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Sample records for adjacent stromal fibroblasts

  1. Nerve regeneration by human corneal stromal keratocytes and stromal fibroblasts

    PubMed Central

    Yam, Gary Hin-Fai; Williams, Geraint P.; Setiawan, Melina; Yusoff, Nur Zahirah Binte M.; Lee, Xiao-wen; Htoon, Hla Myint; Zhou, Lei; Fuest, Matthias; Mehta, Jodhbir S.

    2017-01-01

    Laser refractive surgeries reshape corneal stroma to correct refractive errors, but unavoidably affect corneal nerves. Slow nerve regeneration and atypical neurite morphology cause desensitization and neuro-epitheliopathy. Following injury, surviving corneal stromal keratocytes (CSKs) are activated to stromal fibroblasts (SFs). How these two different cell types influence nerve regeneration is elusive. Our study evaluated the neuro-regulatory effects of human SFs versus CSKs derived from the same corneal stroma using an in vitro chick dorsal root ganglion model. The neurite growth was assessed by a validated concentric circle intersection count method. Serum-free conditioned media (CM) from SFs promoted neurite growth dose-dependently, compared to that from CSKs. We detected neurotrophic and pro-inflammatory factors (interleukin-8, interleukin-15, monocyte chemoattractant protein-1, eotaxin, RANTES) in SFCM by Bio-Plex Human Cytokine assay. More than 130 proteins in SFCM and 49 in CSKCM were identified by nanoLC-MS/MS. Proteins uniquely present in SFCM had reported neuro-regulatory activities and were predicted to regulate neurogenesis, focal adhesion and wound healing. Conclusively, this was the first study showing a physiological relationship between nerve growth and the metabolically active SFs versus quiescent CSKs from the same cornea source. The dose-dependent effect on neurite growth indicated that nerve regeneration could be influenced by SF density. PMID:28349952

  2. Stromal Fibroblasts in Colorectal Liver Metastases Originate From Resident Fibroblasts and Generate an Inflammatory Microenvironment

    PubMed Central

    Mueller, Lars; Goumas, Freya A.; Affeldt, Marianne; Sandtner, Susanne; Gehling, Ursula M.; Brilloff, Silke; Walter, Jessica; Karnatz, Nadia; Lamszus, Katrin; Rogiers, Xavier; Broering, Dieter C.

    2007-01-01

    Cancer-associated stromal fibroblasts (CAFs) are the main cellular constituents of reactive stroma in primary and metastatic cancer. We analyzed phenotypical characteristics of CAFs from human colorectal liver metastases (CLMs) and their role in inflammation and cancer progression. CAFs displayed a vimentin+, α-smooth-muscle actin+, and Thy-1+ phenotype similar to resident portal-located liver fibroblasts (LFs). We demonstrated that CLMs are inflammatory sites showing stromal expression of interleukin-8 (IL-8), a chemokine related to invasion and angiogenesis. In vitro analyses revealed a striking induction of IL-8 expression in CAFs and LFs by tumor necrosis factor-α (TNF-α). The effect of TNF-α on CAFs is inhibited by the nuclear factor-κB inhibitor parthenolide. Conditioned medium of CAFs and LFs similarly stimulated the migration of DLD-1, Colo-678, HuH7 carcinoma cells, and human umbilical vein endothelial cells in vitro. Pretreatment of CAFs with TNF-α increased the chemotaxis of Colo-678 colon carcinoma cells by conditioned medium of CAFs; however, blockage of IL-8 activity showed no inhibitory effect. In conclusion, these data raise the possibility that the majority of CAFs in CLM originate from resident LFs. TNF-α-induced up-regulation of IL-8 via nuclear factor-κB in CAFs is an inflammatory pathway, potentially permissive for cancer invasion that may represent a novel therapeutic target. PMID:17916596

  3. Paracrine effects of uterine leucocytes on gene expression of human uterine stromal fibroblasts.

    PubMed

    Germeyer, Ariane; Sharkey, Andrew Mark; Prasadajudio, Mirari; Sherwin, Robert; Moffett, Ashley; Bieback, Karen; Clausmeyer, Susanne; Masters, Leanne; Popovici, Roxana Maria; Hess, Alexandra Petra; Strowitzki, Thomas; von Wolff, Michael

