontogeny and aging status. Human platelet lysate (hPL) has been recently investigated as valuable alternative to fetal bovine serum (FBS) in cell culture and bone regeneration. A parallel comparison of how all these multiple factors may converge to influence mineralization has yet to be reported. To compare mineralization of human mesenchymal cell types known to differ in extracellular matrix calcification potency, bone marrow-derived mesenchymal stromal cells and dermal fibroblasts from neonatal and adult donors, at both low and high passages, were investigated in an ex vivo experimental model by supplementing the osteogenic induction medium with FBS or with hPL. Four commercial hPL preparations were profiled by liquid chromatography/electrospray ionization quadrupole time-of-flight spectrometry, and mineralization was visualized by von Kossa staining and quantified by morphometric evaluations after 9, 14 and 21 days of culture. Data demonstrate that (i) commercial hPL preparations differ according to mass spectra profiles, (ii) hPL variously influences mineral deposition depending on cell line and possibly on platelet product preparation methods, (iii) donor age modifies mineral deposition in the presence of the same hPL and (iv) reduced in vitro proliferative capacity affects osteogenic induction and response to hPL. Despite the standardized procedures applied to obtain commercial hPL, this study highlights the divergent effects of different preparations and emphasizes the importance of cellular ontology, donor age and cell proliferative capacity to optimize the osteogenic induction capabilities of mesenchymal stromal cells and design more effective

Embryonal carcinoma cell induction of miRNA and mRNA changes in co-cultured prostate stromal fibromuscular cells

PubMed Central

VÊNCIO, ENEIDA F.; PASCAL, LAURA E.; PAGE, LAURA S.; DENYER, GARETH; WANG, AMY J.; RUOHOLA-BAKER, HANNELE; ZHANG, SHILE; WANG, KAI; GALAS, DAVID J.; LIU, ALVIN Y.

2014-01-01

The prostate stromal mesenchyme controls organ-specific development. In cancer, the stromal compartment shows altered gene expression compared to non-cancer. The lineage relationship between cancer-associated stromal cells and normal tissue stromal cells is not known. Nor is the cause underlying the expression difference. Previously, the embryonal carcinoma (EC) cell line, NCCIT, was used by us to study the stromal induction property. In the current study, stromal cells from non-cancer (NP) and cancer (CP) were isolated from tissue specimens and co-cultured with NCCIT cells in a trans-well format to preclude heterotypic cell contact. After 3 days, the stromal cells were analyzed by gene arrays for microRNA (miRNA) and mRNA expression. In co-culture, NCCIT cells were found to alter the miRNA and mRNA expression of NP stromal cells to one like that of CP stromal cells. In contrast, NCCIT had no significant effect on the gene expression of CP stromal cells. We conclude that the gene expression changes in stromal cells can be induced by diffusible factors synthesized by EC cells, and suggest that cancer-associated stromal cells represent a more primitive or less differentiated stromal cell type. PMID:20945389

High intratumoral expression of fibroblast activation protein (FAP) in colon cancer is associated with poorer patient prognosis.

PubMed

Wikberg, Maria L; Edin, Sofia; Lundberg, Ida V; Van Guelpen, Bethany; Dahlin, Anna M; Rutegård, Jörgen; Stenling, Roger; Oberg, Ake; Palmqvist, Richard

2013-04-01

An active stroma is important for cancer cell invasion and metastasis. We investigated the expression of fibroblast activation protein (FAP) in relation to patient prognosis in colorectal cancer. Colorectal cancer specimens from 449 patients were immunohistochemically stained with a FAP antibody and evaluated in the tumor center and tumor front using a semiquantitative four-level scale. FAP was expressed by fibroblasts in 85-90 % of the tumors examined. High versus no/low expression in the tumor center was associated with poor prognosis (multivariate hazard ratio, HR = 1.72; 95 % CI 1.07-2.77, p = 0.025). FAP expression in the tumor front, though more frequent than in the tumor center, was not associated with prognosis. FAP expression in the tumor center was more common in specimens with positive microsatellite instability (MSI) screening status and in patients with high CpG island methylator phenotype (CIMP) status. However, inclusion of MSI screening status and CIMP status in the multivariate analysis strengthened the risk estimates for high FAP expression in the tumor center (HR = 1.89; 95 % CI 1.13-3.14; p = 0.014), emphasizing the role of FAP as an independent prognostic factor. Stromal FAP expression is common in colorectal cancer, and we conclude that high FAP expression in the tumor center, but not the tumor front, is an independent negative prognostic factor.

Innate lymphoid cells and their stromal microenvironments.

PubMed

Kellermayer, Zoltán; Vojkovics, Dóra; Balogh, Péter

2017-09-01

In addition to the interaction between antigen presenting cells, T and B lymphocytes, recent studies have revealed important roles for a diverse set of auxiliary cells that profoundly influence the induction and regulation of immune responses against pathogens. Of these the stromal cells composed of various non-hematopoietic constituents are crucial for the creation and maintenance of specialized semi-static three-dimensional lymphoid tissue microenvironment, whereas the more recently described innate lymphoid cells are generated by the diversification of committed lymphoid precursor cells independently from clonally rearranged antigen receptor genes. Recent findings have revealed important contributions by innate lymphoid cells in inflammation and protection against pathogens in a tissue-specific manner. Importantly, lymphoid stromal cells also influence the onset of immune responses in tissue-specific fashion, raising the possibility of tissue-specific stromal - innate lymphoid cell collaboration. In this review we summarize the main features and interactions between these two cells types, with particular emphasis on ILC type 3 cells and their microenvironmental partners. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Polyphyllin I inhibits gastric cancer cell proliferation by downregulating the expression of fibroblast activation protein alpha (FAP) and hepatocyte growth factor (HGF) in cancer-associated fibroblasts.

PubMed

Dong, Ruizeng; Guo, Jianmin; Zhang, Zewei; Zhou, Yimin; Hua, Yonghong

2018-03-18

The aim of this study was to identify the anti-cancer mechanism of Polyphyllin I (PPI) on gastric cancer cells via its activity on cancer-associated fibroblasts (CAFs). We cultured purified gastric CAFs obtained from fresh human gastric cancer tissue and examined the effect of Polyphyllin I on CAF proliferation using a colorimetric viability assay. In addition, we established a nude mouse xenograft model to examine the effect of Polyphyllin I administration on tumorigenesis. Using Western analysis, we quantified protein expression of the CAF-derived cytokines fibroblast activation protein alpha (FAP), secreted protein acidic and cysteine rich (SPARC), stromal cell-derived factor 1 (SDF-1), hepatocyte growth factor tenascin-C (TNC), and hepatocyte growth factor (HGF) in both in vitro and in vivo models. We found that Polyphyllin I inhibits the proliferation of CAFs in a concentration-dependent manner. Following treatment with 2 μg/ml PPI for 24 h in vitro, the expression of FAP, SDF-1 and HGF protein in CAFs was significantly lower than that in the control group, but there was no significant difference in SPARC and TNC protein expression between the two groups. In the nude mouse xenograft model, the tumor inhibition rate was 45.5% when PPI was administered early and 29.4% with administration in the third week. The expression of FAP and HGF in the xenografts was significantly decreased, while the expression of SPARC, SDF-1, and TNC was largely unaltered. Altogether, these data suggest that Polyphyllin I can inhibit the proliferation of gastric cancer cells by downregulating the expression of FAP and HGF in CAFs in vivo. Copyright © 2018 Elsevier Inc. All rights reserved.

Endometrial stromal tumours revisited: an update based on the 2014 WHO classification.

PubMed

Ali, Rola H; Rouzbahman, Marjan

2015-05-01

Endometrial stromal tumours (EST) are rare tumours of endometrial stromal origin that account for less than 2% of all uterine tumours. Recent cytogenetic and molecular advances in this area have improved our understanding of ESTs and helped refine their classification into more meaningful categories. Accordingly, the newly released 2014 WHO classification system recognises four categories: endometrial stromal nodule (ESN), low-grade endometrial stromal sarcoma (LGESS), high-grade endometrial stromal sarcoma (HGESS) and undifferentiated uterine sarcoma (UUS). At the molecular level, these tumours may demonstrate a relatively simple karyotype with a defining chromosomal rearrangement (as in the majority of ESNs, LGESSs and YWHAE-rearranged HGESS) or demonstrate complex cytogenetic aberrations lacking specific rearrangements (as in UUSs). Herein we provide an update on this topic aimed at the practicing pathologist. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Identification of the 64 kilodalton chloroplast stromal phosphoprotein as phosphoglucomutase. [Pisum sativum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Salvucci, M.E.; Drake, R.R.; Broadbent, K.P.

1990-05-01

Phosphorylation of the 64 kilodalton stromal phosphoprotein by incubation of pea (Pisum sativum) chloroplast extracts with ({gamma}-{sup 32}P)ATP decreased in the presence of Glc-6-P and Glc-1,6-P{sub 2}, but was stimulated by glucose. Two-dimensional gel electrophoresis following incubation of intact chloroplasts and stromal extracts with ({gamma}-{sup 32}P)ATP, or incubation of stromal extracts and partially purified phosphoglucomutase (EC 2.7.5.1) with ({sup 32}P)Glc-1-P showed that the identical 64 kilodalton polypeptide was labeled. A 62 kilodalton polypeptide was phosphorylated by incubation of tobacco (Nicotiana sylvestris) stromal extracts with either ({gamma}-{sup 32}P)ATP or ({sup 32}P)Glc-1-P. In contrast, an analogous polypeptide was not phosphorylated in extractsmore » from a tobacco mutant deficient in plastid phosphoglucomutase activity. The results indicate that the 64 (or 62) kilodalton chloroplast stromal phosphoprotein is phosphoglucomutase.« less

Cancer-associated fibroblasts affect breast cancer cell gene expression, invasion and angiogenesis.

PubMed

Eiro, Noemi; González, Lucía; Martínez-Ordoñez, Anxo; Fernandez-Garcia, Belen; González, Luis O; Cid, Sandra; Dominguez, Francisco; Perez-Fernandez, Román; Vizoso, Francisco J

2018-03-01

It has been reported that stromal cell features may affect the clinical outcome of breast cancer patients. Cancer associated fibroblasts (CAFs) represent one of the most abundant cell types within the breast cancer stroma. Here, we aimed to explore the influence of CAFs on breast cancer gene expression, as well as on invasion and angiogenesis. qRT-PCR was used to evaluate the expression of several cancer progression related genes (S100A4, TGFβ, FGF2, FGF7, PDGFA, PDGFB, VEGFA, IL-6, IL-8, uPA, MMP2, MMP9, MMP11 and TIMP1) in the human breast cancer-derived cell lines MCF-7 and MDA-MB-231, before and after co-culture with CAFs. Stromal mononuclear inflammatory cell (MIC) MMP11 expression was used to stratify primary tumors. In addition, we assessed the in vitro effects of CAFs on both MDA-MB-231 breast cancer cell invasion and endothelial cell (HUVEC) tube formation. We found that the expression levels of most of the genes tested were significantly increased in both breast cancer-derived cell lines after co-culture with CAFs from either MMP11+ or MMP11- MIC tumors. IL-6 and IL-8 showed an increased expression in both cancer-derived cell lines after co-culture with CAFs from MMP11+ MIC tumors. We also found that the invasive and angiogenic capacities of, respectively, MDA-MB-231 and HUVEC cells were increased after co-culture with CAFs, especially those from MMP11+ MIC tumors. Our data indicate that tumor-derived CAFs can induce up-regulation of genes involved in breast cancer progression. Our data additionally indicate that CAFs, especially those derived from MMP11+ MIC tumors, can promote breast cancer cell invasion and angiogenesis.

Microencapsulated VEGF gene-modified umbilical cord mesenchymal stromal cells promote the vascularization of tissue-engineered dermis: an experimental study.

PubMed

Han, Yanfu; Tao, Ran; Han, Yanqing; Sun, Tianjun; Chai, Jiake; Xu, Guang; Liu, Jing

2014-02-01

Tissue-engineered dermis (TED) is thought to be the best treatment for skin defect wounds; however, lack of vascular structures in these products can cause slow vascularization or even transplant failure. We assessed the therapeutic potential of microencapsulated human umbilical cord mesenchymal stromal cells (hUCMSCs) expressing vascular endothelial growth factor (VEGF) in vascularization of TED. hUCMSCs were isolated by means of enzymatic digestion and identified by means of testing biological characteristics. hUCMSCs were induced to differentiate into dermal fibroblasts in conditioned induction media. Collagen-chitosan laser drilling acellular dermal matrix (ADM) composite scaffold was prepared by means of the freeze dehydration and dehydrothermal cross-linking method. hUCMSC-derived fibroblasts were implanted on composite scaffolds to construct TED. TED with microencapsulated VEGF gene-modified hUCMSCs was then transplanted into skin defect wounds in pigs. The angiogenesis of TED at 1 week and status of wound healing at 3 weeks were observed. The collagen-chitosan laser ADM composite has a uniform microporous structure. This composite has been used to grow hUCMSC-derived fibroblasts in vitro and to successfully construct stem cell-derived TED. Microencapsulated VEGF gene-modified hUCMSCs were prepared with the use of a sodium alginate-barium chloride one-step encapsulation technology. Seven days after the transplantation of the stem cell-derived TED and microencapsulated VEGF gene-modified hUCMSCs into the skin defect wounds on the backs of miniature pigs, the VEGF expression increased and the TED had a higher degree of vascularization. Re-epithelialization of the wound was completed after 3 weeks. Microencapsulated VEGF gene-modified hUCMSCs can effectively improve the vascularization of TED and consequently the quality of wound healing. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Protection of Brain Injury by Amniotic Mesenchymal Stromal Cell-Secreted Metabolites.

PubMed

Pischiutta, Francesca; Brunelli, Laura; Romele, Pietro; Silini, Antonietta; Sammali, Eliana; Paracchini, Lara; Marchini, Sergio; Talamini, Laura; Bigini, Paolo; Boncoraglio, Giorgio B; Pastorelli, Roberta; De Simoni, Maria-Grazia; Parolini, Ornella; Zanier, Elisa R

2016-11-01

To define the features of human amniotic mesenchymal stromal cell secretome and its protective properties in experimental models of acute brain injury. Prospective experimental study. Laboratory research. C57Bl/6 mice. Mice subjected to sham or traumatic brain injury by controlled cortical impact received human amniotic mesenchymal stromal cells or phosphate-buffered saline infused intracerebroventricularly or intravenously 24 hours after injury. Organotypic cortical brain slices exposed to ischemic injury by oxygen-glucose deprivation were treated with human amniotic mesenchymal stromal cells or with their secretome (conditioned medium) in a transwell system. Traumatic brain injured mice receiving human amniotic mesenchymal stromal cells intravenously or intracerebroventricularly showed early and lasting functional and anatomical brain protection. cortical slices injured by oxigen-glucose deprivation and treated with human amniotic mesenchymal stromal cells or conditioned medium showed comparable protective effects (neuronal rescue, promotion of M2 microglia polarization, induction of trophic factors) indicating that the exposure of human amniotic mesenchymal stromal cells to the injured tissue is not necessary for the release of bioactive factors. Using sequential size-exclusion and gel-filtration chromatography, we identified a conditioned medium subfraction, which specifically displays these highly protective properties and we found that this fraction was rich in bioactive molecules with molecular weight smaller than 700 Da. Quantitative RNA analysis and mass spectrometry-based peptidomics showed that the active factors are not proteins or RNAs. The metabolomic profiling of six metabolic classes identified a list of molecules whose abundance was selectively elevated in the active conditioned medium fraction. Human amniotic mesenchymal stromal cell-secreted factors protect the brain after acute injury. Importantly, a fraction rich in metabolites, and

Rho A and the Rho kinase pathway regulate fibroblast contraction: Enhanced contraction in constitutively active Rho A fibroblast cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nobe, Koji, E-mail: kojinobe@pharm.showa-u.ac.jp; Nobe, Hiromi; Department of Physical Therapy, Bunkyo-Gakuin University

Research highlights: {yields} Mechanisms of fibroblast cell contraction in collagen matrix. {yields} Assessed an isometric force development using 3D-reconstituted-fibroblast fiber. {yields} Constitutively active Rho A induced the over-contraction of fibroblast cells. {yields} Rho A and Rho kinase pathway has a central role in fibroblast cell contraction. -- Abstract: Fibroblast cells play a central role in the proliferation phase of wound healing processes, contributing to force development. The intracellular signaling pathways regulating this non-muscle contraction are only partially understood. To study the relations between Rho A and contractile responses, constitutively active Rho A (CA-Rho A) fibroblast cells were reconstituted into fibersmore » and the effects of calf serum (CS) on isometric force were studied. CS-induced force in CA-Rho A fibroblast fibers was twice as large as that in wild type (NIH 3T3) fibroblast fibers. During this response, the translocation of Rho A from the cytosol to the membrane was detected by Rho A activity assays and Western blot analysis. Pre-treatment with a Rho specific inhibitor (C3-exoenzyme) suppressed translocation as well as contraction. These results indicate that Rho A activation is essential for fibroblast contraction. The Rho kinase inhibitor ( (Y27632)) inhibited both NIH 3T3 and CA-Rho A fibroblast fiber contractions. Activation of Rho A is thus directly coupled with Rho kinase activity. We conclude that the translocation of Rho A from the cytosol to the membrane and the Rho kinase pathway can regulate wound healing processes mediated by fibroblast contraction.« less

Dendritic cells control fibroblastic reticular network tension and lymph node expansion.

PubMed

Acton, Sophie E; Farrugia, Aaron J; Astarita, Jillian L; Mourão-Sá, Diego; Jenkins, Robert P; Nye, Emma; Hooper, Steven; van Blijswijk, Janneke; Rogers, Neil C; Snelgrove, Kathryn J; Rosewell, Ian; Moita, Luis F; Stamp, Gordon; Turley, Shannon J; Sahai, Erik; Reis e Sousa, Caetano

2014-10-23

After immunogenic challenge, infiltrating and dividing lymphocytes markedly increase lymph node cellularity, leading to organ expansion. Here we report that the physical elasticity of lymph nodes is maintained in part by podoplanin (PDPN) signalling in stromal fibroblastic reticular cells (FRCs) and its modulation by CLEC-2 expressed on dendritic cells. We show in mouse cells that PDPN induces actomyosin contractility in FRCs via activation of RhoA/C and downstream Rho-associated protein kinase (ROCK). Engagement by CLEC-2 causes PDPN clustering and rapidly uncouples PDPN from RhoA/C activation, relaxing the actomyosin cytoskeleton and permitting FRC stretching. Notably, administration of CLEC-2 protein to immunized mice augments lymph node expansion. In contrast, lymph node expansion is significantly constrained in mice selectively lacking CLEC-2 expression in dendritic cells. Thus, the same dendritic cells that initiate immunity by presenting antigens to T lymphocytes also initiate remodelling of lymph nodes by delivering CLEC-2 to FRCs. CLEC-2 modulation of PDPN signalling permits FRC network stretching and allows for the rapid lymph node expansion--driven by lymphocyte influx and proliferation--that is the critical hallmark of adaptive immunity.

GPER-mediated proliferation and estradiol production in breast cancer-associated fibroblasts

PubMed Central

Luo, Haojun; Yang, Guanglun; Yu, Tenghua; Luo, Shujuan; Wu, Chengyi; Sun, Yan; Liu, Manran; Tu, Gang

2014-01-01

Cancer-associated fibroblasts (CAFs) are crucial co-mediators of breast cancer progression. Estrogen is the predominant driving force in the cyclic regulation of the mammary extracellular matrix, thus potentially affecting the tumor-associated stroma. Recently, a third estrogen receptor, estrogen (G-protein-coupled) receptor (GPER), has been reported to be expressed in breast CAFs. In this study, GPER was detected by immunohistochemical analysis in stromal fibroblasts of 41.8% (59/141) of the primary breast cancer samples. GPER expression in CAFs isolated from primary breast cancer tissues was confirmed by immunostaining and RT-PCR analyses. Tamoxifen (TAM) in addition to 17β-estradiol (E2) and the GPER agonist G1 activated GPER, resulting in transient increases in cell index, intracellular calcium, and ERK1/2 phosphorylation. Furthermore, TAM, E2, and G1 promoted CAF proliferation and cell-cycle progression, both of which were blocked by GPER interference, the selective GPER antagonist G15, the epidermal growth factor receptor (EGFR) inhibitor AG1478, and the ERK1/2 inhibitor U0126. Importantly, TAM as well as G1 increased E2 production in breast CAFs via GPER/EGFR/ERK signaling when the substrate of E2, testosterone, was added to the medium. GPER-induced aromatase upregulation was probably responsible for this phenomenon, as TAM- and G1-induced CYP19A1 gene expression was reduced by GPER knockdown and G15, AG1478, and U0126 administration. Accordingly, GPER-mediated CAF-dependent estrogenic effects on the tumor-associated stroma are conceivable, and CAF is likely to contribute to breast cancer progression, especially TAM resistance, via a positive feedback loop involving GPER/EGFR/ERK signaling and E2 production. PMID:24481325

Lewis lung carcinoma progression is facilitated by TIG-3 fibroblast cells.

PubMed

Yamauchi, Yoshikane; Izumi, Yotaro; Asakura, Keisuke; Kawai, Kenji; Wakui, Masatoshi; Ohmura, Mitsuyo; Suematsu, Makoto; Nomori, Hiroaki

2013-09-01

The interactions of tumor cells with stromal fibroblasts influence tumor biology, but the exact mechanisms involved are still unclear. In the present study, we evaluated the effects of a human lung fibroblast cell line, TIG-3, on Lewis lung carcinoma (LLC) cells both in vitro and in vivo. LLC and TIG-3 cells were co-cultured/co-implanted in vitro and in vivo. Cell invasion was assayed. Local tumor growth, as well as lung metastasis, were evaluated after subcutaneous cell co-implantation into NOD/SCID/γ-null (NOG) mice. LLC, and TIG-3 cells were pre-treated with either SB431542, a small molecule TGF-β receptor antagonist, or siRNA for transforming growth factor (TGF)-β before co-culture or co-implantation, and the effects of pre-treatments were compared both in cell culture and in mice. Subcutaneous LLC tumor growth (L group) in NOG mice was significantly increased by co-implantation of TIG-3 cells (L+T group) at four weeks. The number of macroscopic lung metastases was also significantly increased in the L+T group in comparison to the L group. In vitro cell invasion was significantly increased in the L+T group in comparison to the L group. In vitro expression of phosphorylated-SMAD3 was significantly increased in the L+T group in comparison to the L group. Furthermore, pre-treatment with either SB431542 or siRNA for TGF-β reduced the invasiveness both in culture and in mice. This study suggested that in vitro as well as in vivo progression of LLC was facilitated by co-culture/co-implantation with TIG-3 cells, and that this process was at least in part dependent on TGF-β-mediated interactions.