    2009-01-01

    The endometrium contains a distinct population of immune cells that undergo cyclic changes during the menstrual cycle and implantation. The majority of these leucocytes are uterine NK (uNK) cells, however how these cells interact with uterine stromal fibroblasts remains unclear. We therefore investigated the paracrine effect of medium conditioned by uterine decidual leucocytes (which are enriched for uNK cells) on the gene expression profile of endometrial stromal fibroblasts in vitro using a cDNA microarray. Our results, verified by real-time PCR, ELISA and FACS analysis, reveal that soluble factors from uterine leucocytes substantially alter endometrial stromal fibroblast gene expression. The largest group of up-regulated genes found was chemokines and cytokines. These include IL-8, CCL8 and CXCL1, which have also been shown to be stimulated by contact of stromal fibroblasts with trophoblast, suggesting that uNK cells work synergistically to support trophoblast migration during implantation. The decidual leucocytes also up-regulated IL-15 and IL-15Ralpha in stromal fibroblasts which could produce a niche for uNK cells allowing proliferation within and recruitment into the uterus, as seen in bone marrow. Overall this study demonstrates, for the first time, the paracrine communication between uterine leucocytes and uterine stromal fibroblasts, and adds to the understanding of how the uterine immune system contributes to the changes seen within the cycling endometrium.

  4. Immunohistological study of the endometrial stromal fibroblasts in the opossum, Monodelphis domestica: evidence for homology with eutherian stromal fibroblasts.

    PubMed

    Kin, Koryu; Maziarz, Jamie; Wagner, Günter P

    2014-05-01

    Molecular phylogenetic studies suggest that the hemochorial placentation and decidualization are ancestral traits of eutherian mammals. While the origin of the placental tissue is well understood, the origin of the decidual cells is unclear. Here we address the origin of decidual cells by examining the expression patterns of six transcription factors (TFs) as well as four structural proteins in the endometrium of a marsupial, Monodelphis domestica, and compared them with the patterns known from eutherian species. We found a mesenchymal cell population in the subepithelial compartment of the opossum endometrium. These cells express a set of TFs, such as homeobox A11 (HOXA11), CCAAT/enhancer-binding protein beta (CEBPB), and progesterone receptor (PGR), that are important for eutherian endometrial stromal cells. On the other hand, we did not find the expression of a decidual cell marker desmin (DES) or of TFs that are important for decidual cell differentiation, such as forkhead box O1 (FOXO1), in those cells. Based on these results, we propose that opossum has cells homologous to eutherian endometrial fibroblasts but no decidual cells. In addition, we describe cellular changes associated with the progression of pregnancy: nuclear localization of CEBPB in luminal epithelial cells as early as 8 days postcoitum, expansion of endometrial glands, nuclear localization of FOXO1 in glandular epithelial cells, and expression of smooth muscle actin in luminal epithelial cells. These data show that the luminal and glandular epithelium react to the presence of the preplacentation conceptus and suggest a limited form of pregnancy recognition.

  5. Glycolytic cancer associated fibroblasts promote breast cancer tumor growth, without a measurable increase in angiogenesis: evidence for stromal-epithelial metabolic coupling.

    PubMed

    Migneco, Gemma; Whitaker-Menezes, Diana; Chiavarina, Barbara; Castello-Cros, Remedios; Pavlides, Stephanos; Pestell, Richard G; Fatatis, Alessandro; Flomenberg, Neal; Tsirigos, Aristotelis; Howell, Anthony; Martinez-Outschoorn, Ubaldo E; Sotgia, Federica; Lisanti, Michael P

    2010-06-15

    Previously, we proposed a new model for understanding the Warburg effect in tumorigenesis and metastasis. In this model, the stromal fibroblasts would undergo aerobic glycolysis (a.k.a., the Warburg effect)--producing and secreting increased pyruvate/lactate that could then be used by adjacent epithelial cancer cells as "fuel" for the mitochondrial TCA cycle, oxidative phosphorylation, and ATP production. To test this model more directly, here we used a matched set of metabolically well-characterized immortalized fibroblasts that differ in a single gene. CL3 fibroblasts show a shift towards oxidative metabolism, and have an increased mitochondrial mass. In contrast, CL4 fibroblasts show a shift towards aerobic glycolysis, and have a reduced mitochondrial mass. We validated these differences in CL3 and CL4 fibroblasts by performing an unbiased proteomics analysis, showing the functional upregulation of 4 glycolytic enzymes, namely ENO1, ALDOA, LDHA and TPI1, in CL4 fibroblasts. Many of the proteins that were upregulated in CL4 fibroblasts, as seen by unbiased proteomics, were also transcriptionally upregulated in the stroma of human breast cancers, especially in the patients that were prone to metastasis. Importantly, when CL4 fibroblasts were co-injected with human breast cancer cells (MDA-MB-231) in a xenograft model, tumor growth was dramatically enhanced. CL4 fibroblasts induced a > 4-fold increase in tumor mass, and a near 8-fold increase in tumor volume, without any measurable increases in tumor angiogenesis. In parallel, CL3 and CL4 fibroblasts both failed to form tumors when they were injected alone, without epithelial cancer cells. Mechanistically, under co-culture conditions, CL4 glycolytic fibroblasts increased mitochondrial activity in adjacent breast cancer cells (relative to CL3 cells), consistent with the "Reverse Warburg Effect". Notably, Western blot analysis of CL4 fibroblasts revealed a significant reduction in caveolin-1 (Cav-1) protein levels

  6. Immunization of stromal cell targeting fibroblast activation protein providing immunotherapy to breast cancer mouse model.

    PubMed

    Meng, Mingyao; Wang, Wenju; Yan, Jun; Tan, Jing; Liao, Liwei; Shi, Jianlin; Wei, Chuanyu; Xie, Yanhua; Jin, Xingfang; Yang, Li; Jin, Qing; Zhu, Huirong; Tan, Weiwei; Yang, Fang; Hou, Zongliu

    2016-08-01

    Unlike heterogeneous tumor cells, cancer-associated fibroblasts (CAF) are genetically more stable which serve as a reliable target for tumor immunotherapy. Fibroblast activation protein (FAP) which is restrictively expressed in tumor cells and CAF in vivo and plays a prominent role in tumor initiation, progression, and metastasis can function as a tumor rejection antigen. In the current study, we have constructed artificial FAP(+) stromal cells which mimicked the FAP(+) CAF in vivo. We immunized a breast cancer mouse model with FAP(+) stromal cells to perform immunotherapy against FAP(+) cells in the tumor microenvironment. By forced expression of FAP, we have obtained FAP(+) stromal cells whose phenotype was CD11b(+)/CD34(+)/Sca-1(+)/FSP-1(+)/MHC class I(+). Interestingly, proliferation capacity of the fibroblasts was significantly enhanced by FAP. In the breast cancer-bearing mouse model, vaccination with FAP(+) stromal cells has significantly inhibited the growth of allograft tumor and reduced lung metastasis indeed. Depletion of T cell assays has suggested that both CD4(+) and CD8(+) T cells were involved in the tumor cytotoxic immune response. Furthermore, tumor tissue from FAP-immunized mice revealed that targeting FAP(+) CAF has induced apoptosis and decreased collagen type I and CD31 expression in the tumor microenvironment. These results implicated that immunization with FAP(+) stromal cells led to the disruption of the tumor microenvironment. Our study may provide a novel strategy for immunotherapy of a broad range of cancer.

  7. Stromal fibroblasts facilitate cancer cell invasion by a novel invadopodia-independent matrix degradation process

    PubMed Central

    Krueger, Eugene W.; Chen, Jing; Qiang, Li; McNiven, Mark A.

    2015-01-01

    Metastatic invasion of tumors into peripheral tissues is known to rely upon protease-mediated degradation of the surrounding stroma. This remodeling process utilizes complex, actin-based, specializations of the plasma membrane termed invadopodia that act both to sequester and release matrix metalloproteinases. Here we report that cells of mesenchymal origin, including tumor-associated fibroblasts, degrade substantial amounts of surrounding matrix by a mechanism independent of conventional invadopodia. These degradative sites lack the punctate shape of conventional invadopodia to spread along the cell base and are reticular and/or fibrous in character. In marked contrast to invadopodia, this degradation does not require the action of Src kinase, Cdc42, or Dyn2. Rather, inhibition of Dyn2 causes a dramatic upregulation of stromal matrix degradation. Further, expression and activity of matrix metalloproteinases are differentially regulated between tumor cells and stromal fibroblasts. This matrix remodeling by fibroblasts increases the invasive capacity of tumor cells, thereby illustrating how the tumor microenvironment can contribute to metastasis. These findings provide evidence for a novel matrix remodeling process conducted by stromal fibroblasts that is substantially more effective than conventional invadopodia, distinct in structural organization, and regulated by disparate molecular mechanisms. PMID:25982272

  8. Mucosal stromal fibroblasts markedly enhance HIV infection of CD4+ T cells

    PubMed Central

    Kohgadai, Nargis; Müller, Janis A.; Laustsen, Anders; Thavachelvam, Karthiga; Stürzel, Christina M.; Jones, Jennifer J.; Somsouk, Ma; Garcia, Maurice M.; Smith, James F.; Greenblatt, Ruth M.; Münch, Jan; Jakobsen, Martin R.; Giudice, Linda C.; Greene, Warner C.; Roan, Nadia R.