Epidermal regulation of dermal fibroblast activity.

PubMed

Garner, W L

1998-07-01

Although the association between delayed burn wound healing and subsequent hypertrophic scar formation is well-established, the mechanism for this relationship is unknown. Unhealed burn wounds lack an epidermis, suggesting a possible regulatory role for the epidermis in controlling dermal fibroblast matrix synthesis. Therefore, we examined the effect of epidermal cells and media conditioned by epidermal cells on fibroblast collagen synthesis and replication. Purified fibroblast and keratinocyte cell strains were developed from discarded normal adult human skin. Conditioned media were created by incubation of cytokine-free and serum-free medium with either confluent fibroblast or keratinocyte cultures for 18 hours (n = 3). Nearly confluent fibroblast cultures were exposed for 48 hours to graded concentrations of either unconditioned medium (control), conditioned medium, or varying numbers of keratinocytes. Replication was quantified by the incorporation of 3H-thymidine. Collagen synthesis was measured by the incorporation of 3H-proline into collagenase-sensitive protein. Data were compared using analysis of variance (ANOVA) and linear regression. Keratinocyte conditioned medium induced a significant increase in replication (n = 3) (p = 0.004) and a decrease in collagen synthesis (n = 6) (p < 0.001). In contrast, neither fibroblast conditioned medium nor control medium had an effect on fibroblast replication or collagen synthesis. Co-culture of fibroblast with a graded number of keratinocytes similarly decreased collagen synthesis (n = 6) (p < 0.001). Dermal fibroblast collagen synthesis appears to be regulated by a soluble keratinocyte product. This result suggests a mechanism for the clinical observation that unhealed burn wounds, which lack the epidermis, demonstrate excess collagen production and scar. Clinical strategies to decrease hypertrophic scar should include an attempt at early wound closure with skin grafting or the application of cultured epithelial

Stromal vascular cells and adipogenesis: Cells within adipose depots regulate adipogenesis

USDA-ARS?s Scientific Manuscript database

A collection of investigations indicate the importance of adipose tissue stromal/stem cells to vasculogenesis and angiogenesis during adipogenesis. Early in development the stromal-vascular (S-V) elements control and dictate the extent of adipogenesis in a depot dependent manner. For instance, the...

Stromal matrix metalloproteinase-11 is involved in the mammary gland postnatal development.

PubMed

Tan, J; Buache, E; Alpy, F; Daguenet, E; Tomasetto, C-L; Ren, G-S; Rio, M-C

2014-07-31

MMP-11 is a bad prognosis paracrine factor in invasive breast cancers. However, its mammary physiological function remains largely unknown. In the present study we have investigated MMP-11 function during postnatal mammary gland development and function using MMP-11-deficient (MMP-11-/-) mice. Histological and immunohistochemical analyses as well as whole-mount mammary gland staining show alteration of the mammary gland in the absence of MMP-11, where ductal tree, alveolar structures and milk production are reduced. Moreover, a series of transplantation experiments allowed us to demonstrate that MMP-11 exerts an essential local paracrine function that favors mammary gland branching and epithelial cell outgrowth and invasion through adjacent connective tissues. Indeed, MMP-11-/- cleared fat pads are not permissive for wild-type epithelium development, whereas MMP-11-/- epithelium transplants grow normally when implanted in wild-type cleared fat pads. In addition, using primary mammary epithelial organoids, we show in vitro that this MMP-11 pro-branching effect is not direct, suggesting that MMP-11 acts via production/release of stroma-associated soluble factor(s). Finally, the lack of MMP-11 leads to decreased periductal collagen content, suggesting that MMP-11 has a role in collagen homeostasis. Thus, local stromal MMP-11 might also regulate mammary epithelial cell behavior mechanically by promoting extracellular matrix stiffness. Collectively, the present data indicate that MMP-11 is a paracrine factor involved during postnatal mammary gland morphogenesis, and support the concept that the stroma strongly impact epithelial cell behavior. Interestingly, stromal MMP-11 has previously been reported to favor malignant epithelial cell survival and promote cancer aggressiveness. Thus, MMP-11 has a paracrine function during mammary gland development that might be harnessed to promote tumor progression, exposing a new link between development and malignancy.

CXCL14 and NOS1 expression in specimens from patients with stage I-IIIA nonsmall cell lung cancer after curative resection.

PubMed

Ji, Xiaoqin; Shen, Zetian; Zhao, Benxin; Yuan, Xi; Zhu, Xixu

2018-03-01

Many studies show that CXC chemokine ligand 14 (CXCL14) is highly expressed in tumor-associated stromal cells, promoting tumor cell growth, and invasion. Because of its unclear receptors, CXCL14-initiated intracellular signal cascades remain largely unknown. However, CXCL14 can regulate nitric oxide synthase 1 (NOS1) as its intracellular molecular target. In this paper, we investigated the expression of CXCL14 and NOS1 in specimens from patients with stage I-IIIA nonsmall cell lung cancer (NSCLC) after curative resection, and evaluated the prognostic significance of this gene expression in stromal fibroblasts and cancer cells.Immunohistochemistry was used to detect the expression of CXCL14 and NOS1 in 106 formalin fixed, paraffin-embedded specimens from patients with stage I-IIIA NSCLC. The chi-square test was performed to examine the correlation of CXCL14 and NOS1 expression level with clinicopathological features. The effects of the expression of CXCL14 or NOS1 on progression-free survival (PFS) and overall survival (OS) were determined by Kaplan-Meier and Cox hazard proportional model.The percentages of high CXCL14 expression in stromal fibroblasts and that in cancer cells were 46.2% (49/106) and 23.6% (25/106), respectively. The positive expression rates of NOS1 in cancer cells were 42.5% (45/106). The result indicated that there was a significant positive correlation between CXCL14 expression level in stromal fibroblasts and that in cancer cells (χ = 4.158, P = .041). In addition, the expression of CXCL14 in stromal fibroblasts was significantly correlated with NOS1 expression in cancer cells (χ = 16.156, P < .001). The 5-year PFS rates with low and high CXCL14 expression in stromal fibroblasts were 66.7% and 14.3% (χ = 44.008, P < .001), respectively, and the 5-year OS rates with those were 87.1% and 43.5% (χ = 21.531, P < .001), respectively. The 5-year PFS rates with negative and positive expression of NOS1 in cancer

Rationale for the potential use of mesenchymal stromal cells in liver transplantation

PubMed Central

Vandermeulen, Morgan; Grégoire, Céline; Briquet, Alexandra; Lechanteur, Chantal; Beguin, Yves; Detry, Olivier

2014-01-01

Mesenchymal stromal cells (MSCs) are multipotent and self-renewing cells that reside essentially in the bone marrow as a non-hematopoietic cell population, but may also be isolated from the connective tissues of most organs. MSCs represent a heterogeneous population of adult, fibroblast-like cells characterized by their ability to differentiate into tissues of mesodermal lineages including adipocytes, chondrocytes and osteocytes. For several years now, MSCs have been evaluated for their in vivo and in vitro immunomodulatory and ‘tissue reconstruction’ properties, which could make them interesting in various clinical settings, and particularly in organ transplantation. This paper aims to review current knowledge on the properties of MSCs and their use in pre-clinical and clinical studies in solid organ transplantation, and particularly in the field of liver transplantation. The first available clinical data seem to show that MSCs are safe to use, at least in the medium-term, but more time is needed to evaluate the potential adverse effects of long-term use. Many issues must be resolved on the correct use of MSCs. Intensive in vitro and pre-clinical research are the keys to a better understanding of the way that MSCs act, and to eventually lead to clinical success. PMID:25469010

3D Functional Corneal Stromal Tissue Equivalent Based on Corneal Stromal Stem Cells and Multi-Layered Silk Film Architecture.

PubMed

Ghezzi, Chiara E; Marelli, Benedetto; Omenetto, Fiorenzo G; Funderburgh, James L; Kaplan, David L

2017-01-01

The worldwide need for human cornea equivalents continues to grow. Few clinical options are limited to allogenic and synthetic material replacements. We hypothesized that tissue engineered human cornea systems based on mechanically robust, patterned, porous, thin, optically clear silk protein films, in combination with human corneal stromal stem cells (hCSSCs), would generate 3D functional corneal stroma tissue equivalents, in comparison to previously developed 2D approaches. Silk film contact guidance was used to control the alignment and distribution of hCSSCs on RGD-treated single porous silk films, which were then stacked in an orthogonally, multi-layered architecture and cultured for 9 weeks. These systems were compared similar systems generated with human corneal fibroblasts (hCFs). Both cell types were viable and preferentially aligned along the biomaterial patterns for up to 9 weeks in culture. H&E histological sections showed that the systems seeded with the hCSSCs displayed ECM production throughout the entire thickness of the constructs. In addition, the ECM proteins tested positive for keratocyte-specific tissue markers, including keratan sulfate, lumican, and keratocan. The quantification of hCSSC gene expression of keratocyte-tissue markers, including keratocan, lumican, human aldehyde dehydrogenase 3A1 (ALDH3A1), prostaglandin D2 synthase (PTDGS), and pyruvate dehydrogenase kinase, isozyme 4 (PDK4), within the 3D tissue systems demonstrated upregulation when compared to 2D single silk films and to the systems generated with the hCFs. Furthermore, the production of ECM from the hCSSC seeded systems and subsequent remodeling of the initial matrix significantly improved cohesiveness and mechanical performance of the constructs, while maintaining transparency after 9 weeks.

Changes in the gene expression of co-cultured human fibroblast cells and osteosarcoma cells: the role of microenvironment.

PubMed

Salvatore, Viviana; Focaroli, Stefano; Teti, Gabriella; Mazzotti, Antonio; Falconi, Mirella

2015-10-06

The progression of malignant tumors does not depend exclusively on the autonomous properties of cancer cells; it is also influenced by tumor stroma reactivity and is under strict microenvironmental control. By themselves, stromal cells are not malignant, and they maintain normal tissue structure and function. However, through intercellular interactions or by paracrine secretions from cancer cells, normal stromal cells acquire abnormal phenotypes that sustain cancer cell growth and tumor progression. In their dysfunctional state, fibroblast and immune cells produce chemokines and growth factors that stimulate cancer cell growth and invasion. In our previous work, we established an in vitro model based on a monolayer co-culture system of healthy human fibroblasts (HFs) and human osteosarcoma cells (the MG-63 cell line) that simulates the microenvironment of tumor cells and healthy cells. The coexistence between MG-63 cells and HFs allowed us to identify the YKL-40 protein as the main marker for verifying the influence of tumor cells grown in contact with healthy cells. In this study, we evaluated the interactions of HFs and MG-63 cells in a transwell co-culture system over 24 h, 48 h, 72 h, and 96 h. We analyzed the contributions of these populations to the tumor microenvironment during cancer progression, as measured by multiple markers. We examined the effect of siRNA knockdown of YKL-40 by tracking the subsequent changes in gene expression within the co-culture. We validated the expression of several genes, focusing on those involved in cancer cell invasion, inflammatory responses, and angiogenesis: TNF alpha, IL-6, MMP-1, MMP-9, and VEGF. We compared the results to those from a transwell co-culture without the YKL-40 knockdown. In a pro-inflammatory environment promoted by TNF alpha and IL-6, siRNA knockdown of YKL-40 caused a down-regulation of VEGF and MMP-1 expression in HFs. These findings demonstrated that the tumor microenvironment has an influence on the

A novel role of EMMPRIN/CD147 in transformation of quiescent fibroblasts to cancer-associated fibroblasts by breast cancer cells

PubMed Central

Xu, Jing; Lu, Yang; Qiu, Songbo; Chen, Zhi-Nan; Fan, Zhen

2013-01-01

We tested the novel hypothesis that EMMPRIN/CD147, a transmembrane glycoprotein overexpressed in breast cancer cells, has a previously unknown role in transforming fibroblasts to cancer-associated fibroblasts, and that cancer-associated fibroblasts in turn induce epithelial-to-mesenchymal transition of breast cancer cells. Co-culture of fibroblasts with breast cancer cells or treatment of fibroblasts with breast cancer cell conditioned culture medium or recombinant EMMPRIN/CD147 induced expression of α-SMA in the fibroblasts in an EMMPRIN/CD147-dependent manner and promoted epithelial-to-mesenchymal transition of breast cancer cells and enhanced cell migration potential. These findings support a novel role of EMMPRIN/CD147 in regulating the interaction between cancer and stroma. PMID:23474495

Stromal cells in chronic inflammation and tertiary lymphoid organ formation.

PubMed

Buckley, Christopher D; Barone, Francesca; Nayar, Saba; Bénézech, Cecile; Caamaño, Jorge

2015-01-01

Inflammation is an unstable state. It either resolves or persists. Why inflammation persists and the factors that define tissue tropism remain obscure. Increasing evidence suggests that tissue-resident stromal cells not only provide positional memory but also actively regulate the differential accumulation of inflammatory cells within inflamed tissues. Furthermore, at many sites of chronic inflammation, structures that mimic secondary lymphoid tissues are observed, suggesting that chronic inflammation and lymphoid tissue formation share common activation programs. Similarly, blood and lymphatic endothelial cells contribute to tissue homeostasis and disease persistence in chronic inflammation. This review highlights our increasing understanding of the role of stromal cells in inflammation and summarizes the novel immunological role that stromal cells exert in the persistence of inflammatory diseases.

Alteration of Skin Properties with Autologous Dermal Fibroblasts

PubMed Central

Thangapazham, Rajesh L.; Darling, Thomas N.; Meyerle, Jon

2014-01-01

Dermal fibroblasts are mesenchymal cells found between the skin epidermis and subcutaneous tissue. They are primarily responsible for synthesizing collagen and glycosaminoglycans; components of extracellular matrix supporting the structural integrity of the skin. Dermal fibroblasts play a pivotal role in cutaneous wound healing and skin repair. Preclinical studies suggest wider applications of dermal fibroblasts ranging from skin based indications to non-skin tissue regeneration in tendon repair. One clinical application for autologous dermal fibroblasts has been approved by the Food and Drug Administration (FDA) while others are in preclinical development or various stages of regulatory approval. In this context, we outline the role of fibroblasts in wound healing and discuss recent advances and the current development pipeline for cellular therapies using autologous dermal fibroblasts. The microanatomic and phenotypic differences of fibroblasts occupying particular locations within the skin are reviewed, emphasizing the therapeutic relevance of attributes exhibited by subpopulations of fibroblasts. Special focus is provided to fibroblast characteristics that define regional differences in skin, including the thick and hairless skin of the palms and soles as compared to hair-bearing skin. This regional specificity and functional identity of fibroblasts provides another platform for developing regional skin applications such as the induction of hair follicles in bald scalp or alteration of the phenotype of stump skin in amputees to better support their prosthetic devices. PMID:24828202

Progressive Enrichment of Stemness Features and Tumor Stromal Alterations in Multistep Hepatocarcinogenesis.

PubMed

Yoo, Jeong Eun; Kim, Young-Joo; Rhee, Hyungjin; Kim, Haeryoung; Ahn, Ei Yong; Choi, Jin Sub; Roncalli, Massimo; Park, Young Nyun

2017-01-01

Cancer stem cells (CSCs), a subset of tumor cells, contribute to an aggressive biological behavior, which is also affected by the tumor stroma. Despite the role of CSCs and the tumor stroma in hepatocellular carcinoma (HCC), features of stemness have not yet been studied in relation to tumor stromal alterations in multistep hepatocarcinogenesis. We investigated the expression status of stemness markers and tumor stromal changes in B viral carcinogenesis, which is the main etiology of HCC in Asia. Stemness features of tumoral hepatocytes (EpCAM, K19, Oct3/4, c-KIT, c-MET, and CD133), and tumor stromal cells expressing α-smooth muscle actin (α-SMA), CD68, CD163, and IL-6 were analyzed in 36 low grade dysplastic nodules (DNs), 48 high grade DNs, 30 early HCCs (eHCCs), and 51 progressed HCCs (pHCCs) by immunohistochemistry or real-time PCR. Stemness features (EpCAM and K19 in particular) were progressively acquired during hepatocarcinogenesis in combination with enrichment of stromal cells (CAFs, TAMs, IL-6+ cells). Stemness features were seen sporadically in DNs, more consistent in eHCCs, and peaked in pHCCs. Likewise, stromal cells were discernable in DNs, showed up as consistent cell densities in eHCCs and peaked in pHCCs. The stemness features and tumor stromal alterations also peaked in less differentiated or larger HCCs. In conclusion, progression of B viral multistep hepatocarcinogenesis is characterized by an enrichment of stemness features of neoplastic hepatocytes and a parallel alteration of the tumor stroma. The modulation of neoplastic hepatocytes and stromal cells was at low levels in precancerous lesions (DNs), consistently increased in incipient cancer (eHCCs) and peaked in pHCCs. Thus, in B viral hepatocarcinogenesis, interactions between CSCs and the tumor stroma, although starting early, seem to play a major role in tumor progression.

Influence of stromal refractive index and hydration on corneal laser refractive surgery.

PubMed

de Ortueta, Diego; von Rüden, Dennis; Magnago, Thomas; Arba Mosquera, Samuel

2014-06-01

To evaluate the influence of the stromal refractive index and hydration on postoperative outcomes in eyes that had corneal laser refractive surgery using the Amaris laser system. Augenzentrum Recklinghausen, Recklinghausen, Germany. Comparative case series. At the 6-month follow-up, right eyes were retrospectively analyzed. The effect of the stromal refractive index and hydration on refractive outcomes was assessed using univariate linear and multilinear correlations. Sixty eyes were analyzed. Univariate linear analyses showed that the stromal refractive index and hydration were correlated with the thickness of the preoperative exposed stroma and was statistically different for laser in situ keratomileusis and laser-assisted subepithelial keratectomy treatments. Univariate multilinear analyses showed that the spherical equivalent (SE) was correlated with the attempted SE and stromal refractive index (or hydration). Analyses suggest overcorrections for higher stromal refractive index values and for lower hydration values. The stromal refractive index and hydration affected postoperative outcomes in a subtle, yet significant manner. An adjustment toward greater attempted correction in highly hydrated corneas and less intended correction in low hydrated corneas might help optimize refractive outcomes. Mr. Magnago and Dr. Arba-Mosquera are employees of and Dr. Diego de Ortueta is a consultant to Schwind eye-tech-solutions GmbH & Co. KG. Mr. Rüden has no financial or proprietary interest in any material or method mentioned. Copyright © 2014 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

Stimulatory effects of histamine on migration of nasal fibroblasts.

PubMed

Hong, Sung-Moon; Park, Il-Ho; Um, Ji-Young; Shin, Jae-Min; Lee, Heung-Man

2015-10-01

Fibroblast migration is crucial for normal wound repair after sinonasal surgery. Histamine is known to be involved in wound healing by its effects on cell proliferation and migration. This study aimed to determine whether histamine affects the migration of nasal fibroblasts and to investigate the mechanism of action of histamine on nasal fibroblasts. Primary cultures of nasal fibroblasts were established from inferior turbinate samples. Fibroblast migration was evaluated with scratch assays. Cells were treated with histamine and/or histamine receptor-selective antagonists. U-73122 and pertussis toxin, which are selective inhibitors of the lower signaling pathway of H1R and H4R, were used to confirm the modulation of nasal fibroblast migration by histamine. Fibroblast cytoskeletal structures were visualized with immunocytochemistry. Histamine significantly stimulated the migration of nasal fibroblasts. Antagonists selective for HR1 and HR4 significantly reduced nasal fibroblast migration. In immunocytochemical staining, histamine treatment increased membrane ruffling and pyrilamine, diphenhydramine, fexofenadine, and JNJ7777120 decreased histamine-induced membrane ruffling. U-73122 and pertussis toxin also decreased histamine-induced migration of fibroblasts. Histamine maintains its stimulatory effects on fibroblast migration in the presence of mitomycin C, which blocks proliferation of cells. We showed that histamine stimulates fibroblast migration in nasal fibroblasts. This effect appeared to be mediated by HR1 and HR4. However, because fibroblast migration also can be involved in scaring and fibrosis, more research is necessary to determine the effects of antihistamine on wound healing after sinus surgery. © 2015 ARS-AAOA, LLC.

Stromal Expression of Fibroblast Activation Protein Alpha (FAP) Predicts Platinum Resistance and Shorter Recurrence in patients with Epithelial Ovarian Cancer.

PubMed

Mhawech-Fauceglia, Paulette; Yan, Li; Sharifian, Maryam; Ren, Xing; Liu, Song; Kim, Grace; Gayther, Simon A; Pejovic, Tanja; Lawrenson, Kate

2015-04-01

The microenvironment plays an important role in tumorigenesis. Fibroblast activation protein alpha (FAP) is overexpressed by fibroblasts present in the microenvironment of many tumors. High FAP expression is a negative prognostic factor in several malignancies, but this has not been investigated in epithelial ovarian cancer (EOC). The aim of this study is to define the value of FAP in EOC. Immunohistochemical staining using an anti-FAP antibody was performed on 338 EOC tissues. mRNA levels in cancer cell lines and FAP silencing using siRNA was also done. FAP immunoexpression by tumor stroma was a significant predictive factor for platinum resistance (p = 0.0154). In survival analysis of days to recurrence, FAP stoma (+) was associated with shorter recurrence than those with FAP (-) stroma (p = 0.0247). In 21.8 % of tumors, FAP protein was expressed by the tumor epithelium, and FAP mRNA was more highly expressed in tumors (n = 489) than in normal tissues (n = 8) (p = 3.88 × 10(-4)). In vitro, addition of FAP to EOC cells induced a 10-12 % increase in cell viability both in the presence and absence of cisplatin. Conversely, siRNA silencing of FAP resulted in ~10 % reduction in EOC cell proliferation. We have shown that FAP expression in EOC is associated with poorer clinical outcomes. FAP may have novel cell-autonomous effects suggesting that targeting FAP could have pleiotropic anti-tumor effects, and anti-FAP therapy could be a highly effective novel treatment for EOC, especially in cisplatinum-resistant cases.

Drugs Approved for Gastrointestinal Stromal Tumors

Cancer.gov

This page lists cancer drugs approved by the Food and Drug Administration (FDA) for gastrointestinal stromal tumors (GIST). The list includes generic names and brand names. The drug names link to NCI's Cancer Drug Information summaries.

The gene for fibroblast activation protein {alpha} (FAP), a putative cell surface-bound serine protease expressed in cancer stroma and wound healing, maps to chromosome band 2q23

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mathew, S.; Murty, V.V.V.S.; Chaganti, R.S.K.

The human fibroblast activation protein {alpha} (FAP{alpha}) is an inducible cell surface glycoprotein of M{sub r} 95,000 recognized by a number of monoclonal antibodies (mAbs), including the prototype mAb F19. Immunohistochemical studies have shown that FAP{alpha} expression in vivo is tightly regulated, with transient expression in some fetal mesenchymal tissues but absence of expression in most normal adult tissues. Reexpression of FAP{alpha} is observed in the reactive stromal fibroblasts of several common types of epithelial cancers, including >90% of breast, colorectal, and lung carcinomas and healing wounds. Cloning and sequence analysis of an FAP{alpha}-specific cDNA has revealed that the moleculemore » is encoded by a novel gene, FAP, which shows sequence similarity to members of the serine protease family of integral membrane proteins, namely dipeptidyl peptidase IV (DPPIV, also known as lymphocyte activation antigen, CD26, or adenosine dearoinase binding protein) and DPPX, a DPPIV-related molecule of unknown function. 15 refs., 1 fig.« less

Feedback Activation of Basic Fibroblast Growth Factor Signaling via the Wnt/β-Catenin Pathway in Skin Fibroblasts

PubMed Central

Wang, Xu; Zhu, Yuting; Sun, Congcong; Wang, Tao; Shen, Yingjie; Cai, Wanhui; Sun, Jia; Chi, Lisha; Wang, Haijun; Song, Na; Niu, Chao; Shen, Jiayi; Cong, Weitao; Zhu, Zhongxin; Xuan, Yuanhu; Li, Xiaokun; Jin, Litai

2017-01-01

Skin wound healing is a complex process requiring the coordinated behavior of many cell types, especially in the proliferation and migration of fibroblasts. Basic fibroblast growth factor (bFGF) is a member of the FGF family that promotes fibroblast migration, but the underlying molecular mechanism remains elusive. The present RNA sequencing study showed that the expression levels of several canonical Wnt pathway genes, including Wnt2b, Wnt3, Wnt11, T-cell factor 7 (TCF7), and Frizzled 8 (FZD8) were modified by bFGF stimulation in fibroblasts. Enzyme-linked immunosorbent assay (ELISA) analysis also showed that Wnt pathway was activated under bFGF treatment. Furthermore, treatment of fibroblasts with lithium chloride or IWR-1, an inducer and inhibitor of the Wnt signaling pathway, respectively, promoted and inhibited cell migration. Also, levels of cytosolic glycogen synthase kinase 3 beta phosphorylated at serine9 (pGSK3β Ser9) and nuclear β-catenin were increased upon exposure to bFGF. Molecular and biochemical assays indicated that phosphoinositide 3-kinase (PI3K) signaling activated the GSK3β/β-catenin/Wnt signaling pathway via activation of c-Jun N-terminal kinase (JNK), suggesting that PI3K and JNK act at the upstream of β-catenin. In contrast, knock-down of β-catenin delayed fibroblast cell migration even under bFGF stimulation. RNA sequencing analysis of β-catenin knock-down fibroblasts demonstrated that β-catenin positively regulated the transcription of bFGF and FGF21. Moreover, FGF21 treatment activated AKT and JNK, and accelerated fibroblast migration to a similar extent as bFGF does. In addition, ELISA analysis demonstrated that both of bFGF and FGF21 were auto secretion factor and be regulated by Wnt pathway stimulators. Taken together, our analyses define a feedback regulatory loop between bFGF (FGF21) and Wnt signaling acting through β-catenin in skin fibroblasts. PMID:28217097

Cardiac Fibroblast: The Renaissance Cell

PubMed Central

Souders, Colby A.; Bowers, Stephanie L.K.; Baudino, Troy A.

2012-01-01

The permanent cellular constituents of the heart include cardiac fibroblasts, myocytes, endothelial cells and vascular smooth muscle cells. Previous studies have demonstrated that there are undulating changes in cardiac cell populations during embryonic development, through neonatal development and into the adult. Transient cell populations include lymphocytes, mast cells and macrophages, which can interact with these permanent cell types to affect cardiac function. It has also been observed that there are marked differences in the makeup of the cardiac cell populations depending on the species, which may be important when examining myocardial remodeling. Current dogma states that the fibroblast makes up the largest cell population of the heart; however, this appears to vary for different species, especially mice. Cardiac fibroblasts play a critical role in maintaining normal cardiac function, as well as in cardiac remodeling during pathological conditions such as myocardial infarct and hypertension. These cells have numerous functions, including synthesis and deposition of extracellular matrix, cell-cell communication with myocytes, cell-cell signaling with other fibroblasts, as well as with endothelial cells. These contacts affect the electrophysiological properties, secretion of growth factors and cytokines, as well as potentiating blood vessel formation. While a plethora of information is known about several of these processes, relatively little is understood about fibroblasts and their role in angiogenesis during development or cardiac remodeling. In this review we provide insight into the various properties of cardiac fibroblasts that helps illustrate their importance in maintaining proper cardiac function, as well as their critical role in the remodeling heart. PMID:19959782

[Gastric stromal tumor treated by laparoscopic surgery].

PubMed

Alecu, L; Costan, I; Vitalariu, A; Obrocea, F; Păcuraru, Elena; Gulinescu, L

2002-01-01

The authors describe a 59 years old female patient, with a gastrointestinal stromal tumor located on the posterior wall of the gastric funds, who was treated successfully by laparoscopic wedge resection (with clear resection margins), through an anteriorly placed gastrotomy, thus allowing an endoscopic linear cutter Endo GIA, to excise the tumor with a cuff of normal gastric tissue. The anterior gastrotomy was performed with Ultra-Shears. Delivery of the tumor through the gastrotomy is essential for success. The operative time was 110 minutes. The tumor was diagnosed as a gastrointestinal stromal submucosal tumor (of low-grade malignancy) and immunohistochemicaly, this tumor was positive for CD 34. Posterior gastric tumor can be removed using laparoscopic surgery.

Characterization of interleukin-4-stimulated nasal polyp fibroblasts.

PubMed

Steinke, John W; Crouse, Charles D; Bradley, Dewayne; Hise, Kathleen; Lynch, Kevin; Kountakis, Stilianos E; Borish, Larry

2004-02-01

Chronic hyperplastic eosinophilic sinusitis is an inflammatory disease that results in the accumulation of eosinophils, fibroblasts, mast cells, and goblet cells at the site of injury. A common feature of this disease is the presence of nasal polyposis (NP). The current studies were designed to assess the contribution of interleukin (IL)-4 to fibroblast-mediated inflammation in chronic hyperplastic eosinophilic sinusitis/NP. In addition, we hypothesized that cysteinyl leukotrienes (CysLT) may directly influence fibroblast-mediated fibrotic and remodeling pathways in this disorder. Fibroblasts were isolated from NP tissue. All fibroblast lines expressed the IL-4 receptor. IL-4 induced changes in mRNA and protein expression of fibrotic (transforming growth factor-beta1 and -beta2) and inflammatory cytokines and chemokines (IL-6 and CCL11) by fibroblasts as measured by semiquantitative and quantitative polymerase chain reaction, RNase protection assay, and enzyme-linked immunosorbent assay. The expression of CysLT and other proinflammatory lipid receptors on fibroblasts was evaluated. CysLT1 and CysLT2 receptors were not expressed on fibroblasts; however, LPA(1) receptor was constitutively expressed and LPA(2) receptor expression was upregulated by IL-4. The metabolic cascade involved in CysLT synthesis was not expressed in fibroblasts and could not be induced by IL-4 treatment.

The fibroblast surface markers FAP, anti-fibroblast, and FSP are expressed by cells of epithelial origin and may be altered during epithelial-to-mesenchymal transition.

PubMed

Kahounová, Zuzana; Kurfürstová, Daniela; Bouchal, Jan; Kharaishvili, Gvantsa; Navrátil, Jiří; Remšík, Ján; Šimečková, Šárka; Študent, Vladimír; Kozubík, Alois; Souček, Karel

2017-04-06

The identification of fibroblasts and cancer-associated fibroblasts from human cancer tissue using surface markers is difficult, especially because the markers used currently are usually not expressed solely by fibroblasts, and the identification of fibroblast-specific surface molecules is still under investigation. It was aimed to compare three commercially available antibodies in the detection of different surface epitopes of fibroblasts (anti-fibroblast, fibroblast activation protein α, and fibroblast surface protein). The specificity of their expression, employing fibroblast cell lines and tumor-derived fibroblasts from breast and prostate tissues was investigated. Both the established fibroblast cell line HFF-1 and ex vivo primary fibroblasts isolated from breast and prostate cancer tissues expressed the tested surface markers to different degrees. Surprisingly, those markers were expressed also by permanent cell lines of epithelial origin, both benign and cancer-derived (breast-cell lines MCF 10A, HMLE and prostate-cell lines BPH-1, DU 145, and PC-3). The expression of fibroblast activation protein α increased on the surface of previously described models of epithelial cells undergoing epithelial-to-mesenchymal transition in response to treatment with TGF-β1. To prove the co-expression of the fibroblast markers on cells of epithelial origin, we used freshly dissociated human prostate and breast cancer tissues. The results confirmed the co-expression of anti-fibroblast and fibroblast surface protein on CD31/CD45-negative/EpCAM-positive epithelial cells. In summary, our data support the findings that the tested fibroblast markers are not fibroblast specific and may be expressed also by cells of epithelial origin (e.g., cells undergoing EMT). Therefore, the expression of these markers should be interpreted with caution, and the combination of several epitopes for both positive (anti-fibroblast or fibroblast activation protein α) and negative (Ep

Endometrial stromal sarcoma diagnosed after uterine morcellation in laparoscopic supracervical hysterectomy.

PubMed

Della Badia, Carl; Karini, Homa

2010-01-01

Endometrial stromal sarcoma is a rare uterine cancer with no reliable method for preoperative diagnosis. A 30-year-old parous woman underwent laparoscopic supracervical hysterectomy because of a leiomyoma. The uterus was removed from the abdominal cavity with an electric morcellator with a spinning blade. The pathology report revealed low-grade endometrial stromal sarcoma. Two months after the initial surgery, a second laparoscopic procedure was performed. The final pathology report confirmed low-grade endometrial stromal sarcoma involving the ovary, fallopian tube, and ovarian artery. It was concluded that morcellation of leiomyomas at laparoscopic supracervical hysterectomy may potentially increase metastasis if the tumor is a sarcoma. Copyright © 2010 AAGL. Published by Elsevier Inc. All rights reserved.

Metronomic chemotherapy prevents therapy-induced stromal activation and induction of tumor-initiating cells

PubMed Central

Chan, Tze-Sian; Pai, Vincent C.; Tan, Kok-Tong; Yen, Chia-Jui; Hsu, Shu-Ching; Chen, Wei-Yu; Shan, Yan-Shen; Lee, Michael T.; Chu, Jui-Mei

2016-01-01

Although traditional chemotherapy kills a fraction of tumor cells, it also activates the stroma and can promote the growth and survival of residual cancer cells to foster tumor recurrence and metastasis. Accordingly, overcoming the host response induced by chemotherapy could substantially improve therapeutic outcome and patient survival. In this study, resistance to treatment and metastasis has been attributed to expansion of stem-like tumor-initiating cells (TICs). Molecular analysis of the tumor stroma in neoadjuvant chemotherapy–treated human desmoplastic cancers and orthotopic tumor xenografts revealed that traditional maximum-tolerated dose chemotherapy, regardless of the agents used, induces persistent STAT-1 and NF-κB activity in carcinoma-associated fibroblasts. This induction results in the expression and secretion of ELR motif–positive (ELR+) chemokines, which signal through CXCR-2 on carcinoma cells to trigger their phenotypic conversion into TICs and promote their invasive behaviors, leading to paradoxical tumor aggression after therapy. In contrast, the same overall dose administered as a low-dose metronomic chemotherapy regimen largely prevented therapy-induced stromal ELR+ chemokine paracrine signaling, thus enhancing treatment response and extending survival of mice carrying desmoplastic cancers. These experiments illustrate the importance of stroma in cancer therapy and how its impact on treatment resistance could be tempered by altering the dosing schedule of systemic chemotherapy. PMID:27881732

Fibroblastic connective tissue nevus.

PubMed

Velez, Moises J; Billings, Steven D; Weaver, Joshua A

2016-01-01

Fibroblastic connective tissue nevus (FCTN) is a newly recognized, benign cutaneous mesenchymal lesion of fibroblasts/myofibroblastic lineage, which expands the classification of connective tissue nevi. We present three cases of FCTN and discuss significant clinical, morphologic and immunophenotypic overlap with dermatomyofibroma. Our cases were from young women, aged 32, 24 and 10, and presented as 1.2 and 1 cm nodules on the posterior neck and right upper flank, respectively while presenting as a linear plaque of the right posterior thigh in the latter case. The lesions showed a poorly circumscribed proliferation of hypercellular spindle cells arranged in short to longer intersecting fascicles entrapping adnexal structures. Superficial adipose tissue was also entrapped in one case. The spindle cells had fibroblastic features with pale eosinophilic cytoplasmic extensions and inconspicuous nucleoli. The spindle cells were positive for CD34 in two cases. One case was negative for CD34, smooth muscle actin (SMA), desmin and S100. The overall features were consistent with a diagnosis of FCTN. In two cases, we further elucidated the fibroblastic differentiation of the spindle cells in FCTN with electron microscopy, which has not been previously described. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Interacting resident epicardium-derived fibroblasts and recruited bone marrow cells form myocardial infarction scar.

PubMed

Ruiz-Villalba, Adrián; Simón, Ana M; Pogontke, Cristina; Castillo, María I; Abizanda, Gloria; Pelacho, Beatriz; Sánchez-Domínguez, Rebeca; Segovia, José C; Prósper, Felipe; Pérez-Pomares, José M

2015-05-19

Although efforts continue to find new therapies to regenerate infarcted heart tissue, knowledge of the cellular and molecular mechanisms involved remains poor. This study sought to identify the origin of cardiac fibroblasts (CFs) in the infarcted heart to better understand the pathophysiology of ventricular remodeling following myocardial infarction (MI). Permanent genetic tracing of epicardium-derived cell (EPDC) and bone marrow-derived blood cell (BMC) lineages was established using Cre/LoxP technology. In vivo gene and protein expression studies, as well as in vitro cell culture assays, were developed to characterize EPDC and BMC interaction and properties. EPDCs, which colonize the cardiac interstitium during embryogenesis, massively differentiate into CFs after MI. This response is disease-specific, because angiotensin II-induced pressure overload does not trigger significant EPDC fibroblastic differentiation. The expansion of epicardial-derived CFs follows BMC infiltration into the infarct site; the number of EPDCs equals that of BMCs 1 week post-infarction. BMC-EPDC interaction leads to cell polarization, packing, massive collagen deposition, and scar formation. Moreover, epicardium-derived CFs display stromal properties with respect to BMCs, contributing to the sustained recruitment of circulating cells to the damaged zone and the cardiac persistence of hematopoietic progenitors/stem cells after MI. EPDCs, but not BMCs, are the main origin of CFs in the ischemic heart. Adult resident EPDC contribution to the CF compartment is time- and disease-dependent. Our findings are relevant to the understanding of post-MI ventricular remodeling and may contribute to the development of new therapies to treat this disease. Copyright © 2015 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Mesenchymal Stem Cells Derived from Human Limbal Niche Cells

PubMed Central

Li, Gui-Gang; Zhu, Ying-Ting; Xie, Hua-Tao; Chen, Szu-Yu; Tseng, Scheffer C. G.

2012-01-01

Purpose. We investigated whether human limbal niche cells generate mesenchymal stem cells. Methods. Limbal niche cells were isolated from the limbal stroma by collagenase alone or following dispase removal of the limbal epithelium (D/C), and cultured on plastic in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), or coated or three-dimensional Matrigel in embryonic stem cell medium with leukemia inhibitory factor and basic fibroblast growth factor. Expression of cell markers, colony-forming units-fibroblast, tri-lineage differentiation, and ability of supporting limbal epithelial stem/progenitor cells were compared to limbal residual stromal cells. Results. Stromal cells expressing angiogenesis markers were found perivascularly, subjacent to limbal basal epithelial cells, and in D/C and limbal residual stromal cells. When seeded in three-dimensional Matrigel, D/C but not limbal residual stromal cells yielded spheres of angiogenesis progenitors that stabilized vascular networks. Similar to collagenase-isolated cells, D/C cells could be expanded on coated Matrigel for more than 12 passages, yielding spindle cells expressing angiogenesis and mesenchymal stem cells markers, and possessing significantly higher colony-forming units-fibroblast and more efficient tri-lineage differentiation than D/C and limbal residual stromal cells expanded on plastic in DMEM with 10% FBS, of which both lost the pericyte phenotype while limbal residual stromal cells turned into myofibroblasts. Upon reunion with limbal epithelial stem/progenitor cells to form spheres, D/C cells expanded on coated Matrigel maintained higher expression of p63α and lower expression of cytokeratin 12 than those expanded on plastic in DMEM with 10% FBS, while spheres formed with human corneal fibroblasts expressed cytokeratin 12 without p63α. Conclusions. In the limbal stroma, cells subjacent to limbal basal epithelial cells serve as niche cells, and generate progenitors with

CT features, mimics and atypical presentations of gastrointestinal stromal tumor (GIST)

PubMed Central

Sripathi, Smiti; Rajagopal, KV; Srivastava, Rajendra Kumar; Ayachit, Anurag

2011-01-01

The term stromal tumor was coined in 1983 by Clark and Mazur for smooth muscle neoplasm of the gastrointestinal tract (GIT). Gastrointestinal stromal tumors (GIST) are nonepithelial tumors arising from the interstitial cells of Cajal, which express KIT protein-CD117 on immunohistochemistry. GIST can arise anywhere in the GIT, including the mesentery, omentum, and retroperitoneum. PMID:22013291

CT features, mimics and atypical presentations of gastrointestinal stromal tumor (GIST).

PubMed

Sripathi, Smiti; Rajagopal, Kv; Srivastava, Rajendra Kumar; Ayachit, Anurag

2011-07-01

The term stromal tumor was coined in 1983 by Clark and Mazur for smooth muscle neoplasm of the gastrointestinal tract (GIT). Gastrointestinal stromal tumors (GIST) are nonepithelial tumors arising from the interstitial cells of Cajal, which express KIT protein-CD117 on immunohistochemistry. GIST can arise anywhere in the GIT, including the mesentery, omentum, and retroperitoneum.

The influence of genistein on free radicals in normal dermal fibroblasts and keloid fibroblasts examined by EPR spectroscopy.

PubMed

Jurzak, Magdalena; Ramos, Paweł; Pilawa, Barbara

2017-01-01

Normal and keloid fibroblasts were examined using X-band (9.3 GHz) electron paramagnetic resonance spectroscopy. The effect of genistein on the concentration of free radicals in both normal dermal and keloid fibroblasts after ultraviolet irradiation was investigated. The highest concentration of free radicals was seen in keloid fibroblasts, with normal fibroblasts containing a lower concentration. The concentration of free radicals in both normal and keloid fibroblasts was altered in a concentration-dependent manner by the presence of genistein. The change in intra-cellular free radical concentration after the ultraviolet irradiation of both normal and keloid fibroblasts is also discussed. The antioxidant properties of genistein, using its 1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging activity as a model, were tested, and the effect of ultraviolet irradiation on its interaction with free radicals was examined. The electron paramagnetic resonance spectra of DPPH showed quenching by genistein. The interaction of genistein with DPPH free radicals in the absence of ultraviolet irradiation was shown to be slow, but this interaction was much faster under ultraviolet irradiation. Ultraviolet irradiation enhanced the free radical-scavenging activity of genistein.

Mesenchymal stromal cells support the viability and differentiation of thymocytes through direct contact in autologous co-cultures.

PubMed

Azghadi, Seyed Mohammad Reza; Suciu, Maria; Gruia, Alexandra Teodora; Barbu-Tudoran, Lucian; Cristea, Mirabela Iustina; Mic, Ani Aurora; Muntean, Danina; Nica, Dragos Vasile; Mic, Felix Aurel

2016-08-01

The development of thymocytes and generation of mature T cells is a complex process that requires spatio-temporal interactions of thymocytes with the other cells of the thymus microenvironment. Recently, mesenchymal stromal cells were isolated from the neonatal human thymus and differentiated into chondrogenic, osteogenic, and adipogenic lineages, just like their bone marrow counterparts. However, their function in thymocyte homeostasis is unknown. In our autologous co-cultures of rat mesenchymal stromal cells and thymocytes, the stromal cells preserve the viability of cultured thymocytes and stimulate the development of CD4-CD8- double-negative and the maturation of mainly CD4+ single-positive thymocytes. Thymocytes also influence the stemness of bone marrow mesenchymal stromal cells, as their expression of CD44, a marker associated with cellular proliferation and migration, is reduced in co-cultures. Mesenchymal stromal cells' influence on thymocyte development requires direct physical contact between the two cells and is not mediated by a soluble factor. When the two types of cells were physically separated, the stimulative effects of mesenchymal stromal cells on thymocytes did not occur. Electron microscopy confirmed the close contact between the membranes of thymocytes and mesenchymal stromal cells. Our experiments suggest that membrane exchanges could occur between mesenchymal stromal cells and thymocytes, such as the transfer of CD44 from mesenchymal stromal cells to the thymocytes, but its functional significance for thymocytes development remains to be established. These results suggest that mesenchymal stromal cells could normally be a part of the in vivo thymic microenvironment and form a niche that could sustain and guide the development of thymocytes.

Gap junction blockade induces apoptosis in human endometrial stromal cells.

PubMed

Yu, Jie; Berga, Sarah L; Zou, Wei; Sun, He-Ying; Johnston-MacAnanny, Erika; Yalcinkaya, Tamer; Sidell, Neil; Bagchi, Indrani C; Bagchi, Milan K; Taylor, Robert N

2014-07-01

One of the most dynamic adult human tissues is the endometrium. Through coordinated, cyclical proliferation, differentiation, leukocyte recruitment, apoptosis, and desquamation, the uterine lining is expanded and shed monthly, unless pregnancy is established. Errors in these steps potentially cause endometrial dysfunction, abnormal uterine bleeding, failed embryonic implantation, infertility, or endometrial carcinoma. Our prior studies showed that gap junctions comprised of Gap junction alpha-1 (GJA1) protein, also known as connexin 43 (CX43), subunits are critical to endometrial stromal cell differentiation. The current studies were undertaken to explore the mechanism of endometrial dysfunction when gap junction intercellular communication (GJIC) is disrupted. Gap junction blockade by two distinct GJIC inhibitors, 18α-glycyrrhetinic acid (AGA) and octanol (OcOH), suppressed proliferation and induced apoptosis in endometrial stromal cells, as manifested by reduced biomarkers of cell viability, increased TUNEL staining, caspase-3 activation, sub-G1 chromosomal DNA complement, as well as shortened telomere length. Unexpectedly, we also observed that the chemical inhibitors blocked CX43 gene expression. Moreover, when endometrial stromal cells were induced to undergo hormonal decidualization, following a 7-day exposure to 10 nM 17β-estradiol + 100 nM progesterone + 0.5 mM dibutyryl cAMP, characteristic epithelioid changes in cell shape and secretion of prolactin were blunted in the presence of AGA or OcOH, recapitulating effects of RNA interference of CX43. Our findings indicate that endometrial stromal cell proliferation and maintenance of decidualized endometrial function are GJIC-dependent, and that disruption of gap junctions induces endometrial stromal cell apoptosis. These observations may have important implications for several common clinical endometrial pathologies. © 2014 Wiley Periodicals, Inc.