    2017-01-01

    Understanding early events of HIV transmission within mucosal tissues is vital for developing effective prevention strategies. Here, we report that primary stromal fibroblasts isolated from endometrium, cervix, foreskin, male urethra, and intestines significantly increase HIV infection of CD4+ T cells–by up to 37-fold for R5-tropic HIV and 100-fold for X4-tropic HIV–without themselves becoming infected. Fibroblasts were more efficient than dendritic cells at trans-infection and mediate this response in the absence of the DC-SIGN and Siglec-1 receptors. In comparison, mucosal epithelial cells secrete antivirals and inhibit HIV infection. These data suggest that breaches in the epithelium allow external or luminal HIV to escape an antiviral environment to access the infection-favorable environment of the stromal fibroblasts, and suggest that resident fibroblasts have a central, but previously unrecognized, role in HIV acquisition at mucosal sites. Inhibiting fibroblast-mediated enhancement of HIV infection should be considered as a novel prevention strategy. PMID:28207890

  9. Stromal Fibroblasts in Tertiary Lymphoid Structures: A Novel Target in Chronic Inflammation

    PubMed Central

    Barone, Francesca; Gardner, David H.; Nayar, Saba; Steinthal, Nathalie; Buckley, Christopher D.; Luther, Sanjiv A.

    2016-01-01

    Tertiary lymphoid structures (TLS) are organized aggregates of lymphocytes, myeloid, and stromal cells that provide ectopic hubs for acquired immune responses. TLS share phenotypical and functional features with secondary lymphoid organs (SLO); however, they require persistent inflammatory signals to arise and are often observed at target sites of autoimmune disease, chronic infection, cancer, and organ transplantation. Over the past 10 years, important progress has been made in our understanding of the role of stromal fibroblasts in SLO development, organization, and function. A complex and stereotyped series of events regulate fibroblast differentiation from embryonic life in SLOs to lymphoid organ architecture observed in adults. In contrast, TLS-associated fibroblasts differentiate from postnatal, locally activated mesenchyme, predominantly in settings of inflammation and persistent antigen presentation. Therefore, there are critical differences in the cellular and molecular requirements that regulate SLO versus TLS development that ultimately impact on stromal and hematopoietic cell function. These differences may contribute to the pathogenic nature of TLS in the context of chronic inflammation and malignant transformation and offer a window of opportunity for therapeutic interventions in TLS associated pathologies. PMID:27877173

  10. Marrow stromal fibroblastic cell cultivation in vitro on decellularized bone marrow extracellular matrix.

    PubMed

    Dutra, Timothy F; French, Samuel W

    2010-02-01

    The in vitro biocompatibility of decellularized bone marrow extracellular matrix was evaluated. Following a freeze-thaw cycle, sectioned discs of fresh frozen rat metaphyseal bone were sequentially incubated in solutions of hypertonic, then hypotonic Ringer's solution, followed by deoxycholic acid, then DNAase I. The adequacy of decellularization of marrow stroma was examined by light microscopy. Marrow stromal fibroblastic cells were harvested by dispersion of rat long bone marrow, followed by concentration by discontinuous Ficoll-Paque gradient centrifugation. The fibroblastic cells were expanded by in vitro cultivation, and second passage cells were cryopreserved until needed. Cryopreserved marrow stromal cells were applied dropwise to sections of decellularized bone marrow extracellular matrix, and cultured in BJGb medium with 20% fetal bovine serum for ten days. Mature cultures were formalin fixed, decalcified, and embedded in paraffin. Light microscopy of hematoxylin and eosin stained sections showed individual spindle cells invading the upper portion of the decellularized extracellular matrix, and also a monolayer of spindle cells on the upper surfaces of exposed trabecular and cortical bone. This experiment showed that decellularized marrow extracellular matrix is a biocompatible three dimensional in vitro substrate for marrow stromal fibroblastic cells.

  11. Stromal Fibroblasts in Tertiary Lymphoid Structures: A Novel Target in Chronic Inflammation.

    PubMed

    Barone, Francesca; Gardner, David H; Nayar, Saba; Steinthal, Nathalie; Buckley, Christopher D; Luther, Sanjiv A

    2016-01-01

    Tertiary lymphoid structures (TLS) are organized aggregates of lymphocytes, myeloid, and stromal cells that provide ectopic hubs for acquired immune responses. TLS share phenotypical and functional features with secondary lymphoid organs (SLO); however, they require persistent inflammatory signals to arise and are often observed at target sites of autoimmune disease, chronic infection, cancer, and organ transplantation. Over the past 10 years, important progress has been made in our understanding of the role of stromal fibroblasts in SLO development, organization, and function. A complex and stereotyped series of events regulate fibroblast differentiation from embryonic life in SLOs to lymphoid organ architecture observed in adults. In contrast, TLS-associated fibroblasts differentiate from postnatal, locally activated mesenchyme, predominantly in settings of inflammation and persistent antigen presentation. Therefore, there are critical differences in the cellular and molecular requirements that regulate SLO versus TLS development that ultimately impact on stromal and hematopoietic cell function. These differences may contribute to the pathogenic nature of TLS in the context of chronic inflammation and malignant transformation and offer a window of opportunity for therapeutic interventions in TLS associated pathologies.