Fibrogenic Lung Injury Induces Non-Cell-Autonomous Fibroblast Invasion.

PubMed

Ahluwalia, Neil; Grasberger, Paula E; Mugo, Brian M; Feghali-Bostwick, Carol; Pardo, Annie; Selman, Moisés; Lagares, David; Tager, Andrew M

2016-06-01

Pathologic accumulation of fibroblasts in pulmonary fibrosis appears to depend on their invasion through basement membranes and extracellular matrices. Fibroblasts from the fibrotic lungs of patients with idiopathic pulmonary fibrosis (IPF) have been demonstrated to acquire a phenotype characterized by increased cell-autonomous invasion. Here, we investigated whether fibroblast invasion is further stimulated by soluble mediators induced by lung injury. We found that bronchoalveolar lavage fluids from bleomycin-challenged mice or patients with IPF contain mediators that dramatically increase the matrix invasion of primary lung fibroblasts. Further characterization of this non-cell-autonomous fibroblast invasion suggested that the mediators driving this process are produced locally after lung injury and are preferentially produced by fibrogenic (e.g., bleomycin-induced) rather than nonfibrogenic (e.g., LPS-induced) lung injury. Comparison of invasion and migration induced by a series of fibroblast-active mediators indicated that these two forms of fibroblast movement are directed by distinct sets of stimuli. Finally, knockdown of multiple different membrane receptors, including platelet-derived growth factor receptor-β, lysophosphatidic acid 1, epidermal growth factor receptor, and fibroblast growth factor receptor 2, mitigated the non-cell-autonomous fibroblast invasion induced by bronchoalveolar lavage from bleomycin-injured mice, suggesting that multiple different mediators drive fibroblast invasion in pulmonary fibrosis. The magnitude of this mediator-driven fibroblast invasion suggests that its inhibition could be a novel therapeutic strategy for pulmonary fibrosis. Further elaboration of the molecular mechanisms that drive non-cell-autonomous fibroblast invasion consequently may provide a rich set of novel drug targets for the treatment of IPF and other fibrotic lung diseases.

Decidualized Human Endometrial Stromal Cells Mediate Hemostasis, Angiogenesis, and Abnormal Uterine Bleeding

PubMed Central

Lockwood, Charles J.; Krikun, Graciela; Hickey, Martha; Huang, S. Joseph; Schatz, Frederick

2011-01-01

Factor VII binds trans-membrane tissue factor to initiate hemostasis by forming thrombin. Tissue factor expression is enhanced in decidualized human endometrial stromal cells during the luteal phase. Long-term progestin only contraceptives elicit: 1) abnormal uterine bleeding from fragile vessels at focal bleeding sites, 2) paradoxically high tissue factor expression at bleeding sites; 3) reduced endometrial blood flow promoting local hypoxia and enhancing reactive oxygen species levels; and 4) aberrant angiogenesis reflecting increased stromal cell-expressed vascular endothelial growth factor, decreased Angiopoietin-1 and increased endothelial cell-expressed Angiopoietin-2. Aberrantly high local vascular permeability enhances circulating factor VII to decidualized stromal cell-expressed tissue factor to generate excess thrombin. Hypoxia-thrombin interactions augment expression of vascular endothelial growth factor and interleukin-8 by stromal cells. Thrombin, vascular endothelial growth factor and interlerukin-8 synergis-tically augment angiogenesis in a milieu of reactive oxygen species-induced endothelial cell activation. The resulting enhanced vessel fragility promotes abnormal uterine bleeding. PMID:19208784

Multiple gastrointestinal stromal tumors in type I neurofibromatosis: a pathologic and molecular study.

PubMed

Yantiss, Rhonda K; Rosenberg, Andrew E; Sarran, Lisa; Besmer, Peter; Antonescu, Cristina R

2005-04-01

Multiple gastrointestinal stromal tumors typically occur in familial form associated with KIT receptor tyrosine kinase or platelet-derived growth factor receptor-alpha (PDGFRA) germline mutations, but may also develop in the setting of type 1 neurofibromatosis. The molecular abnormalities of gastrointestinal stromal tumors arising in neurofibromatosis have not been extensively studied. We identified three patients with type 1 neuro-fibromatosis and multiple small intestinal stromal tumors. Immunostains for CD117, CD34, desmin, actins, S-100 protein, and keratins were performed on all of the tumors. DNA was extracted from representative paraffin blocks from separate tumor nodules in each case and subjected to a nested polymerase chain reaction, using primers for KIT exons 9, 11, 13, and 17 and PDGFRA exons 12 and 18, followed by direct sequencing. The mean patient age was 56 years (range: 37-86 years, male/female ratio: 2/1). One patient had three tumors, one had five, and one had greater than 10 tumor nodules, all of which demonstrated histologic features characteristic of gastrointestinal stromal tumors and stained strongly for CD117 and CD34. One patient died of disease at 35 months, one was disease free at 12 months and one was lost to follow-up. DNA extracts from 10 gastrointestinal stromal tumors (three from each of two patients and four from one patient) were subjected to polymerase chain reactions and assessed for mutations. All of the tumors were wild type for KIT exons 9, 13, and 17 and PDGFRA exons 12 and 18. Three tumors from one patient had identical point mutations in KIT exon 11, whereas the other tumors were wild type at this locus. We conclude that, although most patients with type 1 neurofibromatosis and gastrointestinal stromal tumors do not have KIT or PDGFRA mutations, KIT germline mutations might be implicated in the pathogenesis of gastrointestinal stromal tumors in some patients.

Human serum reduces mitomycin-C cytotoxicity in human tenon's fibroblasts.

PubMed

Crowston, Jonathan G; Wang, Xiao Y; Khaw, Peng T; Zoellner, Hans; Healey, Paul R

2006-03-01

To determine the effect of human serum factors on mitomycin-C (MMC) cytotoxicity in cultured human subconjunctival Tenon's capsule fibroblasts. Fibroblast monolayers were treated with 5-minute applications of mitomycin-C (0.4 mg/mL) and incubated in culture medium with or without additional human serum. Fibroblast apoptosis was quantified by direct cell counts based on nuclear morphology, flow cytometry with annexin-V/propidium iodide, and a lactate dehydrogenase release assay. The number of viable fibroblasts and fibroblast proliferation were measured with a colorimetric MTT assay and by bromodeoxyuridine (BrdU) labeling. Mitomycin-C induced significant levels of fibroblast apoptosis. The addition of human serum resulted in a 40% reduction in MMC-induced fibroblast apoptosis (range, 31.3%-55.3%; P = 0.021) as determined by nuclear morphology and a 32.4% reduction measured by annexin-V/PI. There was a corresponding dose-dependent increase in the number of viable fibroblasts. Serum did not restore proliferation in MMC-treated fibroblasts. Factors present in human serum reduce MMC cytotoxicity in cultured human Tenon's fibroblasts. Human serum increased the number of viable fibroblasts by inhibiting MMC-induced fibroblast apoptosis. Serum factors access aqueous humor after trabeculectomy and may therefore influence the clinical outcome of MMC treatment.

Immortalized Cancer-associated Fibroblasts Promote Prostate Cancer Carcinogenesis, Proliferation and Invasion.

PubMed

Yu, Shengqiang; Jiang, Yingjuan; Wan, Fengchun; Wu, Jitao; Gao, Zhenli; Liu, Dongfu

2017-08-01

Cancer-associated fibroblasts (CAFs) are dominant components of the prostate cancer (PCa) stroma. However, the contrasting effects of CAFs and adjacent normal prostate fibroblasts (NPFs) are still poorly defined. The senescence of non-immortalized CAFs after subculture may limit the cell number and influence experimental results of in vitro studies. In this study, we immortalized CAFs to study their role in PCa carcinogenesis, proliferation, and invasion. We cultured and immortalized CAFs and NPFs, then compared their effect on epithelial malignant transformation by using in vitro co-culture, soft agar assay, and a mouse renal capsule xenograft model. We also compared their roles in PCa progression by using in vitro co-culture, cell viability assays, invasion assays, and a mouse xenograft model. For the mechanistic study, we screened a series of growth factors by using real-time polymerase chain reaction. The CAFs and NPFs were successfully cultured, immortalized, and characterized. The CAFs were able to transform prostate epithelial cells into malignant cells, but NPFs were not. The CAFs were more active in promoting proliferation of and invasion by PCa cells, and in secreting higher levels of a series of growth factors. The immortalized CAFs were more supportive of PCa carcinogenesis and progression. Targeting CAFs might be a potential option for PCa therapy. Immortalized CAFs and NPFs will also be valuable resources for future experimental exploration. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Activation of NK Cells in Mixed Cultures of Wharton's Jelly Mesenchymal Stromal Cells and Peripheral Blood Lymphocytes.

PubMed

Svirshchevskaya, E V; Poltavtsev, A M; Os'mak, G Zh; Poltavtseva, R A

2018-01-01

Mesenchymal stromal cells possess immunosuppressive properties that might be used for the therapy of inflammatory diseases of various geneses. The effects of mesenchymal stromal cells depend on their lifetime in the recipient tissues. During heterologous transplantation, mesenchymal stromal cells are eliminated by NK cells. We studied NK cell formation in mixed cultures of Wharton's jelly mesenchymal stromal cells and peripheral blood lymphocytes from an autologous donor. Lymphocytes were activated by a mitogen or IL-2. The lifetime of mesenchymal stromal cells was estimated by MTT test. Cytotoxic activity and phenotype of NK cells were evaluated by flow cytometry. It was found that activation of NK cells depended on IL-2 and was registered on day 2 of incubation with IL-2. In cultures with mitogen-activated lymphocytes, cytotoxicity was observed after 5-6 days. Cytotoxicity of NK correlated with significant decrease in CD16+ and increase in CD56+ NK and with reduction of mesenchymal stromal cell viability. Thus, the main mechanism of elimination of mesenchymal stromal cells is cytotoxicity of NK cells that depended on IL-2 production.

Osteogenic Differentiation of Mesenchymal Stromal Cells: A Comparative Analysis Between Human Subcutaneous Adipose Tissue and Dental Pulp.

PubMed

D'Alimonte, Iolanda; Mastrangelo, Filiberto; Giuliani, Patricia; Pierdomenico, Laura; Marchisio, Marco; Zuccarini, Mariachiara; Di Iorio, Patrizia; Quaresima, Raimondo; Caciagli, Francesco; Ciccarelli, Renata

2017-06-01

White adipose tissue is a source of mesenchymal stromal/stem cells (MSCs) that are actively studied for their possible therapeutic use in bone tissue repair/remodeling. To better appreciate the osteogenic potential of these cells, we compared some properties of MSCs from human subcutaneous adipose tissue [subcutaneous-adipose stromal cells (S-ASCs)] and dental pulp stem cell (DPSCs) of third-impacted molars, the latter representing a well-established MSC source. Both undifferentiated cell types showed similar fibroblast-like morphology and mesenchymal marker expression. However, undifferentiated S-ASCs displayed a faster doubling time coupled to greater proliferation and colony-forming ability than DPSCs. Also, the osteogenic differentiation of S-ASCs was greater than that of DPSCs, as evaluated by the higher levels of expression of early osteogenic markers Runt-related transcription factor-2 (RUNX2) and alkaline phosphatase at days 3-14 and of extracellular matrix mineralization at days 14-21. Moreover, S-ASCs showed a better colonization of the titanium scaffold. In addition, we investigated whether S-ASC osteogenic commitment was enhanced by adenosine A1 receptor (A1R) stimulation, as previously shown for DPSCs. Although A1R expression was constant during DPSC differentiation, it increased in S-ASC at day 21 from osteogenesis induction. Accordingly, A1R stimulation by the agonist 2-chloro-N 6 -cyclopentyl-adenosine, added to the cultures at each medium change, stimulated proliferation only in differentiating DPSC and enhanced the osteogenic differentiation earlier in DPSCs than in S-ASCs. These effects were counteracted by cell pretreatment with a selective A1R antagonist. Thus, our findings suggest that S-ASCs could be advantageously used in regenerative orthopedics/dentistry, and locally released or exogenously added purines may play a role in bone repair/remodeling, even though this aspect should be more thoroughly evaluated.

Evolution of Cancer Stem-like Cells in Endocrine-Resistant Metastatic Breast Cancers Is Mediated by Stromal Microvesicles.

PubMed

Sansone, Pasquale; Berishaj, Marjan; Rajasekhar, Vinagolu K; Ceccarelli, Claudio; Chang, Qing; Strillacci, Antonio; Savini, Claudia; Shapiro, Lauren; Bowman, Robert L; Mastroleo, Chiara; De Carolis, Sabrina; Daly, Laura; Benito-Martin, Alberto; Perna, Fabiana; Fabbri, Nicola; Healey, John H; Spisni, Enzo; Cricca, Monica; Lyden, David; Bonafé, Massimiliano; Bromberg, Jacqueline

2017-04-15

The hypothesis that microvesicle-mediated miRNA transfer converts noncancer stem cells into cancer stem cells (CSC) leading to therapy resistance remains poorly investigated. Here we provide direct evidence supporting this hypothesis, by demonstrating how microvesicles derived from cancer-associated fibroblasts (CAF) transfer miR-221 to promote hormonal therapy resistance (HTR) in models of luminal breast cancer. We determined that CAF-derived microvesicles horizontally transferred miR-221 to tumor cells and, in combination with hormone therapy, activated an ER lo /Notch hi feed-forward loop responsible for the generation of CD133 hi CSCs. Importantly, microvesicles from patients with HTR metastatic disease expressed high levels of miR-221. We further determined that the IL6-pStat3 pathway promoted the biogenesis of onco-miR-221 hi CAF microvesicles and established stromal CSC niches in experimental and patient-derived breast cancer models. Coinjection of patient-derived CAFs from bone metastases led to de novo HTR tumors, which was reversed with IL6R blockade. Finally, we generated patient-derived xenograft (PDX) models from patient-derived HTR bone metastases and analyzed tumor cells, stroma, and microvesicles. Murine and human CAFs were enriched in HTR tumors expressing high levels of CD133 hi cells. Depletion of murine CAFs from PDX restored sensitivity to HT, with a concurrent reduction of CD133 hi CSCs. Conversely, in models of CD133 neg , HT-sensitive cancer cells, both murine and human CAFs promoted de novo HT resistance via the generation of CD133 hi CSCs that expressed low levels of estrogen receptor alpha. Overall, our results illuminate how microvesicle-mediated horizontal transfer of genetic material from host stromal cells to cancer cells triggers the evolution of therapy-resistant metastases, with potentially broad implications for their control. Cancer Res; 77(8); 1927-41. ©2017 AACR . ©2017 American Association for Cancer Research.

Breast tumors educate stromal tissue with individualized but coordinated proteomic signatures

PubMed Central

Wang, Xuya; Mooradian, Arshag D.; Erdmann-Gilmore, Petra; Zhang, Qiang; Viner, Rosa; Davies, Sherri R.; Huang, Kuan-lin; Bomgarden, Ryan; Van Tine, Brian A.; Shao, Jieya; Ding, Li; Li, Shunqiang; Ellis, Matthew J.; Rogers, John C.; Townsend, R. Reid; Fenyö, David; Held, Jason M.

2017-01-01

Cancer forms specialized microenvironmental niches that promote local invasion and colonization. Engrafted patient-derived xenografts (PDXs) locally invade and colonize naïve stroma, while enabling unambiguous molecular discrimination of human proteins in the tumor from mouse proteins in the microenvironment. To characterize how patient breast tumors form a niche and educate naïve stroma, subcutaneous breast cancer PDXs were globally profiled using species-specific quantitative proteomics. Regulation of PDX stromal proteins by breast tumors was extensive, with thirty-five percent of the stromal proteome consistently altered by tumors across different animals and passages. Differentially regulated proteins in the stroma clustered into six signatures that included both known and novel contributors to tumor invasion and colonization. Stromal proteomes were coordinately regulated, though the sets of proteins altered by each tumor were highly distinct. Integrated analysis of tumor and stromal proteins, a comparison possible in xenograft models, indicated that the known hallmarks of cancer contribute pleiotropically to establishing and maintaining the tumor’s microenvironmental niche. Tumor education of the stroma is therefore an intrinsic property of breast tumors that is highly individualized, yet proceeds by consistent, non-random and defined tumor-promoting molecular alterations. PMID:28790197

Changing the Properties of Multipotent Mesenchymal Stromal Cells by IFNγ Administration.

PubMed

Petinati, N A; Kapranov, N M; Bigil'deev, A E; Popova, M D; Davydova, Yu O; Gal'tseva, I V; Drize, N I; Kuz'mina, L A; Parovichnikova, E N; Savchenko, V G

2017-06-01

We studied changes in the population of human multipotent mesenchymal stromal cells activated by IFNγ. The cells were cultured under standard conditions; IFNγ was added in various concentrations for 4 h or over 2 passages. It was shown that the total cell production significantly decreased after long-term culturing with IFNγ, but 4-h exposure did not affect this parameter. After 4-h culturing, the expression levels of IDO1, CSF1, and IL-6 increased by 300, 7, and 2.4 times, respectively, and this increase persisted 1 and 2 days after removal of IFNγ from the culture medium. The expression of class I and II MHC (HLA) on cell surface practically did not change immediately after exposure to IFNγ, but during further culturing, HLA-ABC (MHC I) and HLA-DR (MHC II) expression significantly increased, which abolished the immune privilege in these cells, the property allowing clinical use of allogenic multipotent mesenchymal stromal cells. Multipotent mesenchymal stromal cells can suppress proliferation of lymphocytes. The degree of this suppression depends on individual properties of multipotent mesenchymal stromal cell donor. Treatment with IFNγ did not significantly affect the intensity of inhibition of lymphocyte proliferation by these cells.

Legumain Regulates Differentiation Fate of Human Bone Marrow Stromal Cells and Is Altered in Postmenopausal Osteoporosis.

PubMed

Jafari, Abbas; Qanie, Diyako; Andersen, Thomas L; Zhang, Yuxi; Chen, Li; Postert, Benno; Parsons, Stuart; Ditzel, Nicholas; Khosla, Sundeep; Johansen, Harald Thidemann; Kjærsgaard-Andersen, Per; Delaisse, Jean-Marie; Abdallah, Basem M; Hesselson, Daniel; Solberg, Rigmor; Kassem, Moustapha

2017-02-14

Secreted factors are a key component of stem cell niche and their dysregulation compromises stem cell function. Legumain is a secreted cysteine protease involved in diverse biological processes. Here, we demonstrate that legumain regulates lineage commitment of human bone marrow stromal cells and that its expression level and cellular localization are altered in postmenopausal osteoporotic patients. As shown by genetic and pharmacological manipulation, legumain inhibited osteoblast (OB) differentiation and in vivo bone formation through degradation of the bone matrix protein fibronectin. In addition, genetic ablation or pharmacological inhibition of legumain activity led to precocious OB differentiation and increased vertebral mineralization in zebrafish. Finally, we show that localized increased expression of legumain in bone marrow adipocytes was inversely correlated with adjacent trabecular bone mass in a cohort of patients with postmenopausal osteoporosis. Our data suggest that altered proteolytic activity of legumain in the bone microenvironment contributes to decreased bone mass in postmenopausal osteoporosis. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Inflammatory response of a prostate stromal cell line induced by Trichomonas vaginalis.

PubMed

Im, S J; Han, I H; Kim, J H; Gu, N Y; Seo, M Y; Chung, Y H; Ryu, J S

2016-04-01

While Trichomonas vaginalis, a cause of sexually transmitted infection, is known as a surface-dwelling protozoa, trichomonads have been detected in prostatic tissue from benign prostatic hyperplasia and prostatitis by immunoperoxidase assay or PCR. However, the immune response of prostate stromal cells infected with T. vaginalis has not been investigated. Our objective was to investigate whether T. vaginalis could induce an inflammatory response in prostate stromal cells. Incubation of a human prostate stromal myofibroblast cells (WPMY-1) with live T. vaginalis T016 increased expression of the inflammatory chemokines CXCL8 and CCL2. In addition, TLR4, ROS, MAPK and NF-κB expression increased, while inhibitors of TLR4, ROS, MAPKs and NF-κB reduced CXCL8 and CCL2 production. Medium conditioned by incubation of WPMY-1 cells with T. vaginalis stimulated the migration of human neutrophils and monocytes (THP-1 cells). We conclude that T. vaginalis increases CXCL8 and CCL2 production by human prostate stromal cells by activating TLR4, ROS, MAPKs and NF-κB, and this in turn attracts neutrophils and monocytes and leads to an inflammatory response. This study is the first attempt to demonstrate an inflammatory reaction in prostate stromal cells caused by T. vaginalis. © 2016 John Wiley & Sons Ltd.

Novel culture system of mesenchymal stromal cells from human subcutaneous adipose tissue.

PubMed

Iwashima, Shigejiro; Ozaki, Takenori; Maruyama, Shoichi; Saka, Yousuke; Kobori, Masato; Omae, Kaoru; Yamaguchi, Hirotake; Niimi, Tomoaki; Toriyama, Kazuhiro; Kamei, Yuzuru; Torii, Shuhei; Murohara, Toyoaki; Yuzawa, Yukio; Kitagawa, Yasuo; Matsuo, Seiichi

2009-05-01

Accumulating evidence suggests that the delivery of human adipose tissue-derived stromal cells (hASCs) has great potential as regenerative therapy. This was performed to develop a method for expanding hASCs by reducing the amount of serum required. We demonstrate that hASCs were able to expand efficiently in media containing 2% serum and fibroblast growth factor-2. These cells, or low serum cultured hASCs (hLASCs), expressed cell surface markers similar to those on bone marrow-derived mesenchymal stem cells, and could be differentiated into cells of mesenchymal lineage. Of interest, hLASCs secreted higher levels of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) than hASCs cultured in 20% serum (hHASCs). Moreover, hLASC-conditioned media significantly increased endothelial cell (EC) proliferation and decreased EC apoptosis compared to that obtained from hHASCs or control media only. Antibodies against VEGF and HGF virtually negated these effects. When hASCs were administered into the ischemic hindlimbs of nude rats, hLASCs improved blood flow, increased capillary density, and raised the levels of VEGF and HGF in the muscles as compared with hHASCs. In conclusion, we demonstrate a novel low serum culture system for hASCs, which may have great potential in regenerative cell therapy for damaged organs in the clinical setting.

A New Paradigm for Ovarian Sex Cord-Stromal Tumor Development

DTIC Science & Technology

2016-05-01

1 AWARD NUMBER: W81XWH-15-1-0082 TITLE: A New Paradigm for Ovarian Sex Cord-Stromal Tumor Development PRINCIPAL INVESTIGATOR: Qinglei Li...5a. CONTRACT NUMBER A New Paradigm for Ovarian Sex Cord-Stromal Tumor Development 5b. GRANT NUMBER W81XWH-15-1-0082 5c. PROGRAM ELEMENT NUMBER 6...role of sustained activation of oocyte TGFBR1 in ovarian tumor development using an in vitro approach. 2. Keywords Ovarian tumor, Sex cord

Matrix Stiffness Corresponding to Strictured Bowel Induces a Fibrogenic Response in Human Colonic Fibroblasts

PubMed Central

Johnson, Laura A.; Rodansky, Eva S.; Sauder, Kay L.; Horowitz, Jeffrey C.; Mih, Justin D.; Tschumperlin, Daniel J.; Higgins, Peter D.

2013-01-01

Background Crohn’s disease is characterized by repeated cycles of inflammation and mucosal healing which ultimately progress to intestinal fibrosis. This inexorable progression towards fibrosis suggests that fibrosis becomes inflammation-independent and auto-propagative. We hypothesized that matrix stiffness regulates this auto-propagation of intestinal fibrosis. Methods The stiffness of fresh ex vivo samples from normal human small intestine, Crohn’s disease strictures, and the unaffected margin were measured with a microelastometer. Normal human colonic fibroblasts were cultured on physiologically normal or pathologically stiff matrices corresponding to the physiological stiffness of normal or fibrotic bowel. Cellular response was assayed for changes in cell morphology, α-smooth muscle actin (αSMA) staining, and gene expression. Results Microelastometer measurements revealed a significant increase in colonic tissue stiffness between normal human colon and Crohn’s strictures as well as between the stricture and adjacent tissue margin. In Ccd-18co cells grown on stiff matrices corresponding to Crohn’s strictures, cellular proliferation increased. Pathologic stiffness induced a marked change in cell morphology and increased αSMA protein expression. Growth on a stiff matrix induced fibrogenic gene expression, decreased matrix metalloproteinase and pro-inflammatory gene expression, and was associated with nuclear localization of the transcriptional cofactor MRTF-A. Conclusions Matrix stiffness, representative of the pathological stiffness of Crohn’s strictures, activates human colonic fibroblasts to a fibrogenic phenotype. Matrix stiffness affects multiple pathways suggesting the mechanical properties of the cellular environment are critical to fibroblast function and may contribute to autopropagation of intestinal fibrosis in the absence of inflammation, thereby contributing to the intractable intestinal fibrosis characteristic of Crohn’s disease. PMID

Identification of Keratinocyte Growth Factor as a Target of microRNA-155 in Lung Fibroblasts: Implication in Epithelial-Mesenchymal Interactions

PubMed Central

Chevalier, Benoit; Puisségur, Marie-Pierre; Lebrigand, Kevin; Robbe-Sermesant, Karine; Bertero, Thomas; Lino Cardenas, Christian L.; Courcot, Elisabeth; Rios, Géraldine; Fourre, Sandra; Lo-Guidice, Jean-Marc; Marcet, Brice; Cardinaud, Bruno; Barbry, Pascal; Mari, Bernard

2009-01-01

Background Epithelial-mesenchymal interactions are critical in regulating many aspects of vertebrate embryo development, and for the maintenance of homeostatic equilibrium in adult tissues. The interactions between epithelium and mesenchyme are believed to be mediated by paracrine signals such as cytokines and extracellular matrix components secreted from fibroblasts that affect adjacent epithelia. In this study, we sought to identify the repertoire of microRNAs (miRNAs) in normal lung human fibroblasts and their potential regulation by the cytokines TNF-α, IL-1β and TGF-β. Methodology/Principal Findings MiR-155 was significantly induced by inflammatory cytokines TNF-α and IL-1β while it was down-regulated by TGF-β. Ectopic expression of miR-155 in human fibroblasts induced modulation of a large set of genes related to “cell to cell signalling”, “cell morphology” and “cellular movement”. This was consistent with an induction of caspase-3 activity and with an increase in cell migration in fibroblasts tranfected with miR-155. Using different miRNA bioinformatic target prediction tools, we found a specific enrichment for miR-155 predicted targets among the population of down-regulated transcripts. Among fibroblast-selective targets, one interesting hit was keratinocyte growth factor (KGF, FGF-7), a member of the fibroblast growth factor (FGF) family, which owns two potential binding sites for miR-155 in its 3′-UTR. Luciferase assays experimentally validated that miR-155 can efficiently target KGF 3′-UTR. Site-directed mutagenesis revealed that only one out of the 2 potential sites was truly functional. Functional in vitro assays experimentally validated that miR-155 can efficiently target KGF 3′-UTR. Furthermore, in vivo experiments using a mouse model of lung fibrosis showed that miR-155 expression level was correlated with the degree of lung fibrosis. Conclusions/Significance Our results strongly suggest a physiological function of miR-155 in

Bevacizumab in Treating Patients With Recurrent Sex Cord-Stromal Tumors of the Ovary

ClinicalTrials.gov

2018-03-20

Malignant Ovarian Epithelial Tumor; Ovarian Granulosa Cell Tumor; Ovarian Gynandroblastoma; Ovarian Sertoli-Leydig Cell Tumor; Ovarian Sex Cord Tumor With Annular Tubules; Ovarian Sex Cord-Stromal Tumor; Ovarian Sex Cord-Stromal Tumor of Mixed or Unclassified Cell Types; Ovarian Steroid Cell Tumor

Size cues and the adjacency principle.

DOT National Transportation Integrated Search

1963-11-01

The purpose of the present study was to apply the adjacency principle to the perception of relative depth from size cues. In agreement with the adjacency principle, it was found that the size cue between adjacent objects was more effective than the s...

Prevalence and histopathological characteristics of corneal stromal dystrophies in Saudi Arabia.

PubMed

Alzuhairy, Sultan; Alkatan, Hind M; Al-Rajhi, Ali A

2015-01-01

The aim was to determine the frequency and describe the main histopathologic features of corneal stromal dystrophy in Saudi Arabia. A single-center, retrospective analysis of 193 corneal specimens diagnosed with stromal dystrophy. All samples were retrieved from the Histopathology Department at King Khaled Eye Specialist Hospital over a 10-year period (2002 to December 31, 2011). Cases of stromal dystrophy undergoing keratoplasty were included in the study. Routine histopathologic stains and specific stains were used to determine a diagnosis. The corresponding demographic data and basic clinical/surgical information were collected via chart review. The study sample was comprised of 193 eyes. The final diagnoses were macular corneal dystrophy (MCD) in 180 (93.26%) eyes, granular corneal dystrophy (GCD) in 9 (4.66%) and lattice corneal dystrophy (LCD) in 4 (2.07%) eyes. The mean age at presentation was 27.03 years for MCD, 26.33 years for GCD and 53.75 years for LCD. The interval between diagnosis and surgical intervention was not statistically different between the macular and granular groups (P = 0.141). There was a positive family history for the MCD (37.22%) and GCD (44.44%) groups. All eyes underwent penetrating keratoplasty (PKP) except 10 MCD cases that underwent lamellar keratoplasty. Diffuse stromal deposits were present in 87.2% of MCD corneas and 66.67% of GCD corneas. Seventeen eyes with MCD were misdiagnosed as GCD. None of the LCD cases were clinically identified since all of these cases were diagnosed as corneal scarring. In eyes with MCD that underwent PKP, there was diffuse stromal involvement (in 87.22% eyes) and changes in Descemet's membrane (in 53.5% eyes). This pathological study suggested that MCD was the most common corneal stromal dystrophy that required keratoplasty in Saudi Arabia. Patient with MCD and GCD presented at a significantly younger age than LCD. The clinical diagnosis of MCD is not achieved in all cases likely due to a more

Prevalence and Histopathological Characteristics of Corneal Stromal Dystrophies in Saudi Arabia

PubMed Central

Alzuhairy, Sultan; Alkatan, Hind M.; Al-Rajhi, Ali A.

2015-01-01

Purpose: The aim was to determine the frequency and describe the main histopathologic features of corneal stromal dystrophy in Saudi Arabia. Methods: A single-center, retrospective analysis of 193 corneal specimens diagnosed with stromal dystrophy. All samples were retrieved from the Histopathology Department at King Khaled Eye Specialist Hospital over a 10-year period (2002 to December 31, 2011). Cases of stromal dystrophy undergoing keratoplasty were included in the study. Routine histopathologic stains and specific stains were used to determine a diagnosis. The corresponding demographic data and basic clinical/surgical information were collected via chart review. Results: The study sample was comprised of 193 eyes. The final diagnoses were macular corneal dystrophy (MCD) in 180 (93.26%) eyes, granular corneal dystrophy (GCD) in 9 (4.66%) and lattice corneal dystrophy (LCD) in 4 (2.07%) eyes. The mean age at presentation was 27.03 years for MCD, 26.33 years for GCD and 53.75 years for LCD. The interval between diagnosis and surgical intervention was not statistically different between the macular and granular groups (P = 0.141). There was a positive family history for the MCD (37.22%) and GCD (44.44%) groups. All eyes underwent penetrating keratoplasty (PKP) except 10 MCD cases that underwent lamellar keratoplasty. Diffuse stromal deposits were present in 87.2% of MCD corneas and 66.67% of GCD corneas. Seventeen eyes with MCD were misdiagnosed as GCD. None of the LCD cases were clinically identified since all of these cases were diagnosed as corneal scarring. In eyes with MCD that underwent PKP, there was diffuse stromal involvement (in 87.22% eyes) and changes in Descemet's membrane (in 53.5% eyes). Conclusion: This pathological study suggested that MCD was the most common corneal stromal dystrophy that required keratoplasty in Saudi Arabia. Patient with MCD and GCD presented at a significantly younger age than LCD. The clinical diagnosis of MCD is not achieved

TGFβ1 alters androgenic metabolites and hydroxysteroid dehydrogenase enzyme expression in human prostate reactive stromal primary cells: Is steroid metabolism altered by prostate reactive stromal microenvironment?

PubMed Central

Piao, Yun-shang; Wiesenfeld, Paddy; Sprando, Robert; Arnold, Julia T.

2013-01-01

The inflammatory tissue microenvironment can be an active promoter in preneoplastic cancer lesions. Altered steroid hormone metabolism as induced by the inflammatory microenvironment may contribute to epithelial cancer progression. Dehydroepiandrosterone sulfate (DHEAS) is the most abundant endogenous steroid hormone present in human serum and can be metabolized to DHEA, androgens and/or estrogens in peripheral tissues. We have previously reported that TGFβ1-induced reactive prostate stromal cells increase DHEA metabolism to active androgens and alter prostate cancer cell gene expression. While much of the focus on mechanisms of prostate cancer and steroid metabolism is in the epithelial cancer cells, this study focuses on TGFβ1-induced effects on DHEA metabolic pathways and enzymes in human prostate stromal cells. In DHEA-treated primary prostate stromal cells, TGFβ1 produced time- and dose-dependent increases in metabolism of DHEA to androstenedione and testosterone. Also TGFβ1-treated prostate stromal cells exhibited changes in the gene expression of enzymes involved in steroid metabolism including up-regulation of 3β hydroxysteroid dehydrogenase (HSD), and down-regulation of 17βHSD5, and 17βHSD2. These studies suggest that reactive prostate stroma and the inflammatory microenvironment may contribute to altered steroid metabolism and increased intratumoral androgens. PMID:23770322

TGFβ1 alters androgenic metabolites and hydroxysteroid dehydrogenase enzyme expression in human prostate reactive stromal primary cells: Is steroid metabolism altered by prostate reactive stromal microenvironment?

PubMed

Piao, Yun-shang; Wiesenfeld, Paddy; Sprando, Robert; Arnold, Julia T

2013-11-01

The inflammatory tissue microenvironment can be an active promoter in preneoplastic cancer lesions. Altered steroid hormone metabolism as induced by the inflammatory microenvironment may contribute to epithelial cancer progression. Dehydroepiandrosterone sulfate (DHEAS) is the most abundant endogenous steroid hormone present in human serum and can be metabolized to DHEA, androgens and/or estrogens in peripheral tissues. We have previously reported that TGFβ1-induced reactive prostate stromal cells increase DHEA metabolism to active androgens and alter prostate cancer cell gene expression. While much of the focus on mechanisms of prostate cancer and steroid metabolism is in the epithelial cancer cells, this study focuses on TGFβ1-induced effects on DHEA metabolic pathways and enzymes in human prostate stromal cells. In DHEA-treated primary prostate stromal cells, TGFβ1 produced time- and dose-dependent increases in metabolism of DHEA to androstenedione and testosterone. Also TGFβ1-treated prostate stromal cells exhibited changes in the gene expression of enzymes involved in steroid metabolism including up-regulation of 3β hydroxysteroid dehydrogenase (HSD), and down-regulation of 17βHSD5, and 17βHSD2. These studies suggest that reactive prostate stroma and the inflammatory microenvironment may contribute to altered steroid metabolism and increased intratumoral androgens. Published by Elsevier Ltd.

HOX and TALE signatures specify human stromal stem cell populations from different sources.

PubMed

Picchi, Jacopo; Trombi, Luisa; Spugnesi, Laura; Barachini, Serena; Maroni, Giorgia; Brodano, Giovanni Barbanti; Boriani, Stefano; Valtieri, Mauro; Petrini, Mario; Magli, Maria Cristina

2013-04-01

Human stromal stem cell populations reside in different tissues and anatomical sites, however a critical question related to their efficient use in regenerative medicine is whether they exhibit equivalent biological properties. Here, we compared cellular and molecular characteristics of stromal stem cells derived from the bone marrow, at different body sites (iliac crest, sternum, and vertebrae) and other tissues (dental pulp and colon). In particular, we investigated whether homeobox genes of the HOX and TALE subfamilies might provide suitable markers to identify distinct stromal cell populations, as HOX proteins control cell positional identity and, together with their co-factors TALE, are involved in orchestrating differentiation of adult tissues. Our results show that stromal populations from different sources, although immunophenotypically similar, display distinct HOX and TALE signatures, as well as different growth and differentiation abilities. Stromal stem cells from different tissues are characterized by specific HOX profiles, differing in the number and type of active genes, as well as in their level of expression. Conversely, bone marrow-derived cell populations can be essentially distinguished for the expression levels of specific HOX members, strongly suggesting that quantitative differences in HOX activity may be crucial. Taken together, our data indicate that the HOX and TALE profiles provide positional, embryological and hierarchical identity of human stromal stem cells. Furthermore, our data suggest that cell populations derived from different body sites may not represent equivalent cell sources for cell-based therapeutical strategies for regeneration and repair of specific tissues. Copyright © 2012 Wiley Periodicals, Inc.

Transforming growth factor-beta regulates mammary carcinoma cell survival and interaction with the adjacent microenvironment.

PubMed

Bierie, Brian; Stover, Daniel G; Abel, Ty W; Chytil, Anna; Gorska, Agnieszka E; Aakre, Mary; Forrester, Elizabeth; Yang, Li; Wagner, Kay-Uwe; Moses, Harold L

2008-03-15

Transforming growth factor (TGF)-beta signaling has been associated with early tumor suppression and late tumor progression; however, many of the mechanisms that mediate these processes are not known. Using Cre/LoxP technology, with the whey acidic protein promoter driving transgenic expression of Cre recombinase (WAP-Cre), we have now ablated the type II TGF-beta receptor (T beta RII) expression specifically within mouse mammary alveolar progenitors. Transgenic expression of the polyoma virus middle T antigen, under control of the mouse mammary tumor virus enhancer/promoter, was used to produce mammary tumors in the absence or presence of Cre (T beta RII((fl/fl);PY) and T beta RII((fl/fl);PY;WC), respectively). The loss of TGF-beta signaling significantly decreased tumor latency and increased the rate of pulmonary metastasis. The loss of TGF-beta signaling was significantly correlated with increased tumor size and enhanced carcinoma cell survival. In addition, we observed significant differences in stromal fibrovascular abundance and composition accompanied by increased recruitment of F4/80(+) cell populations in T beta RII((fl/fl);PY;WC) mice when compared with T beta RII((fl/fl);PY) controls. The recruitment of F4/80(+) cells correlated with increased expression of known inflammatory genes including Cxcl1, Cxcl5, and Ptgs2 (cyclooxygenase-2). Notably, we also identified an enriched K5(+) dNp63(+) cell population in primary T beta RII((fl/fl);PY;WC) tumors and corresponding pulmonary metastases, suggesting that loss of TGF-beta signaling in this subset of carcinoma cells can contribute to metastasis. Together, our current results indicate that loss of TGF-beta signaling in mammary alveolar progenitors may affect tumor initiation, progression, and metastasis through regulation of both intrinsic cell signaling and adjacent stromal-epithelial interactions in vivo.

Human brain metastatic stroma attracts breast cancer cells via chemokines CXCL16 and CXCL12.

PubMed

Chung, Brile; Esmaeili, Ali A; Gopalakrishna-Pillai, Sailesh; Murad, John P; Andersen, Emily S; Kumar Reddy, Naveen; Srinivasan, Gayathri; Armstrong, Brian; Chu, Caleb; Kim, Young; Tong, Tommy; Waisman, James; Yim, John H; Badie, Behnam; Lee, Peter P

2017-01-01

The tumor microenvironment is composed of heterogeneous populations of cells, including cancer, immune, and stromal cells. Progression of tumor growth and initiation of metastasis is critically dependent on the reciprocal interactions between cancer cells and stroma. Through RNA-Seq and protein analyses, we found that cancer-associated fibroblasts derived from human breast cancer brain metastasis express significantly higher levels of chemokines CXCL12 and CXCL16 than fibroblasts from primary breast tumors or normal breast. To further understand the interplay between cancer cells and cancer-associated fibroblasts from each site, we developed three-dimensional organoids composed of patient-derived primary or brain metastasis cancer cells with matching cancer-associated fibroblasts. Three-dimensional CAF aggregates generated from brain metastasis promote migration of cancer cells more effectively than cancer-associated fibroblast aggregates derived from primary tumor or normal breast stromal cells. Treatment with a CXCR4 antagonist and/or CXCL16 neutralizing antibody, alone or in combination, significantly inhibited migration of cancer cells to brain metastatic cancer-associated fibroblast aggregates. These results demonstrate that human brain metastasis cancer-associated fibroblasts potently attract breast cancer cells via chemokines CXCL12 and CXCL16, and blocking CXCR6-CXCL16/CXCR4-CXCL12 receptor-ligand interactions may be an effective therapy for preventing breast cancer brain metastasis.

Quiescent Fibroblasts Exhibit High Metabolic Activity

PubMed Central

Lemons, Johanna M. S.; Feng, Xiao-Jiang; Bennett, Bryson D.; Legesse-Miller, Aster; Johnson, Elizabeth L.; Raitman, Irene; Pollina, Elizabeth A.; Rabitz, Herschel A.; Rabinowitz, Joshua D.; Coller, Hilary A.

2010-01-01

Many cells in mammals exist in the state of quiescence, which is characterized by reversible exit from the cell cycle. Quiescent cells are widely reported to exhibit reduced size, nucleotide synthesis, and metabolic activity. Much lower glycolytic rates have been reported in quiescent compared with proliferating lymphocytes. In contrast, we show here that primary human fibroblasts continue to exhibit high metabolic rates when induced into quiescence via contact inhibition. By monitoring isotope labeling through metabolic pathways and quantitatively identifying fluxes from the data, we show that contact-inhibited fibroblasts utilize glucose in all branches of central carbon metabolism at rates similar to those of proliferating cells, with greater overflow flux from the pentose phosphate pathway back to glycolysis. Inhibition of the pentose phosphate pathway resulted in apoptosis preferentially in quiescent fibroblasts. By feeding the cells labeled glutamine, we also detected a “backwards” flux in the tricarboxylic acid cycle from α-ketoglutarate to citrate that was enhanced in contact-inhibited fibroblasts; this flux likely contributes to shuttling of NADPH from the mitochondrion to cytosol for redox defense or fatty acid synthesis. The high metabolic activity of the fibroblasts was directed in part toward breakdown and resynthesis of protein and lipid, and in part toward excretion of extracellular matrix proteins. Thus, reduced metabolic activity is not a hallmark of the quiescent state. Quiescent fibroblasts, relieved of the biosynthetic requirements associated with generating progeny, direct their metabolic activity to preservation of self integrity and alternative functions beneficial to the organism as a whole. PMID:21049082

Hypoxia induces pulmonary fibroblast proliferation through NFAT signaling.

PubMed

Senavirathna, Lakmini Kumari; Huang, Chaoqun; Yang, Xiaoyun; Munteanu, Maria Cristina; Sathiaseelan, Roshini; Xu, Dao; Henke, Craig A; Liu, Lin

2018-02-09

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and typically fatal lung disease with a very low survival rate. Excess accumulation of fibroblasts, myofibroblasts and extracellular matrix creates hypoxic conditions within the lungs, causing asphyxiation. Hypoxia is, therefore, one of the prominent features of IPF. However, there have been few studies concerning the effects of hypoxia on pulmonary fibroblasts. In this study, we investigated the molecular mechanisms of hypoxia-induced lung fibroblast proliferation. Hypoxia increased the proliferation of normal human pulmonary fibroblasts and IPF fibroblasts after exposure for 3-6 days. Cell cycle analysis demonstrated that hypoxia promoted the G1/S phase transition. Hypoxia downregulated cyclin D1 and A2 levels, while it upregulated cyclin E1 protein levels. However, hypoxia had no effect on the protein expression levels of cyclin-dependent kinase 2, 4, and 6. Chemical inhibition of hypoxia-inducible factor (HIF)-2 reduced hypoxia-induced fibroblast proliferation. Moreover, silencing of Nuclear Factor Activated T cell (NFAT) c2 attenuated the hypoxia-mediated fibroblasts proliferation. Hypoxia also induced the nuclear translocation of NFATc2, as determined by immunofluorescence staining. NFAT reporter assays showed that hypoxia-induced NFAT signaling activation is dependent on HIF-2, but not HIF-1. Furthermore, the inhibition or silencing of HIF-2, but not HIF-1, reduced the hypoxia-mediated NFATc2 nuclear translocation. Our studies suggest that hypoxia induces the proliferation of human pulmonary fibroblasts through NFAT signaling and HIF-2.