  12. Tissue engineering of corneal stromal layer with dermal fibroblasts: phenotypic and functional switch of differentiated cells in cornea.

    PubMed

    Zhang, Yan Qing; Zhang, Wen Jie; Liu, Wei; Hu, Xiao Jie; Zhou, Guang Dong; Cui, Lei; Cao, Yilin

    2008-02-01

    Previously, we successfully engineered a corneal stromal layer using corneal stromal cells. However, the limited source and proliferation potential of corneal stromal cells has driven us to search for alternative cell sources for corneal stroma engineering. Based on the idea that the tissue-specific environment may alter cell fate, we proposed that dermal fibroblasts could switch their phenotype to that of corneal stromal cells in the corneal environment. Thus, dermal fibroblasts were harvested from newborn rabbits, seeded on biodegradable polyglycolic acid (PGA) scaffolds, cultured in vitro for 1 week, and then implanted into adult rabbit corneas. After 8 weeks of implantation, nearly transparent corneal stroma was formed, with a histological structure similar to that of its native counterpart. The existence of cells that had been retrovirally labeled with green fluorescence protein (GFP) demonstrated the survival of implanted cells. In addition, all GFP-positive cells that survived expressed keratocan, a specific marker for corneal stromal cells, and formed fine collagen fibrils with a highly organized pattern similar to that of native stroma. However, neither dermal fibroblast-PGA construct pre-incubated in vitro for 3 weeks nor chondrocyte-PGA construct could form transparent stroma. The results demonstrated that neonatal dermal fibroblasts could switch their phenotype in the new tissue environment under restricted conditions. The functional restoration of corneal transparency using dermal fibroblasts suggests that they could be an alternative cell source for corneal stroma engineering.

  13. Evidence for a stromal-epithelial "lactate shuttle" in human tumors: MCT4 is a marker of oxidative stress in cancer-associated fibroblasts.

    PubMed

    Whitaker-Menezes, Diana; Martinez-Outschoorn, Ubaldo E; Lin, Zhao; Ertel, Adam; Flomenberg, Neal; Witkiewicz, Agnieszka K; Birbe, Ruth C; Howell, Anthony; Pavlides, Stephanos; Gandara, Ricardo; Pestell, Richard G; Sotgia, Federica; Philp, Nancy J; Lisanti, Michael P

    2011-06-01

    Recently, we proposed a new mechanism for understanding the Warburg effect in cancer metabolism. In this new paradigm, cancer-associated fibroblasts undergo aerobic glycolysis, and extrude lactate to "feed" adjacent cancer cells, which then drives mitochondrial biogenesis and oxidative mitochondrial metabolism in cancer cells. Thus, there is vectorial transport of energy-rich substrates from the fibroblastic tumor stroma to anabolic cancer cells. A prediction of this hypothesis is that cancer-associated fibroblasts should express MCT4, a mono-carboxylate transporter that has been implicated in lactate efflux from glycolytic muscle fibers and astrocytes in the brain. To address this issue, we co-cultured MCF7 breast cancer cells with normal fibroblasts. Interestingly, our results directly show that breast cancer cells specifically induce the expression of MCT4 in cancer-associated fibroblasts; MCF7 cells alone and fibroblasts alone, both failed to express MCT4. We also show that the expression of MCT4 in cancer-associated fibroblasts is due to oxidative stress, and can be prevented by pre-treatment with the anti-oxidant N-acetyl-cysteine. In contrast to our results with MCT4, we see that MCT1, a transporter involved in lactate uptake, is specifically upregulated in MCF7 breast cancer cells when co-cultured with fibroblasts. Virtually identical results were also obtained with primary human breast cancer samples. In human breast cancers, MCT4 selectively labels the tumor stroma, e.g., the cancer-associated fibroblast compartment. Conversely, MCT1 was selectively expressed in the epithelial cancer cells within the same tumors. Functionally, we show that overexpression of MCT4 in fibroblasts protects both MCF7 cancer cells and fibroblasts against cell death, under co-culture conditions. Thus, we provide the first evidence for the existence of a stromal-epithelial lactate shuttle in human tumors, analogous to the lactate shuttles that are essential for the normal