Comprehensive characterization of mesenchymal stromal cells from patients with Fanconi anaemia.

PubMed

Mantelli, Melissa; Avanzini, Maria Antonia; Rosti, Vittorio; Ingo, Daniela M; Conforti, Antonella; Novara, Francesca; Arrigo, Giulia; Boni, Marina; Zappatore, Rita; Lenta, Elisa; Moretta, Antonia; Acquafredda, Gloria; de Silvestri, Annalisa; Cirillo, Valentina; Cicchetti, Elisa; Algeri, Mattia; Strocchio, Luisa; Vinti, Luciana; Starc, Nadia; Biagini, Simone; Sirleto, Pietro; Bernasconi, Paolo; Zuffardi, Orsetta; Maserati, Emanuela; Maccario, Rita; Zecca, Marco; Locatelli, Franco; Bernardo, Maria Ester

2015-09-01

Fanconi anaemia (FA) is an inherited disorder characterized by pancytopenia, congenital malformations and a predisposition to develop malignancies. Alterations in the haematopoietic microenvironment of FA patients have been reported, but little is known regarding the components of their bone marrow (BM) stroma. We characterized mesenchymal stromal cells (MSCs) isolated from BM of 18 FA patients both before and after allogeneic haematopoietic stem cell transplantation (HSCT). Morphology, fibroblast colony-forming unit (CFU-F) ability, proliferative capacity, immunophenotype, differentiation potential, ability to support long-term haematopoiesis and immunomodulatory properties of FA-MSCs were analysed and compared with those of MSCs expanded from 15 age-matched healthy donors (HD-MSCs). FA-MSCs were genetically characterized through conventional karyotyping, diepoxybutane-test and array-comparative genomic hybridization. FA-MSCs generated before and after HSCT were compared. Morphology, immunophenotype, differentiation potential, ability in vitro to inhibit mitogen-induced T-cell proliferation and to support long-term haematopoiesis did not differ between FA-MSCs and HD-MSCs. CFU-F ability and proliferative capacity of FA-MSCs isolated after HSCT were significantly lower than those of HD-MSCs. FA-MSCs reached senescence significantly earlier than HD-MSCs and showed spontaneous chromosome fragility. Our findings indicate that FA-MSCs are defective in their ability to survive in vitro and display spontaneous chromosome breakages; whether these defects are involved in pathophysiology of BM failure syndromes deserves further investigation. © 2015 John Wiley & Sons Ltd.

A Stromal Cell Niche for Human and Mouse Type 3 Innate Lymphoid Cells.

PubMed

Hoorweg, Kerim; Narang, Priyanka; Li, Zhi; Thuery, Anne; Papazian, Natalie; Withers, David R; Coles, Mark C; Cupedo, Tom

2015-11-01

Adaptive immunity critically depends on the functional compartmentalization of secondary lymphoid organs. Mesenchymal stromal cells create and maintain specialized niches that support survival, activation, and expansion of T and B cells, and integrated analysis of lymphocytes and their niche has been instrumental in understanding adaptive immunity. Lymphoid organs are also home to type 3 innate lymphoid cells (ILC3), innate effector cells essential for barrier immunity. However, a specialized stromal niche for ILC3 has not been identified. A novel lineage-tracing approach now identifies a subset of murine fetal lymphoid tissue organizer cells that gives rise exclusively to adult marginal reticular cells. Moreover, both cell types are conserved from mice to humans and colocalize with ILC3 in secondary lymphoid tissues throughout life. In sum, we provide evidence that fetal stromal organizers give rise to adult marginal reticular cells and form a dedicated stromal niche for innate ILC3 in adaptive lymphoid organs. Copyright © 2015 by The American Association of Immunologists, Inc.

46 CFR 148.445 - Adjacent spaces.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 5 2011-10-01 2011-10-01 false Adjacent spaces. 148.445 Section 148.445 Shipping COAST... THAT REQUIRE SPECIAL HANDLING Additional Special Requirements § 148.445 Adjacent spaces. When... following requirements must be met: (a) Each space adjacent to a cargo hold must be ventilated by natural...

46 CFR 148.445 - Adjacent spaces.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 5 2014-10-01 2014-10-01 false Adjacent spaces. 148.445 Section 148.445 Shipping COAST... THAT REQUIRE SPECIAL HANDLING Additional Special Requirements § 148.445 Adjacent spaces. When... following requirements must be met: (a) Each space adjacent to a cargo hold must be ventilated by natural...

46 CFR 148.445 - Adjacent spaces.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 5 2013-10-01 2013-10-01 false Adjacent spaces. 148.445 Section 148.445 Shipping COAST... THAT REQUIRE SPECIAL HANDLING Additional Special Requirements § 148.445 Adjacent spaces. When... following requirements must be met: (a) Each space adjacent to a cargo hold must be ventilated by natural...

46 CFR 148.445 - Adjacent spaces.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 5 2012-10-01 2012-10-01 false Adjacent spaces. 148.445 Section 148.445 Shipping COAST... THAT REQUIRE SPECIAL HANDLING Additional Special Requirements § 148.445 Adjacent spaces. When... following requirements must be met: (a) Each space adjacent to a cargo hold must be ventilated by natural...

Therapeutic effect of mesenchymal multipotent stromal cells on memory in animals with Alzheimer-type neurodegeneration.

PubMed

Bobkova, N V; Poltavtseva, R A; Samokhin, A N; Sukhikh, G T

2013-11-01

Transplantation of human mesenchymal multipotent stromal cells improved spatial memory in bulbectomized mice with Alzheimer-type neurodegeneration. The positive effect was observed in 1 month after intracerebral transplantation and in 3 months after systemic injection of mesenchymal multipotent stromal cells. No cases of malignant transformation were noted. These findings indicate prospects of using mesenchymal multipotent stromal cells for the therapy of Alzheimer disease and the possibility of their systemic administration for attaining the therapeutic effect.

Enhanced cyclooxygenase-2 expression levels and metalloproteinase 2 and 9 activation by Hexachlorobenzene in human endometrial stromal cells.

PubMed

Chiappini, Florencia; Bastón, Juan Ignacio; Vaccarezza, Agustina; Singla, José Javier; Pontillo, Carolina; Miret, Noelia; Farina, Mariana; Meresman, Gabriela; Randi, Andrea

2016-06-01

Hexachlorobenzene (HCB) is an organochlorine pesticide that induces toxic reproductive effects in laboratory animals. It is a dioxin-like compound and a weak ligand of the aryl hydrocarbon receptor (AhR). Endometriosis is characterized by the presence of functional endometrial tissues outside the uterine cavity. Experimental studies indicate that exposure to organochlorines can interfere with both hormonal regulation and immune function to promote endometriosis. Altered expression of metalloproteinases (MMPs) in patients with endometriosis, suggests that MMPs may play a critical role. In the endometriotic lesions, prostaglandin E2 (PGE2) produced by cyclooxygenase-2 (COX-2), binds to its EP4 receptor (EP4), and via c-Src kinase induces MMPs activation, promoting endometriosis. We examined the HCB action on MMP-2 and MMP-9 activities and expression, COX-2 levels, PGE2 signaling, and the AhR involvement in HCB-induced effects. We have used different in vitro models: (1) human endometrial stromal cell line T-HESC, (2) primary cultures of Human Uterine Fibroblast (HUF), and (3) primary cultures of endometrial stromal cells from eutopic endometrium of control (CESC) and subjects with endometriosis (EESC). Our results show that HCB enhances MMP-2 and MMP-9 activities in T-HESC, HUF and ESC cells. The MMP-9 levels were elevated in all models, while the MMP-2 expression only increased in ESC cells. HCB enhanced COX-2 and EP4 expression, PGE2 secretion and the c-Src kinase activation in T-HESC. Besides, we observed that AhR is implicated in these HCB-induced effects. In conclusion, our results show that HCB exposure could contribute to endometriosis development, affecting inflammation and invasion parameters of human endometrial cells. Copyright © 2016 Elsevier Inc. All rights reserved.

In vitro culture of primary plasmacytomas requires stromal cell feeder layers.

PubMed Central

Degrassi, A; Hilbert, D M; Rudikoff, S; Anderson, A O; Potter, M; Coon, H G

1993-01-01

Attempts to grow primary murine plasmacytomas in vitro have, to date, been largely unsuccessful. In this study, we demonstrate that long-term in vitro growth of primary plasmacytomas is accomplished by using feeder layers composed of stromal cells from the initial site of plasmacytomagenesis. The early neoplastic lines established in this manner are dependent on physical contact with the stromal layer, which is mediated in part by CD44, for growth and survival. The stromal cells provide at least two stimuli for the plasma cells, one being interleukin 6 and the second, of unknown nature, resulting from direct physical interaction that cannot be replaced by soluble factors. These plasma cell lines have been passaged for as long as 20 months yet still maintain characteristics associated with primary plasmacytomas as they will grow in vivo only in pristane-primed animals, indicating a continued dependence on the pristane-induced microenvironment characteristic of early-stage tumors. The ability to grow primary plasmacytomas in culture and maintain their "primary" properties provides a model system for detailed analysis of early events in plasma cell tumor progression involving neoplastic cells completely dependent on physical contact with a stromal feeder layer for survival and expansion. Images Fig. 1 Fig. 2 PMID:8446628

Live Imaging of HIV-1 Transfer across T Cell Virological Synapse to Epithelial Cells that Promotes Stromal Macrophage Infection.

PubMed

Real, Fernando; Sennepin, Alexis; Ganor, Yonatan; Schmitt, Alain; Bomsel, Morgane

2018-05-08

During sexual intercourse, HIV-1 crosses epithelial barriers composing the genital mucosa, a poorly understood feature that requires an HIV-1-infected cell vectoring efficient mucosal HIV-1 entry. Therefore, urethral mucosa comprising a polarized epithelium and a stroma composed of fibroblasts and macrophages were reconstructed in vitro. Using this system, we demonstrate by live imaging that efficient HIV-1 transmission to stromal macrophages depends on cell-mediated transfer of the virus through virological synapses formed between HIV-1-infected CD4 + T cells and the epithelial cell mucosal surface. We visualized HIV-1 translocation through mucosal epithelial cells via transcytosis in regions where virological synapses occurred. In turn, interleukin-13 is secreted and HIV-1 targets macrophages, which develop a latent state of infection reversed by lipopolysaccharide (LPS) activation. The live observation of virological synapse formation reported herein is key in the design of vaccines and antiretroviral therapies aimed at blocking HIV-1 access to cellular reservoirs in genital mucosa. Copyright © 2018. Published by Elsevier Inc.

p110α Inhibition Overcomes Stromal Cell-Mediated Ibrutinib Resistance in Mantle Cell Lymphoma.

PubMed

Guan, Jiyu; Huang, Dan; Yakimchuk, Konstantin; Okret, Sam

2018-05-01

Acquired resistance to cancer drugs is common, also for modern targeted drugs like the Bruton tyrosine kinase (BTK) inhibitor ibrutinib, a new drug approved for the treatment of the highly aggressive and relapsing mantle cell lymphoma (MCL). The tumor microenvironment often impacts negatively on drug response. Here, we demonstrate that stromal cells protect MCL cells from ibrutinib-induced apoptosis and support MCL cell regrowth after drug removal by impairing ibrutinib-mediated downregulation of PI3K/AKT signaling. Importantly, the stromal cell-mediated ibrutinib resistance was overcome in vitro by inhibiting AKT activity using the PI3K catalytic p110α subunit-specific inhibitor BYL719. This was seen both for MCL cell lines and primary MCL cells. Furthermore, inhibition of p110α activity by BYL719 potentiated the ability of ibrutinib to inhibit MCL tumor growth in vivo in a mouse xenograft model. The stromal cell-mediated ibrutinib resistance was found to be due to a direct interaction with MCL cells and involves the integrin VLA-4, as disrupting stromal cell-MCL cell interaction using a VLA-4 blocking antibody abrogated the ibrutinib resistance. This suggests that combined treatment with ibrutinib and a p110α inhibitor, alternatively by disrupting stromal cell-MCL cell interaction, may be a promising therapeutic strategy to overcome stromal cell-mediated ibrutinib resistance in MCL. Mol Cancer Ther; 17(5); 1090-100. ©2018 AACR . ©2018 American Association for Cancer Research.

Matrix metalloproteinase inhibition reduces contraction by dupuytren fibroblasts.

PubMed

Townley, William A; Cambrey, Alison D; Khaw, Peng T; Grobbelaar, Adriaan O

2008-11-01

Dupuytren's disease is a common fibroproliferative condition of the hand characterized by fibrotic lesions (nodules and cords), leading to disability through progressive digital contracture. Although the etiology of the disease is poorly understood, recent evidence suggests that abnormal matrix metalloproteinase (MMP) activity may play a role in cell-mediated collagen contraction and tissue scarring. The aim of this study was to investigate the efficacy of ilomastat, a broad-spectrum MMP inhibitor, in an in vitro model of Dupuytren fibroblast-mediated contraction. Nodule-derived and cord-derived fibroblasts were isolated from Dupuytren patients; carpal ligament-derived fibroblasts acted as control. Stress-release fibroblast-populated collagen lattices (FPCLs) were used as a model of contraction. FPCLs were allowed to develop mechanical stress (48 hours) during treatment with ilomastat (0-100 micromol/L), released, and allowed to contract over a 48-hour period. Contraction was estimated by measuring lattice area compared with untreated cells or treatment with a control peptide. MMP-1, MMP-2, and MT1-MMP levels were assessed by zymography, Western blotting, and enzyme-linked immunosorbent assay. Nodule-derived fibroblasts contracted lattices (69% +/- 2) to a greater extent than did cord-derived (55% +/- 3) or carpal ligament-derived (55% +/- 1) fibroblasts. Exposure to ilomastat led to significant inhibition of lattice contraction by all fibroblasts, although a reduction in lattice contraction by nodule-derived fibroblasts was most prominent (84% +/- 8). In addition, treatment with ilomastat led to a concomitant suppression of MMP-1 and MMP-2 activity, whereas MT1-MMP activity was found to be upregulated. Our results demonstrate that inhibition of MMP activity results in a reduction in extracellular matrix contraction by Dupuytren fibroblasts and suggest that MMP activity may be a critical target in preventing recurrent contracture caused by this disease.

[Immunomorphologic features of epithelial-stromal relationships at hyperplasia and endometrial carcinoma].

PubMed

Bantysh, B B; Paukov, v S; Kogan, E A

2012-01-01

The results of a immunomorphologic comprehensive study of epithelial-stromal relationships in the uterus hyperplasia and endometrial cancer suggest that the suppressor gene of cancer (PTEN) plays a key role in the process of neoplastic transformation of endometrial hyperplasia and adenocarcinoma development. For the first time the existence of two highly differentiated endometrial adenocarcinoma immunophenotype were detected The first one is a PTEN-negative endometrial aedenocarcinoma, characterized by an almost complete inhibition of tumor suppressor gene PTEN in the epithelium of the glands and stromal cell of the tumor The second type is a PTEN-positive endometrial adenocarcinoma, in which epithelial and stromal tumor suppressor gene PTEN activity has retained Based on these results we have formulated a hypothesis about the different types of endometrial hyperplasia morphogenesis and its possible transfer to cervical cancer associated with features of tumor suppressor gene PTEN.

A dendritic cell-stromal axis maintains immune responses in lymph nodes

PubMed Central

Kumar, Varsha; Dasoveanu, Dragos C.; Chyou, Susan; Tzeng, Te-Chen; Rozo, Cristina; Liang, Yong; Stohl, William; Fu, Yang-Xin; Ruddle, Nancy; Lu, Theresa T.

2015-01-01

Summary Within secondary lymphoid tissues, stromal reticular cells support lymphocyte function, and targeting reticular cells is a potential strategy for controlling pathogenic lymphocytes in disease. However, the mechanisms that regulate reticular cell function are not well understood. Here we found that during an immune response in lymph nodes, dendritic cells (DCs) maintain reticular cell survival in multiple compartments. DC-derived lymphotoxin beta receptor (LTβR) ligands were critical mediators, and LTβR signaling on reticular cells mediated cell survival by modulating podoplanin (PDPN). PDPN modulated integrin-mediated cell adhesion, which maintained cell survival. This DC-stromal axis maintained lymphocyte survival and the ongoing immune response. Our findings provide insight into the functions of DCs, LTβR, and PDPN and delineate a DC-stromal axis that can potentially be targeted in autoimmune or lymphoproliferative diseases. PMID:25902483

Metabolic alterations in lung cancer-associated fibroblasts correlated with increased glycolytic metabolism of the tumor

PubMed Central

Chaudhri, Virendra K.; Salzler, Gregory G.; Dick, Salihah A.; Buckman, Melanie S.; Sordella, Raffaella; Karoly, Edward D.; Mohney, Robert; Stiles, Brendon M.; Elemento, Olivier; Altorki, Nasser K.; McGraw, Timothy E.

2013-01-01

SUMMARY Cancer cells undergo a metabolic reprogramming but little is known about metabolic alterations of other cells within tumors. We use mass spectrometry-based profiling and a metabolic pathway-based systems analysis to compare 21 primary human lung tumor cancer-associated fibroblast lines (CAFs) to “normal” fibroblast lines (NFs) generated from adjacent non-neoplastic lung tissue. CAFs are pro-tumorigenic, although the mechanisms by which CAFs support tumors have not been elucidated. We have identified several pathways whose metabolite abundance globally distinguished CAFs from NFs, suggesting that metabolic alterations are not limited to cancer cells. In addition, we found metabolic differences between CAFs from high and low glycolytic tumors that might reflect distinct roles of CAFs related to the tumor’s glycolytic capacity. One such change was an increase of dipeptides in CAFs. Dipeptides primarily arise from the breakdown of proteins. We found in CAFs an increase in basal macroautophagy which likely accounts for the increase in dipeptides. Furthermore, we demonstrate a difference between CAFs and NFs in the induction of autophagy promoted by reduced glucose. In sum, our data suggest increased autophagy may account for metabolic differences between CAFs and NFs and may play additional as yet undetermined roles in lung cancer. PMID:23475953

Three-dimensional spheroid cell culture of umbilical cord tissue-derived mesenchymal stromal cells leads to enhanced paracrine induction of wound healing.

PubMed

Santos, Jorge M; Camões, Sérgio P; Filipe, Elysse; Cipriano, Madalena; Barcia, Rita N; Filipe, Mariana; Teixeira, Mariana; Simões, Sandra; Gaspar, Manuela; Mosqueira, Diogo; Nascimento, Diana S; Pinto-do-Ó, Perpétua; Cruz, Pedro; Cruz, Helder; Castro, Matilde; Miranda, Joana P

2015-05-09

The secretion of trophic factors by mesenchymal stromal cells has gained increased interest given the benefits it may bring to the treatment of a variety of traumatic injuries such as skin wounds. Herein, we report on a three-dimensional culture-based method to improve the paracrine activity of a specific population of umbilical cord tissue-derived mesenchymal stromal cells (UCX®) towards the application of conditioned medium for the treatment of cutaneous wounds. A UCX® three-dimensional culture model was developed and characterized with respect to spheroid formation, cell phenotype and cell viability. The secretion by UCX® spheroids of extracellular matrix proteins and trophic factors involved in the wound-healing process was analysed. The skin regenerative potential of UCX® three-dimensional culture-derived conditioned medium (CM3D) was also assessed in vitro and in vivo against UCX® two-dimensional culture-derived conditioned medium (CM2D) using scratch and tubulogenesis assays and a rat wound splinting model, respectively. UCX® spheroids kept in our three-dimensional system remained viable and multipotent and secreted considerable amounts of vascular endothelial growth factor A, which was undetected in two-dimensional cultures, and higher amounts of matrix metalloproteinase-2, matrix metalloproteinase-9, hepatocyte growth factor, transforming growth factor β1, granulocyte-colony stimulating factor, fibroblast growth factor 2 and interleukin-6, when compared to CM2D. Furthermore, CM3D significantly enhanced elastin production and migration of keratinocytes and fibroblasts in vitro. In turn, tubulogenesis assays revealed increased capillary maturation in the presence of CM3D, as seen by a significant increase in capillary thickness and length when compared to CM2D, and increased branching points and capillary number when compared to basal medium. Finally, CM3D-treated wounds presented signs of faster and better resolution when compared to untreated and CM

Proliferation of Peripheral Blood Lymphocytes and Mesenchymal Stromal Cells Derived from Wharton's Jelly in Mixed and Membrane-Separated Cultures.

PubMed

Poltavtsev, A M; Poltavtseva, R A; Yushina, M N; Pavlovich, S V; Svirshchevskaya, E V

2017-08-01

We studied the effect of mesenchymal stromal cells on proliferation of CFSE-stained T cells in mixed and membrane-separated (Transwell) cultures and in 3D culture of mesenchymal stromal cells from Wharton's jelly. The interaction of mesenchymal stromal cells with mitogen-activated peripheral blood lymphocytes from an allogeneic donor was followed by suppression of T-cell proliferation in a wide range of cell proportions. Culturing in the Transwell system showed the absence of suppression assessed by the fraction of proliferating cells and by the cell cycle analysis. In 3D cultures, contact interaction of mesenchymal stromal cells and lymphocytes was demonstrated that led to accumulation of G2/M phase lymphocytes and G0/G1 phase mesenchymal stromal cells. The suppressive effect of mesenchymal stromal cells from Wharton's jelly is mediated by two mechanisms. The effects are realized within 6 days, which suggests that the therapeutic effects of mesenchymal stromal cells persist until their complete elimination from the body.

Normal Human Fibroblasts Are Resistant to RAS-Induced Senescence

PubMed Central

Benanti, Jennifer A.; Galloway, Denise A.

2004-01-01

Oncogenic stimuli are thought to induce senescence in normal cells in order to protect against transformation and to induce proliferation in cells with altered p53 and/or retinoblastoma (Rb) pathways. In human fibroblasts, RAS initiates senescence through upregulation of the cyclin-dependent kinase inhibitor p16INK4A. We show here that in contrast to cultured fibroblast strains, freshly isolated normal fibroblasts are resistant to RAS-induced senescence and instead show some characteristics of transformation. RAS did not induce growth arrest or expression of senescence-associated β-galactosidase, and Rb remained hyperphosphorylated despite elevated levels of p16. Instead, RAS promoted anchorage-independent growth of normal fibroblasts, although expression of hTert with RAS increased colony formation and allowed normal fibroblasts to bypass contact inhibition. To test the hypothesis that p16 levels determine how cells respond to RAS, we expressed RAS in freshly isolated fibroblasts that expressed very low levels of p16, in hTert-immortalized fibroblasts that had accumulated intermediate levels of p16, and in IMR90 fibroblasts with high levels of p16. RAS induced growth arrest in cells with higher p16 levels, and this effect was reversed by p16 knockdown in the hTert-immortalized fibroblasts. These findings indicate that culture-imposed stress sensitizes cells to RAS-induced arrest, whereas early passage cells do not arrest in response to RAS. PMID:15024073

Mesenchymal stem cells induce dermal fibroblast responses to injury

PubMed Central

Smith, Andria N.; Willis, Elise; Chan, Vincent T.; Muffley, Lara A.; Isik, F. Frank; Gibran, Nicole S.; Hocking, Anne M.

2009-01-01

Although bone marrow-derived mesenchymal stem cells have been shown to promote repair when applied to cutaneous wounds, the mechanism for this response remains to be determined. The aim of this study was to determine the effects of paracrine signaling from mesenchymal stem cells on dermal fibroblast responses to injury including proliferation, migration and expression of genes important in wound repair. Dermal fibroblasts were co-cultured with bone marrow-derived mesenchymal stem cells grown in inserts, which allowed for paracrine interactions without direct cell contact. In this co-culture model, bone marrow-derived mesenchymal stem cells regulate dermal fibroblast proliferation, migration and gene expression. When co-cultured with mesenchymal stem cells, dermal fibroblasts show increased proliferation and accelerated migration in a scratch assay. A chemotaxis assay also demonstrated that dermal fibroblasts migrate towards bone marrow-derived mesenchymal stem cells. A PCR array was used to analyze the effect of mesenchymal stem cells on dermal fibroblast gene expression. In response to mesenchymal stem cells, dermal fibroblasts up-regulate integrin alpha 7 expression and down-regulate expression of ICAM1, VCAM1 and MMP11. These observations suggest that mesenchymal stem cells may provide an important early signal for dermal fibroblast responses to cutaneous injury. PMID:19666021

Mesenchymal stem cells induce dermal fibroblast responses to injury

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Andria N., E-mail: snosmith@u.washington.edu; Willis, Elise, E-mail: elise.willis@gmail.com; Chan, Vincent T.

2010-01-01

Although bone marrow-derived mesenchymal stem cells have been shown to promote repair when applied to cutaneous wounds, the mechanism for this response remains to be determined. The aim of this study was to determine the effects of paracrine signaling from mesenchymal stem cells on dermal fibroblast responses to injury including proliferation, migration and expression of genes important in wound repair. Dermal fibroblasts were co-cultured with bone marrow-derived mesenchymal stem cells grown in inserts, which allowed for paracrine interactions without direct cell contact. In this co-culture model, bone marrow-derived mesenchymal stem cells regulate dermal fibroblast proliferation, migration and gene expression. Whenmore » co-cultured with mesenchymal stem cells, dermal fibroblasts show increased proliferation and accelerated migration in a scratch assay. A chemotaxis assay also demonstrated that dermal fibroblasts migrate towards bone marrow-derived mesenchymal stem cells. A PCR array was used to analyze the effect of mesenchymal stem cells on dermal fibroblast gene expression. In response to mesenchymal stem cells, dermal fibroblasts up-regulate integrin alpha 7 expression and down-regulate expression of ICAM1, VCAM1 and MMP11. These observations suggest that mesenchymal stem cells may provide an important early signal for dermal fibroblast responses to cutaneous injury.« less

Expression of a suicidal gene under control of the human secreted protein acidic and rich in cysteine (SPARC) promoter in tumor or stromal cells led to the inhibition of tumor cell growth

PubMed Central

Lopez, María V.; Blanco, Patricia; Viale, Diego L.; Cafferata, Eduardo G.; Carbone, Cecilia; Gould, David; Chernajovsky, Yuti; Podhajcer, Osvaldo L.

2009-01-01

The successful use of transcriptional targeting for cancer therapy depends on the activity of a given promoter inside the malignant cell. Because solid human tumors evolve as a “cross-talk” between the different cell types within the tumor, we hypothesized that targeting the entire tumor mass might have better therapeutic effect. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein overexpressed in different human cancers malignant melanomas both in the malignant cells compartment as in the stromal one (fibroblasts and endothelial cells). We have shown that expression of the herpes simplex virus-thymidine kinase (TK) gene driven by the SPARC promoter in combination with ganciclovir inhibited human melanoma cell growth in monolayer as well as in multicellular spheroids. This inhibitory effect was observed both in homotypic spheroids composed of melanoma cells alone as well as in spheroids made of melanoma cells and stromal cells. Expression of the TK gene was also efficient to inhibit the in vivo tumor growth of established melanomas when TK was expressed either by the malignant cells themselves or by coadministered endothelial cells. Our data suggest that the use of therapeutic genes driven by SPARC promoter could be a valuable strategy for cancer therapy aiming to target all the cellular components of the tumor mass. PMID:17041094

Effects of neuropeptides on human lung fibroblast proliferation and chemotaxis.

PubMed

Harrison, N K; Dawes, K E; Kwon, O J; Barnes, P J; Laurent, G J; Chung, K F

1995-02-01

An increase in subepithelial mesenchymal cells and associated connective tissue is a feature of bronchial asthma. We determined whether neuropeptides could modulate fibroblast activity, particularly with respect to proliferation and chemotaxis. Human lung fibroblasts were cultured with neurokinin A (NKA), substance P (SP), vasoactive intestinal peptide (VIP), and calcitonin-gene-related peptide (CGRP). After 48 h, fibroblast proliferation was measured by a colorimetric assay based on the uptake and subsequent release of methylene blue. The chemotactic response to neuropeptides was determined with the use of a modified Boyden chamber. Both NKA and SP (10(-7)-10(-4) M) stimulated human lung fibroblast proliferation in HFL1 and IMR-90 fibroblasts. VIP and CGRP had no effect on fibroblast proliferation. NKA alone stimulated fibroblast chemotaxis maximally at 10(-10) M. Neutral endopeptidase (NEP) activity of 0.52 and 5.2 pmol/10(6) cells was assayed in IMR-90 and Hs68 fibroblasts, respectively. Phosphoramidon (5 x 10(-6)-10(-5) M), an NEP inhibitor, enhanced fibroblast proliferation in a dose-dependent manner. Thus neuropeptides have the potential to cause activation of mesenchymal cells, and neuropeptide release may contribute to the structural abnormalities observed in asthmatic airways.

Modulation of Mammary Stromal Cell Lactate Dynamics by Ambient Glucose and Epithelial Factors.

PubMed

Tobar, Nicolas; Porras, Omar; Smith, Patricio C; Barros, L Felipe; Martínez, Jorge

2017-01-01

Hyperglycemia is a risk factor for a variety of human cancers. Increased access to glucose and that tumor metabolize glucose by a glycolytic process even in the presence of oxygen (Warburg effect), provide a framework to analyze a particular set of metabolic adaptation mechanisms that may explain this phenomenon. In the present work, using a mammary stromal cell line derived from healthy tissue that was subjected to a long-term culture in low (5 mM) or high (25 mM) glucose, we analyzed kinetic parameters of lactate transport using a FRET biosensor. Our results indicate that the glucose pre-culture and soluble epithelial factors constitute a stimulus for lactate stromal production, factors that also modify the kinetic parameters and the monocarboxylate transporters expression in stromal cells. We also observed a vectorial flux of lactate from stroma to epithelial cells in a co-culture setting and found that the uptake of lactate by epithelial cells correlates with the degree of malignancy. Glucose preconditioning of the stromal cell stimulated epithelial motility. Our findings suggest that lactate generated by stromal cells in the high glucose condition stimulate epithelial migration. Overall, our results support the notion that glucose not only provides a substrate for tumor nutrition but also behaves as a signal promoting malignancy. J. Cell. Physiol. 232: 136-144, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: a joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT).

PubMed

Bourin, Philippe; Bunnell, Bruce A; Casteilla, Louis; Dominici, Massimo; Katz, Adam J; March, Keith L; Redl, Heinz; Rubin, J Peter; Yoshimura, Kotaro; Gimble, Jeffrey M

2013-06-01

Adipose tissue is a rich and very convenient source of cells for regenerative medicine therapeutic approaches. However, a characterization of the population of adipose-derived stromal and stem cells (ASCs) with the greatest therapeutic potential remains unclear. Under the authority of International Federation of Adipose Therapeutics and International Society for Cellular Therapy, this paper sets out to establish minimal definitions of stromal cells both as uncultured stromal vascular fraction (SVF) and as an adherent stromal/stem cells population. Phenotypic and functional criteria for the identification of adipose-derived cells were drawn from the literature. In the SVF, cells are identified phenotypically by the following markers: CD45-CD235a-CD31-CD34+. Added value may be provided by both a viability marker and the following surface antigens: CD13, CD73, CD90 and CD105. The fibroblastoid colony-forming unit assay permits the evaluation of progenitor frequency in the SVF population. In culture, ASCs retain markers in common with other mesenchymal stromal/stem cells (MSCs), including CD90, CD73, CD105, and CD44 and remain negative for CD45 and CD31. They can be distinguished from bone-marrow-derived MSCs by their positivity for CD36 and negativity for CD106. The CFU-F assay is recommended to calculate population doublings capacity of ASCs. The adipocytic, chondroblastic and osteoblastic differentiation assays serve to complete the cell identification and potency assessment in conjunction with a quantitative evaluation of the differentiation either biochemically or by reverse transcription polymerase chain reaction. The goal of this paper is to provide initial guidance for the scientific community working with adipose-derived cells and to facilitate development of international standards based on reproducible parameters. Copyright © 2013 International Society for Cellular Therapy. All rights reserved.

Physiologically activated mammary fibroblasts promote postpartum mammary cancer

PubMed Central

Guo, Qiuchen; Burchard, Julja; Spellman, Paul

2017-01-01

Women diagnosed with breast cancer within 5 years of childbirth have poorer prognosis than nulliparous or pregnant women. Weaning-induced breast involution is implicated, as the collagen-rich, immunosuppressive microenvironment of the involuting mammary gland is tumor promotional in mice. To investigate the role of mammary fibroblasts, isolated mammary PDGFRα+ cells from nulliparous and postweaning mice were assessed for activation phenotype and protumorigenic function. Fibroblast activation during involution was evident by increased expression of fibrillar collagens, lysyl oxidase, Tgfb1, and Cxcl12 genes. The ability of mammary tumors to grow in an isogenic, orthotopic transplant model was increased when tumor cells were coinjected with involution-derived compared with nulliparous-derived mammary fibroblasts. Mammary tumors in the involution-fibroblast group had increased Ly6C+ monocytes at the tumor border, and decreased CD8+ T cell infiltration and tumor cell death. Ibuprofen treatment suppressed involution-fibroblast activation and tumor promotional capacity, concurrent with decreases in tumor Ly6C+ monocytes, and increases in intratumoral CD8+ T cell infiltration, granzyme levels, and tumor cell death. In total, our data identify a COX/prostaglandin E2 (PGE2)–dependent activated mammary fibroblast within the involuting mammary gland that displays protumorigenic, immunosuppressive activity, identifying fibroblasts as potential targets for the prevention and treatment of postpartum breast cancer. PMID:28352652

Evidence for the involvement of fibroblast growth factor 10 in lipofibroblast formation during embryonic lung development

PubMed Central

Al Alam, Denise; El Agha, Elie; Sakurai, Reiko; Kheirollahi, Vahid; Moiseenko, Alena; Danopoulos, Soula; Shrestha, Amit; Schmoldt, Carole; Quantius, Jennifer; Herold, Susanne; Chao, Cho-Ming; Tiozzo, Caterina; De Langhe, Stijn; Plikus, Maksim V.; Thornton, Matthew; Grubbs, Brendan; Minoo, Parviz; Rehan, Virender K.; Bellusci, Saverio

2015-01-01

Lipid-containing alveolar interstitial fibroblasts (lipofibroblasts) are increasingly recognized as an important component of the epithelial stem cell niche in the rodent lung. Although lipofibroblasts were initially believed merely to assist type 2 alveolar epithelial cells in surfactant production during neonatal life, recent evidence suggests that these cells are indispensable for survival and growth of epithelial stem cells during adulthood. Despite increasing interest in lipofibroblast biology, little is known about their cellular origin or the molecular pathways controlling their formation during embryonic development. Here, we show that a population of lipid-droplet-containing stromal cells emerges in the developing mouse lung between E15.5 and E16.5. This is accompanied by significant upregulation, in the lung mesenchyme, of peroxisome proliferator-activated receptor gamma (master switch of lipogenesis), adipose differentiation-related protein (marker of mature lipofibroblasts) and fibroblast growth factor 10 (previously shown to identify a subpopulation of lipofibroblast progenitors). We also demonstrate that although only a subpopulation of total embryonic lipofibroblasts derives from Fgf10+ progenitor cells, in vivo knockdown of Fgfr2b ligand activity and reduction in Fgf10 expression lead to global reduction in the expression levels of lipofibroblast markers at E18.5. Constitutive Fgfr1b knockouts and mutants with conditional partial inactivation of Fgfr2b in the lung mesenchyme reveal the involvement of both receptors in lipofibroblast formation and suggest a possible compensation between the two receptors. We also provide data from human fetal lungs to demonstrate the relevance of our discoveries to humans. Our results reveal an essential role for Fgf10 signaling in the formation of lipofibroblasts during late lung development. PMID:26511927

Quantitative analysis of corneal stromal riboflavin concentration without epithelial removal.

PubMed

Rubinfeld, Roy S; Stulting, R Doyle; Gum, Glenwood G; Talamo, Jonathan H

2018-02-01

To compare the corneal stromal riboflavin concentration and distribution using 2 transepithelial corneal crosslinking (CXL) systems. Absorption Systems, San Diego, California, USA. Experimental study. The stromal riboflavin concentration of 2 transepithelial CXL systems was compared in rabbit eyes in vivo. The systems were the Paracel/Vibex Xtra, comprising riboflavin 0.25% solution containing TRIS and ethylenediaminetetraacetic acid and an isotonic solution of riboflavin 0.25%, (Group 1) and the CXLO system (Group 2). Manufacturers' Instructions For Use were followed. The intensity of riboflavin fluorescence by slitlamp observation 10, 15, and 20 minutes after instillation was graded on a scale of 0 to 5. The animals were humanely killed and the corneal stromal samples analyzed with liquid chromatography and mass spectrometry. The mean riboflavin fluorescence intensity grades in Group 1 (4 eyes) were 3.8, 4.8, and 4.8 at 10, 15, and 20 minutes, respectively. The mean grades in Group 2 (3 eyes) were 2.0, 2.3, and 2.0, respectively. The riboflavin distribution was uniform in Group 1 but not in Group 2. The mean riboflavin concentration by liquid chromatography and mass spectrometry was 27.0 μg/g stromal tissue in Group 1 and 6.7 μg/g in Group 2. A stromal riboflavin concentration theoretically adequate for CXL, 15 μg/g, was achieved in all eyes in Group 1 and no eyes in Group 2. Slitlamp grading correlated well with liquid chromatography and mass spectrometry concentration (R 2  = 0.940). The system used in Group 1 produced corneal riboflavin concentrations that were theoretically adequate for effective transepithelial CXL (≥15 μg/g), while the system in Group 2 did not. Slitlamp grading successfully estimated the corneal riboflavin concentration and can be used to ensure an adequate concentration of riboflavin in the cornea for transepithelial CXL. Copyright © 2018 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

Bone marrow stromal cells spontaneously produce Flt3-ligand: influence of ionizing radiations and cytokine stimulation.

PubMed

Bertho, Jean Marc; Demarquay, Christelle; Mouiseddine, Moubarak; Douenat, Noémie; Stefani, Johanna; Prat, Marie; Paquet, François

2008-08-01

To define the ability of human bone marrow (BM) stromal cells to produce fms-like tyrosine kinase 3 (Flt3)-ligand (FL), and the effect of irradiation, tumour necrosis factor-alpha (TNFalpha) or tumour growth factor beta (TGFbeta) on FL production. Primary BM stromal cell cultures were irradiated at 2-10 Gy or were stimulated with TNFalpha or TGFbeta1. The presence of FL was tested in culture supernatants and in cell lysate. The presence of a membrane-bound form of FL and the level of gene expression were also tested. Primary BM stromal cells spontaneously released FL. This production was increased by TNFalpha but not by TGFbeta1 or by irradiation. Chemical induction of osteoblastic differentiation from BM stromal cells also induced an increase in FL release. Our results suggest that the observed increase in FL concentration after in vivo irradiation is an indirect effect. The possible implication of BM stromal cells in these mechanisms is discussed.

The Role of ADAM9 in Tumor-Stromal Interactions in Breast Cancer

DTIC Science & Technology

2010-04-01

Tumor-Stromal Interactions in Breast Cancer PRINCIPAL INVESTIGATOR: Jessica L. Fry...The Role of ADAM9 in Tumor-Stromal Interactions in Breast Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-06-1-0460 5c...ADAM9, the development of a model system to investigate the isoforms of ADAM-9 in breast cancer cell migration, and the use of this model system to

Endometrial stromal cell attachment and matrix homeostasis in abdominal wall endometriomas.

PubMed

Itoh, Hiroko; Mogami, Haruta; Bou Nemer, Laurice; Word, Larry; Rogers, David; Miller, Rodney; Word, R Ann

2018-02-01

How does progesterone alter matrix remodeling in abdominal wall endometriomas compared with normal endometrium? Progesterone may prevent attachment of endometrial cells to the abdominal wall, but does not ameliorate abnormal stromal cell responses of abdominal wall endometriomas. Menstruation is a tightly orchestrated physiologic event in which steroid hormones and inflammatory cells cooperatively initiate shedding of the endometrium. Abdominal wall endometriomas represent a unique form of endometriosis in which endometrial cells inoculate fascia or dermis at the time of obstetrical or gynecologic surgery. Invasion of endometrium into ectopic sites requires matrix metalloproteinases (MMPs) for tissue remodeling but endometrium is not shed externally. Observational study in 14 cases and 19 controls. Tissues and stromal cells isolated from 14 abdominal wall endometriomas were compared with 19 normal cycling endometrium using immunohistochemistry, quantitative PCR, gelatin zymography and cell attachment assays. P values < 0.05 were considered significant and experiments were repeated in at least three different cell preps to provide scientific rigor to the conclusions. The results indicate that MMP2 and MMP9 are not increased by TGFβ1 in endometrioma stromal cells. Although progesterone prevents attachment of endometrioma cells to matrix components of the abdominal wall, it does not ameliorate these abnormal stromal cell responses to TGFβ1. N/A. Endometriomas were collected from women identified pre-operatively. Not all endometriomas were collected. Stromal cells from normal endometrium were from different patients, not women undergoing endometrioma resection. This work provides insight into the mechanisms by which progesterone may prevent abdominal wall endometriomas but, once established, are refractory to progesterone treatment. Tissue acquisition was supported by NIH P01HD087150. Authors have no competing interests. © The Author(s) 2017. Published by Oxford

Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy

NASA Technical Reports Server (NTRS)

Askari, Arman T.; Unzek, Samuel; Popovic, Zoran B.; Goldman, Corey K.; Forudi, Farhad; Kiedrowski, Matthew; Rovner, Aleksandr; Ellis, Stephen G.; Thomas, James D.; DiCorleto, Paul E.;

CARFMAP: A Curated Pathway Map of Cardiac Fibroblasts.

PubMed

Nim, Hieu T; Furtado, Milena B; Costa, Mauro W; Kitano, Hiroaki; Rosenthal, Nadia A; Boyd, Sarah E

2015-01-01

The adult mammalian heart contains multiple cell types that work in unison under tightly regulated conditions to maintain homeostasis. Cardiac fibroblasts are a significant and unique population of non-muscle cells in the heart that have recently gained substantial interest in the cardiac biology community. To better understand this renaissance cell, it is essential to systematically survey what has been known in the literature about the cellular and molecular processes involved. We have built CARFMAP (http://visionet.erc.monash.edu.au/CARFMAP), an interactive cardiac fibroblast pathway map derived from the biomedical literature using a software-assisted manual data collection approach. CARFMAP is an information-rich interactive tool that enables cardiac biologists to explore the large body of literature in various creative ways. There is surprisingly little overlap between the cardiac fibroblast pathway map, a foreskin fibroblast pathway map, and a whole mouse organism signalling pathway map from the REACTOME database. Among the use cases of CARFMAP is a common task in our cardiac biology laboratory of identifying new genes that are (1) relevant to cardiac literature, and (2) differentially regulated in high-throughput assays. From the expression profiles of mouse cardiac and tail fibroblasts, we employed CARFMAP to characterise cardiac fibroblast pathways. Using CARFMAP in conjunction with transcriptomic data, we generated a stringent list of six genes that would not have been singled out using bioinformatics analyses alone. Experimental validation showed that five genes (Mmp3, Il6, Edn1, Pdgfc and Fgf10) are differentially regulated in the cardiac fibroblast. CARFMAP is a powerful tool for systems analyses of cardiac fibroblasts, facilitating systems-level cardiovascular research.

Effects of polyhexamethylene guanidine phosphate on human gingival fibroblasts.

PubMed

Vitt, Anton; Slizen, Veronica; Boström, Elisabeth A; Yucel-Lindberg, Tülay; Kats, Anna; Sugars, Rachael V; Gustafsson, Anders; Buhlin, Kåre

2017-10-01

Polyhexamethylene guanidine phosphate (PHMG-P) was compared to chlorhexidine (CHX) in order to determine potential cytotoxic and immune-modulatory effects on human gingival fibroblasts. Cytotoxic effects of PHMG-P and CHX on human gingival fibroblasts were assessed using cell viability assay at various time points and concentrations. The effects of PHMG-P and CHX on the secretion of prostaglandin (PG) E 2 , interleukin (IL)-6, IL-8 and matrix metalloproteinase (MMP)-1 by non-stimulated or IL-1β stimulated fibroblasts were evaluated by enzyme-linked immunosorbent assays. PHMG-P concentration 0.00009% led to the total loss of fibroblast viability within 24 h, whereas inhibition of fibroblast viability by CHX occurred at significantly higher concentrations of 0.0009% (p < .001). Short-term exposure to 0.005% PHMG-P led to loss of fibroblast viability after 5 min, whilst cells exposed to 0.005% CHX survived 30 min of treatment (p < .001). IL-1β stimulation induced an inflammatory response with a significant increase in the secretion of PGE 2 , IL-6, IL-8 and MMP-1. Treatment of IL-1β stimulated fibroblasts in combination with PHMG-P or CHX at concentrations of 0.000045 or 0.0.00009% resulted in significantly decreased PGE 2 , IL-6, IL-8 and MMP-1 levels. PHMG-P or CHX alone did not affect the baseline secretion of PGE 2 , IL-6, IL-8 or MMP-1 by gingival fibroblasts. Cytotoxic effects on gingival fibroblasts were triggered by both PHMG-P and CHX at concentrations below those used in clinical practice. The tested antiseptics did not cause inflammation and reduced IL-1β-induced secretion of inflammatory mediators and collagenase by gingival fibroblasts, which suggests anti-inflammatory properties.