  14. Human foreskin fibroblast-like stromal cells can differentiate into functional hepatocytic cells.

    PubMed

    Huang, Hsing-I; Chen, Shao-Kuan; Wang, Robert Y-L; Shen, Chia-Rui; Cheng, Yu-Che

    2013-12-01

    Foreskin fibroblast-like stromal cells (FDSCs) are progenitors isolated from human tissue that can differentiate into diverse cell types. Many types of stem cells can differentiate into hepatocyte-like cells, which could be used for drug testing or in liver regeneration therapy, but whether FDSCs can be converted into functional hepatocytes is unknown. FDSCs show divergent properties when cultured in distinct media, forming spheres in Dulbecco's modified Eagle's medium (DMEM) containing F12, epidermal growth factor (EGF), and basic fibroblast growth factor (b-FGF), but have fibroblast-like morphology when cultured in DMEM-based growth medium. Both cell populations express the typical mesenchymal stem cell markers CD90, CD105, and CD73, but the p75 neurotrophin receptor (p75NTR) was detected only in FDSC spheres. Both types of FDSCs can differentiate into hepatocyte-like cells, which express typical liver markers, including albumin and hepatocyte paraffin 1 (Hep Par1), along with liver-specific biological activities. When plasmids containing the human hepatitis B virus (HBV) genome were transfected transiently into FDSCs, differentiated hepatocyte-like cells secrete large amounts of HBe and HBs antigens. FDSCs could be used for clinical hepatic therapy and/or serve as a model of HBV.

  15. Stromal fibroblasts support dendritic cells to maintain IL-23/Th17 responses after exposure to ionizing radiation

    PubMed Central

    Malecka, Anna; Wang, Qunwei; Shah, Sabaria; Sutavani, Ruhcha V.; Spendlove, Ian; Ramage, Judith M.; Greensmith, Julie; Franks, Hester A.; Gough, Michael J.; Saalbach, Anja; Patel, Poulam M.; Jackson, Andrew M.

    2016-01-01

    Dendritic cell function is modulated by stromal cells, including fibroblasts. Although poorly understood, the signals delivered through this crosstalk substantially alter dendritic cell biology. This is well illustrated with release of TNF-α/IL-1β from activated dendritic cells, promoting PGE2 secretion from stromal fibroblasts. This instructs dendritic cells to up-regulate IL-23, a key Th17-polarizing cytokine. We previously showed that ionizing radiation inhibited IL-23 production by human dendritic cells in vitro. In the present study, we investigated the hypothesis that dendritic cell-fibroblast crosstalk overcomes the suppressive effect of ionizing radiation to support appropriately polarized Th17 responses. Radiation (1–6 Gy) markedly suppressed IL-23 secretion by activated dendritic cells (P < 0.0001) without adversely impacting their viability and consequently, inhibited the generation of Th17 responses. Cytokine suppression by ionizing radiation was selective, as there was no effect on IL-1β, -6, -10, and -27 or TNF-α and only a modest (11%) decrease in IL-12p70 secretion. Coculture with fibroblasts augmented IL-23 secretion by irradiated dendritic cells and increased Th17 responses. Importantly, in contrast to dendritic cells, irradiated fibroblasts maintained their capacity to respond to TNF-α/IL-1β and produce PGE2, thus providing the key intermediary signals for successful dendritic cell-fibroblasts crosstalk. In summary, stromal fibroblasts support Th17-polarizing cytokine production by dendritic cells that would otherwise be suppressed in an irradiated microenvironment. This has potential ramifications for understanding the immune response to local radiotherapy. These findings underscore the need to account for the impact of microenvironmental factors, including stromal cells, in understanding the control of immunity. PMID:27049023

  16. Melanoma Cells Can Adopt the Phenotype of Stromal Fibroblasts and Macrophages by Spontaneous Cell Fusion in Vitro.

    PubMed

    Kemény, Lajos V; Kurgyis, Zsuzsanna; Buknicz, Tünde; Groma, Gergely; Jakab, Ádám; Zänker, Kurt; Dittmar, Thomas; Kemény, Lajos; Németh, István B

    2016-06-02

    After the removal of primary cutaneous melanoma some patients develop local recurrences, even after having histologically tumor-free re-excision. A potential explanation behind this phenomenon is that tumor cells switch their phenotype, making their recognition via standard histopathological assessments extremely difficult. Tumor-stromal cell fusion has been proposed as a potential mechanism for tumor cells to acquire mesenchymal traits; therefore, we hypothesized that melanoma cells could acquire fibroblast- and macrophage-like phenotypes via cell fusion. We show that melanoma cells spontaneously fuse with human dermal fibroblasts and human peripheral blood monocytes in vitro. The hybrid cells' nuclei contain chromosomes from both parental cells and are indistinguishable from the parental fibroblasts or macrophages based on their morphology and immunophenotype, as they could lose the melanoma specific MART1 marker, but express the fibroblast marker smooth muscle actin or the macrophage marker CD68. Our results suggest that, by spontaneous cell fusion in vitro, tumor cells can adopt the morphology and immunophenotype of stromal cells while still carrying oncogenic, tumor-derived genetic information. Therefore, melanoma-stromal cell fusion might play a role in missing tumor cells by routine histopathological assessments.