[Massive hemorrhage of upper gastrointestinal tract caused by gastrointestinal stromal tumor of the stomach--case report].

PubMed

Lalović, Nenad; Dukić Vladicić, Nikolina; Marić, Radmil; Cuk, Mirjana; Simatović, Milan; Jokanović, Dragana

2012-01-01

Acute bleeding from the upper gastrointestinal system is a medical emergency which is followed by high mortality rate, ranging from 6 to 15% in spite of modern diagnostic methods and treatment. Bleeding from the upper gastrointestinal system may be caused by gastrointestinal stromal tumors of the stomach, which are mainly characterized by occult bleeding, while profuse bleeding rarely occurs accompanied by hemorrhagic shock. Gastrointestinal stromal tumors of stomach are the most common mesenchimal tumors of the gastrointestinal tract. In our study we showed a 60-year-old female patient with profuse bleeding from the stomach and the clinical picture of severe hemorrhagic shock, caused by gastrointestinal stromal tumor. An ovoid junction, raised towards the lumen, covered with ulcerated mucosa in several places and followed by massive arterial bleeding was found intraoperatively, after the performed gastrotomy. Histopathological examination with immunohistochemical analysis confirmed that this was a gastrointestinal stromal tumor of the stomach. Acute bleeding from the digestive system is a sudden and serious condition of the body. Urgent esophagogastroduodenoscopy is a sensitive and specific diagnostic and therapeutic method of choice. Massive bleeding from the upper gastrointestinal tract is very rarely caused by gastrointestinal stromal tumors, whose clinical picture is very heterogeneous and depends on tumor size and location. Abundant bleeding from the tumor is an indication for urgent surgical intervention. According to the literature massive hemorrhage of the upper digestive system can rarely be caused by gastrointestinal stromal tumor of the stomach. It is shown that abundant hemorrhage of the upper digestive tract can be caused with gastric gastrointestinal stromal tumor. Surgical resection is the main form of treatment of gastrointestinal stromal tumors of the digestive system and bleeding from these tumors caused by failure of endoscopic hemostasis.

Optical coherence tomography of the effects of stromal hydration on clear corneal incision architecture.

PubMed

Calladine, Daniel; Tanner, Vaughan

2009-08-01

To evaluate the effects of stromal hydration on clear corneal incision (CCI) architecture immediately after surgery using anterior segment optical coherence tomography (AS-OCT). Department of Ophthalmology, Royal Berkshire Hospital, Reading, United Kingdom. Clear corneal incisions in adult eyes were examined using a Visante AS-OCT imaging system within 1 hour of surgery. Half the CCIs had stromal hydration with a balanced salt solution and half did not. Incisions were made with a 2.75 mm steel keratome. Intraocular pressure (IOP) was measured within 90 minutes after surgery. The CCI length and corneal thickness at the CCI site were measured using software built into the AS-OCT system. Thirty CCIs were evaluated. Stromal hydration significantly increased the measured CCI length (P<.05, t test); this was the result of a trend toward increased corneal thickness at the CCI site with hydration (P<.1, t test). The mean CCI length was 1.69 mm +/- 0.27 (SD) (range 1.31 to 2.32 mm) with hydration and 1.51 +/- 0.23 mm (range 1.30 to 1.95 mm) without hydration. The mean IOP was 20.9 +/- 8.18 mm Hg and 15.8 +/- 8.20 mm Hg, respectively. The IOP tended to be higher with hydration (P<.1, t test). Local detachment of Descemet membrane was more likely with stromal hydration (63%) than without (25%). Stromal hydration significantly increased CCI length and tended to leave the eye with a higher early postoperative IOP, showing the importance of taking stromal hydration into account when designing similar OCT studies of CCI architecture.

AGEs trigger autophagy in diabetic skin tissues and fibroblasts.

PubMed

Sun, Kan; Wang, Wei; Wang, Chuan; Lao, Guojuan; Liu, Dan; Mai, Lifang; Yan, Li; Yang, Chuan; Ren, Meng

2016-03-11

Accumulation of advanced glycation end products (AGEs) contributes to the development of diabetic ulcers. Recent evidence indicates that AGEs administration enhanced autophagy in many cell types. As a positive trigger of autophagy, the effect of AGEs on autophagy in skin tissues and fibroblasts remains unknown. Skin tissues were isolated from Spreqne-Dawley rats and immunohistochemical staining was performed to analyze the location of LC3 and FOXO1 in skin tissues. Then primary cultured foreskin fibroblast cells with treated with AGEs and the effect of AGEs on autophagy was investigated. Protein level expressions of LC3, Beclin-1 and FOXO1 in fibroblasts were analyzed by Western blotting. Autophagic flux is detected with autophagy inhibitor chloroquine and mRFP-GFP-LC3 tandem construct. Compared with skin from normal rats, immunohistochemical staining shows a predominant LC3 localization in fibroblasts cytoplasm in diabetic rats. Elevated expression of FOXO1 also existed in diabetic rats dermis fibroblasts when compared with normal rats in immunohistochemical analysis. In human skin fibroblasts cells, AGEs administration stimulated the autophagy related LC3-II/LC3-I and Beclin-1 expressions and increased autophagy flux. In mRFP-GFP-LC3 puncta formation assays, both autolysosome and autophagosome were increased in human fibroblasts after treatment with AGEs. Fibroblasts exposed to AGEs also have increased FOXO1 expression compared with control group. AGEs could induce autophagy at least in part via regulating the FOXO1 activity in diabetic skin tissues and fibroblasts. Copyright © 2016 Elsevier Inc. All rights reserved.

Transient chloride binding as a contributory factor to corneal stromal swelling in the ox.

PubMed Central

Hodson, S; Kaila, D; Hammond, S; Rebello, G; al-Omari, Y

1992-01-01

1. Investigations were made of the cation exchange capacity of fresh isolated ox corneal stroma (Q, units: mequiv fixed stromal charge/kg stromal fluid) at pH 7.4 over a variety of stomal hydrations (H, units: kg stromal fluid/kg dry tissue) both above and below the physiological hydration of 3.2, whilst the stromas were immersed in a variety of sodium chloride solutions (range 5-1000 mM). 2. At any particular salt concentration, the product QH (dry tissue exchange capacity, units: mequiv/kg dry tissue) appeared constant, over all the hydrations investigated. 3. Dry tissue exchange capacity (QH) varied, however, when the bathing salt concentration was altered. It varied between 55 mequiv/kg dry tissue (e.g. Q = 17 mequiv at H = 3.2) in 5 mM-NaCl to 240 mequiv/kg dry tissue (e.g. Q = 75 mequiv/l at H = 3.2) in 1000 mM-NaCl. 4. The variation of stromal exchange capacity in NaCl solutions of different concentrations was similar when detected by three independent procedures: stromal gel pressure measurements, intrastromal sodium ion distributions, and intrastromal electrical potentials. 5. Intrastromal chloride ion distributions were anomalous. Total chloride (measured by radio-isotopes) was consistently higher than that predicted by Donnan theory. 6. The data were consistent with Elliott's hypothesis that a fraction of intrastromal chloride ions bind to the corneal stromal matrix and in so doing contribute to the fixed negative charge of the stroma. 7. Our observations may be explained by a model of the cation exchange capacity of ox cornea which has two types of components. On is (at constant pH) invariant, and has a dry tissue exchange capacity of about 50 mequiv/kg dry tissue, and is probably generated by the sulphonic and carboxylic acid groups of the glycosaminoglycans. The other is explained by supposing it to consist of a chloride binding ligand which exhibits first order binding, is half occupied at ambient chloride concentrations of 300 mM, and has a total

Dissection of stromal and cancer cell-derived signals in melanoma xenografts before and after treatment with DMXAA

PubMed Central

Henare, K; Wang, L; Wang, L-Cs; Thomsen, L; Tijono, S; Chen, C-Jj; Winkler, S; Dunbar, P R; Print, C; Ching, L-M

2012-01-01

Background: The non-malignant cells of the tumour stroma have a critical role in tumour biology. Studies dissecting the interplay between cancer cells and stromal cells are required to further our understanding of tumour progression and methods of intervention. For proof-of-principle of a multi-modal approach to dissect the differential effects of treatment on cancer cells and stromal cells, we analysed the effects of the stromal-targeting agent 5,6-dimethylxanthenone-4-acetic acid on melanoma xenografts. Methods: Flow cytometry and multi-colour immunofluorescence staining was used to analyse leukocyte numbers in xenografts. Murine-specific and human-specific multiplex cytokine panels were used to quantitate cytokines produced by stromal and melanoma cells, respectively. Human and mouse Affymetrix microarrays were used to separately identify melanoma cell-specific and stromal cell-specific gene expression. Results: 5,6-Dimethylxanthenone-4-acetic acid activated pro-inflammatory signalling pathways and cytokine expression from both stromal and cancer cells, leading to neutrophil accumulation and haemorrhagic necrosis and a delay in tumour re-growth of 26 days in A375 melanoma xenografts. Conclusion: 5,6-Dimethylxanthenone-4-acetic acid and related analogues may potentially have utility in the treatment of melanoma. The experimental platform used allowed distinction between cancer cells and stromal cells and can be applied to investigate other tumour models and anti-cancer agents. PMID:22415295

Inhibition of Microsomal Prostaglandin E Synthase-1 in Cancer-Associated Fibroblasts Suppresses Neuroblastoma Tumor Growth.

PubMed

Kock, Anna; Larsson, Karin; Bergqvist, Filip; Eissler, Nina; Elfman, Lotta H M; Raouf, Joan; Korotkova, Marina; Johnsen, John Inge; Jakobsson, Per-Johan; Kogner, Per

2018-06-01

Despite recent progress in diagnosis and treatment, survival for children with high-risk metastatic neuroblastoma is still poor. Prostaglandin E 2 (PGE 2 )-driven inflammation promotes tumor growth, immune suppression, angiogenesis and resistance to established cancer therapies. In neuroblastoma, cancer-associated fibroblasts (CAFs) residing in the tumor microenvironment are the primary source of PGE 2 . However, clinical targeting of PGE 2 with current non-steroidal anti-inflammatory drugs or cyclooxygenase inhibitors has been limited due to risk of adverse side effects. By specifically targeting microsomal prostaglandin E synthase-1 (mPGES-1) activity with a small molecule inhibitor we could block CAF-derived PGE 2 production leading to reduced tumor growth, impaired angiogenesis, inhibited CAF migration and infiltration, reduced tumor cell proliferation and a favorable shift in the M1/M2 macrophage ratio. In this study, we provide proof-of-principle of the benefits of targeting mPGES-1 in neuroblastoma, applicable to a wide variety of tumors. This non-toxic single drug treatment targeting infiltrating stromal cells opens up for combination treatment options with established cancer therapies. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Effects of mitomycin-C on normal dermal fibroblasts.

PubMed

Chen, Theodore; Kunnavatana, Shaun S; Koch, R James

2006-04-01

To evaluate the effects of mitomycin-C on the growth and autocrine growth factor production of human dermal fibroblasts from the face. In vitro study using normal adult dermal fibroblast cell lines in a serum-free model. Cell cultures were exposed to 4 mg/mL, 0.4 mg/mL, 0.04 mg/mL, 0.004 mg/mL, and 0.0004 mg/mL concentrations of mitomycin-C solution. Cell counts were performed, and the cell-free supernatants were collected at 0, 1, 3, and 5 days after the initial exposure. Population doubling times were calculated and supernatants were quantitatively assayed for basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-beta1. Continuous exposure to mitomycin-C caused fibroblast cell death by day 7 at all tested concentrations. A 4 minute exposure to mitomycin-C at 4 mg/mL caused rapid fibroblast cell death. A 4-minute exposure to mitomycin-C at either 0.4 mg/mL or 0.04 mg/mL resulted in decreased fibroblast proliferation. A 4 minute exposure to mitomycin-C at 0.4 mg/mL resulted in a marked increase in the production of both bFGF and TGF-beta1. A clinically ideal concentration of mitomycin-C would slow fibroblast proliferation yet not cause cell death to allow for a wound healing response. Mitomycin-C 0.4 mg/mL for 4 minutes satisfies the above criteria in vitro.

Clonal precursor of bone, cartilage, and hematopoietic niche stromal cells

PubMed Central

Chan, Charles K. F.; Lindau, Paul; Jiang, Wen; Chen, James Y.; Zhang, Lillian F.; Chen, Ching-Cheng; Seita, Jun; Sahoo, Debashis; Kim, Jae-Beom; Lee, Andrew; Park, Sujin; Nag, Divya; Gong, Yongquan; Kulkarni, Subhash; Luppen, Cynthia A.; Theologis, Alexander A.; Wan, Derrick C.; DeBoer, Anthony; Seo, Eun Young; Vincent-Tompkins, Justin D.; Loh, Kyle; Walmsley, Graham G.; Kraft, Daniel L.; Wu, Joseph C.; Longaker, Michael T.; Weissman, Irving L.

2013-01-01

Organs are composites of tissue types with diverse developmental origins, and they rely on distinct stem and progenitor cells to meet physiological demands for cellular production and homeostasis. How diverse stem cell activity is coordinated within organs is not well understood. Here we describe a lineage-restricted, self-renewing common skeletal progenitor (bone, cartilage, stromal progenitor; BCSP) isolated from limb bones and bone marrow tissue of fetal, neonatal, and adult mice. The BCSP clonally produces chondrocytes (cartilage-forming) and osteogenic (bone-forming) cells and at least three subsets of stromal cells that exhibit differential expression of cell surface markers, including CD105 (or endoglin), Thy1 [or CD90 (cluster of differentiation 90)], and 6C3 [ENPEP glutamyl aminopeptidase (aminopeptidase A)]. These three stromal subsets exhibit differential capacities to support hematopoietic (blood-forming) stem and progenitor cells. Although the 6C3-expressing subset demonstrates functional stem cell niche activity by maintaining primitive hematopoietic stem cell (HSC) renewal in vitro, the other stromal populations promote HSC differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells. PMID:23858471

Fibrin glue as the cell-delivery vehicle for mesenchymal stromal cells in regenerative medicine.

PubMed

Wu, Xiuwen; Ren, Jianan; Li, Jieshou

2012-05-01

The use of tissue-engineering techniques such as stem-cell therapy to renew injured tissues is a promising strategy in regenerative medicine. As a cell-delivery vehicle, fibrin glues (FG) facilitate cell attachment, growth and differentiation and, ultimately, tissue formation and organization by its three-dimensional structure. Numerous studies have provided evidence that stromal cells derived from bone marrow (bone marrow stromal cells; BMSC) and adipose tissue (adipose-derived stromal cells; ADSC) contain a population of adult multipotent mesenchymal stromal cells (MSC) and endothelial progenitor cells that can differentiate into several lineages. By combining MSC with FG, the implantation could take advantage of the mutual benefits. Researchers and physicians have pinned their hopes on stem cells for developing novel approaches in regenerative medicine. This review focuses on the therapeutic potential of MSC with FG in bone defect reconstruction, cartilage and tendon injury repair, ligament, heart and nerve regeneration, and, furthermore, wound healing.

Novel signatures of cancer-associated fibroblasts.

PubMed

Bozóky, Benedek; Savchenko, Andrii; Csermely, Péter; Korcsmáros, Tamás; Dúl, Zoltán; Pontén, Fredrik; Székely, László; Klein, George

2013-07-15

Increasing evidence indicates the importance of the tumor microenvironment, in particular cancer-associated fibroblasts, in cancer development and progression. In our study, we developed a novel, visually based method to identify new immunohistochemical signatures of these fibroblasts. The method employed a protein list based on 759 protein products of genes identified by RNA profiling from our previous study, comparing fibroblasts with differential growth-modulating effect on human cancers cells, and their first neighbors in the human protein interactome. These 2,654 proteins were analyzed in the Human Protein Atlas online database by comparing their immunohistochemical expression patterns in normal versus tumor-associated fibroblasts. Twelve new proteins differentially expressed in cancer-associated fibroblasts were identified (DLG1, BHLHE40, ROCK2, RAB31, AZI2, PKM2, ARHGAP31, ARHGAP26, ITCH, EGLN1, RNF19A and PLOD2), four of them can be connected to the Rho kinase signaling pathway. They were further analyzed in several additional tumor stromata and revealed that the majority showed congruence among the different tumors. Many of them were also positive in normal myofibroblast-like cells. The new signatures can be useful in immunohistochemical analysis of different tumor stromata and may also give us an insight into the pathways activated in them in their true in vivo context. The method itself could be used for other similar analysis to identify proteins expressed in other cell types in tumors and their surrounding microenvironment. Copyright © 2013 UICC.

Site-Specific Differentiation of Fibroblasts in Normal and Scleroderma Skin

DTIC Science & Technology

2010-06-01

SITE-SPECIFIC DIFFERENTIATION OF FIBROBLASTS IN NORMAL AND SCLERODERMA SKIN PRINCIPAL INVESTIGATOR: Howard Y. Chang, M.D., Ph.D...2010 4. TITLE AND SUBTITLE Site-Specific Differentiation of Fibroblasts in Normal and 5a. CONTRACT NUMBER Scleroderma Skin 5b. GRANT NUMBER...activated fibroblasts from SSc. 15. SUBJECT TERMS Scleroderma , fibroblasts, gene expression 16. SECURITY CLASSIFICATION OF: U 17. LIMITATION OF

Long pentraxin-3 as an epithelial-stromal fibroblast growth factor-targeting inhibitor in prostate cancer.

PubMed

Ronca, Roberto; Alessi, Patrizia; Coltrini, Daniela; Di Salle, Emanuela; Giacomini, Arianna; Leali, Daria; Corsini, Michela; Belleri, Mirella; Tobia, Chiara; Garlanda, Cecilia; Bonomi, Elisa; Tardanico, Regina; Vermi, William; Presta, Marco

2013-06-01

Fibroblast growth factors (FGFs) exert autocrine/paracrine functions in prostate cancer by stimulating angiogenesis and tumour growth. Here dihydrotestosterone (DHT) up-regulates FGF2 and FGF8b production in murine TRAMP-C2 prostate cancer cells, activating a FGF-dependent autocrine loop of stimulation. The soluble pattern recognition receptor long pentraxin-3 (PTX3) acts as a natural FGF antagonist that binds FGF2 and FGF8b via its N-terminal domain. We demonstrate that recombinant PTX3 protein and the PTX3-derived pentapeptide Ac-ARPCA-NH2 abolish the mitogenic response of murine TRAMP-C2 cells and human LNCaP prostate cancer cells to DHT and FGFs. Also, PTX3 hampers the angiogenic activity of DHT-activated TRAMP-C2 cells on the chick embryo chorioallantoic membrane (CAM). Accordingly, human PTX3 overexpression inhibits the mitogenic activity exerted by DHT or FGFs on hPTX3_TRAMP-C2 cell transfectants and their angiogenic activity. Also, hPTX3_TRAMP-C2 cells show a dramatic decrease of their angiogenic and tumourigenic potential when grafted in syngeneic or immunodeficient athymic male mice. A similar inhibitory effect is observed when TRAMP-C2 cells overexpress only the FGF-binding N-terminal PTX3 domain. In keeping with the anti-tumour activity of PTX3 in experimental prostate cancer, immunohistochemical analysis of prostate needle biopsies from primary prostate adenocarcinoma patients shows that parenchymal PTX3 expression, abundant in basal cells of normal glands, is lost in high-grade prostatic intraepithelial neoplasia and in invasive tumour areas. These results identify PTX3 as a potent FGF antagonist endowed with anti-angiogenic and anti-neoplastic activity in prostate cancer. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Microsporidial Stromal Keratitis: Successful Treatment with Topical Voriconazole and Oral Itraconazole

PubMed Central

Kim, James; Shenoy, Sudhir; Chévez-Barrios, Patricia; Kapur, Manuj

2016-01-01

We report a case of microsporidial stromal keratitis successfully treated with topical voriconazole and oral itraconazole. A 30-year-old Hispanic male construction worker who wears contacts lenses presented with left eye erythematous, epiphora, and mild pain increasing over few days after failing previous antibiotics treatment. His best corrected visual acuity in the left eye was count fingers at three feet, and the slit lamp examination showed 3+ conjunctival injection, a circular central corneal ulcer 3.2 mm in diameter, stromal thinning, and an anterior chamber with white cells, flair, and 0.1 mm hypopyon. A cornea punch biopsy identified microsporidial organisms with some features suggestive of Vittaforma corneae. After treatment with topical voriconazole and oral itraconazole for eight weeks, the patient had complete resolution with no recurrence for over 12 months of follow-up. To our knowledge, this is the first reported case of successful treatment of microsporidial stromal keratitis with antifungals. PMID:28123915

Inhibition of 5-alpha-reductase activity induces stromal remodeling and smooth muscle de-differentiation in adult gerbil ventral prostate.

PubMed

Corradi, Lara S; Góes, Rejane M; Carvalho, Hernandes F; Taboga, Sebastião R

2004-06-01

Prostatic differentiation during embryogenesis and its further homeostatic state maintenance during adult life depend on androgens. Dihydrotestosterone, which is synthesized from testosterone by 5 alpha-reductase (5 alpha-r), is the active molecule triggering androgen action within the prostate. In the present work, we examined the effects of 5 alpha-reductase inhibition by finasteride in the ventral prostate (VP) of the adult gerbil, employing histochemical and electron microscopy techniques to demonstrate the morphological and organizational changes of the organ. After 10 days of finasteride treatment at a dose of 100 mg/kg/day, the prostatic complex (VP and dorsolateral prostate) absolute weight was reduced to about 18%. The epithelial cells became short and cuboidal, with less secretory blebs and reduced acid phosphatase activity. The luminal sectional area diminished, suggestive of decreased secretory activity. The stromal/epithelial ratio increased, the stroma becoming thicker but less cellular. There was a striking accumulation of collagen fibrils, which was accompanied by an increase in deposits of amorphous granular material adjacent to the basal lamina and in the clefts between smooth muscle cells (SMC). Additionally, the periacinar smooth muscle became loosely packed. Some SMC were atrophic and showed a denser array of the cytoskeleton, whereas other SMC had a highly irregular outline with numerous spine-like projections. The present data indicate that 5 alpha-r inhibition causes epithelial and stromal changes by affecting intra-prostatic hormone levels. These alterations are probably the result of an imbalance of the homeostatic interaction between the epithelium and the underlying stroma.