  17. Melanoma Cells Can Adopt the Phenotype of Stromal Fibroblasts and Macrophages by Spontaneous Cell Fusion in Vitro

    PubMed Central

    Kemény, Lajos V.; Kurgyis, Zsuzsanna; Buknicz, Tünde; Groma, Gergely; Jakab, Ádám; Zänker, Kurt; Dittmar, Thomas; Kemény, Lajos; Németh, István B.

    2016-01-01

    After the removal of primary cutaneous melanoma some patients develop local recurrences, even after having histologically tumor-free re-excision. A potential explanation behind this phenomenon is that tumor cells switch their phenotype, making their recognition via standard histopathological assessments extremely difficult. Tumor-stromal cell fusion has been proposed as a potential mechanism for tumor cells to acquire mesenchymal traits; therefore, we hypothesized that melanoma cells could acquire fibroblast- and macrophage-like phenotypes via cell fusion. We show that melanoma cells spontaneously fuse with human dermal fibroblasts and human peripheral blood monocytes in vitro. The hybrid cells’ nuclei contain chromosomes from both parental cells and are indistinguishable from the parental fibroblasts or macrophages based on their morphology and immunophenotype, as they could lose the melanoma specific MART1 marker, but express the fibroblast marker smooth muscle actin or the macrophage marker CD68. Our results suggest that, by spontaneous cell fusion in vitro, tumor cells can adopt the morphology and immunophenotype of stromal cells while still carrying oncogenic, tumor-derived genetic information. Therefore, melanoma–stromal cell fusion might play a role in missing tumor cells by routine histopathological assessments. PMID:27271591

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

    SciTech Connect

    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-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}.

  19. Pyruvate kinase expression (PKM1 and PKM2) in cancer-associated fibroblasts drives stromal nutrient production and tumor growth.

    PubMed

    Chiavarina, Barbara; Whitaker-Menezes, Diana; Martinez-Outschoorn, Ubaldo E; Witkiewicz, Agnieszka K; Birbe, Ruth; Howell, Anthony; Pestell, Richard G; Smith, Johanna; Daniel, Rene; Sotgia, Federica; Lisanti, Michael P

    2011-12-15

    We have previously demonstrated that enhanced aerobic glycolysis and/or autophagy in the tumor stroma supports epithelial cancer cell growth and aggressive behavior, via the secretion of high-energy metabolites. These nutrients include lactate and ketones, as well as chemical building blocks, such as amino acids (glutamine) and nucleotides. Lactate and ketones serve as fuel for cancer cell oxidative metabolism, and building blocks sustain the anabolic needs of rapidly proliferating cancer cells. We have termed these novel concepts the "Reverse Warburg Effect," and the "Autophagic Tumor Stroma Model of Cancer Metabolism." We have also identified a loss of stromal caveolin-1 (Cav-1) as a marker of stromal glycolysis and autophagy. The aim of the current study was to provide genetic evidence that enhanced glycolysis in stromal cells favors tumorigenesis. To this end, normal human fibroblasts were genetically-engineered to express the two isoforms of pyruvate kinase M (PKM1 and PKM2), a key enzyme in the glycolytic pathway. In a xenograft model, fibroblasts expressing PKM1 or PKM2 greatly promoted the growth of co-injected MDA-MB-231 breast cancer cells, without an increase in tumor angiogenesis. Interestingly, PKM1 and PKM2 promoted tumorigenesis by different mechanism(s). Expression of PKM1 enhanced the glycolytic power of stromal cells, with increased output of lactate. Analysis of tumor xenografts demonstrated that PKM1 fibroblasts greatly induced tumor inflammation, as judged by CD45 staining. In contrast, PKM2 did not lead to lactate accumulation, but triggered a "pseudo-starvation" response in stromal cells, with induction of an NFκB-dependent autophagic program, and increased output of the ketone body 3-hydroxy-buryrate. Strikingly, in situ evaluation of Complex IV activity in the tumor xenografts demonstrated that stromal PKM2 expression drives mitochondrial respiration specifically in tumor cells. Finally, immuno-histochemistry analysis of human breast

  20. Zoledronic acid impairs stromal reactivity by inhibiting M2-macrophages polarization and prostate cancer-associated fibroblasts

    PubMed Central

    Comito, Giuseppina; Segura, Coral Pons; Taddei, Maria Letizia; Lanciotti, Michele; Serni, Sergio; Morandi, Andrea; Chiarugi, Paola; Giannoni, Elisa

    2017-01-01

    Zoledronic acid (ZA) is a biphosphonate used for osteoporosis treatment and also proved to be effective to reduce the pain induced by bone metastases when used as adjuvant therapy in solid cancers. However, it has been recently proposed that ZA could have direct anti-tumour effects, although the molecular mechanism is unknown. We herein unravel a novel anti-tumour activity of ZA in prostate cancer (PCa), by targeting the pro-tumorigenic properties of both stromal and immune cells. Particularly, we demonstrate that ZA impairs PCa-induced M2-macrophages polarization, reducing their pro-invasive effect on tumour cells and their pro-angiogenic features. Crucially, ZA administration reverts cancer associated fibroblasts (CAFs) activation by targeting the mevalonate pathway and RhoA geranyl-geranylation, thereby impairing smooth muscle actin-α fibers organization, a prerequisite of fibroblast activation. Moreover, ZA prevents the M2 macrophages-mediated activation of normal fibroblast, highlighting the broad efficacy of this drug on tumour microenvironment. These results are confirmed in a metastatic xenograft PCa mouse model in which ZA-induced stromal normalization impairs cancer-stromal cells crosstalk, resulting in a significant reduction of primary tumour growth and metastases. Overall these findings reinforce the efficacy of ZA as a potential therapeutic approach to reduce cancer aggressiveness, by abrogating the supportive role of tumour microenvironment. PMID:27223431

  1. Enhancement of terminal B lymphocyte differentiation in vitro by fibroblast-like stromal cells from human spleen.

    PubMed

    Skibinski, G; Skibinska, A; Stewart, G D; James, K

    1998-12-01

    Stromal elements are major components of lymphoid tissues contributing to both tissue architecture and function. In this study we report on the phenotype and function of fibroblast-like stromal cells obtained from human spleen. These cells express high levels of CD44 and ICAM-1 and moderate levels of VLA-4, VCAM, CD40 and CD21. They fail to express endothelial, epithelial, lymphocyte and monocyte/macrophage markers. We show that these cells interact with B cell blasts induced in vitro by anti-CD40 and anti-mu stimulation. As a result of these interactions both IL-6 and IgG secretion into culture medium is increased. The enhanced secretion of IgG is partly inhibited by abolishing B cell blaststromal cell contact or by anti-IL-6, anti-VCAM or anti-CD49d antibodies. Our studies also suggest that the ability of stromal cells to promote B cell survival is most likely the underlying mechanism of the enhanced immunoglobulin secretion. Comparison of stromal cells from different lymphoid and non-lymphoid organs revealed that bone marrow- and spleen-derived stromal cells are the most effective in promoting B cell blast differentiation.

  2. Antioxidant function of corneal ALDH3A1 in cultured stromal fibroblasts.

    PubMed

    Lassen, Natalie; Pappa, Aglaia; Black, William J; Jester, James V; Day, Brian J; Min, Elysia; Vasiliou, Vasilis

    2006-11-01

    Aldehyde dehydrogenase 3A1 (ALDH3A1) is highly expressed in epithelial cells and stromal keratocytes of mammalian cornea and is believed to play an important role in cellular defense. To explore a potential protective role against oxidative damage, a rabbit corneal fibroblastic cell line (TRK43) was stably transfected with the human ALDH3A1 and subjected to oxidative stress induced by H(2)O(2), mitomycin C (MMC), or etoposide (VP-16). ALDH3A1-transfected cells were more resistant to H(2)O(2,) MMC, and VP-16 compared to the vector-transfected cells. All treatments induced apoptosis only in vector-transfected cells, which was associated with increased levels of 4-hydroxy-2-nonenal (4-HNE)-adducted proteins. Treatment with H(2)O(2) resulted in a rise in reduced glutathione (GSH) levels in all groups but was more pronounced in the ALDH3A1-expressing cells. Treatment with the DNA-damaging agents led to GSH depletion in control groups, although the depletion was significantly less in ALDH3A1-expressing cells. Increased carbonylation of ALDH3A1 but not significant decline in enzymatic activity was observed after all treatments. In conclusion, our results suggest that ALDH3A1 may act to protect corneal cells against cellular oxidative damage by metabolizing toxic lipid peroxidation products (e.g., 4-HNE), maintaining cellular GSH levels and redox balance, and operating as an antioxidant.

  3. TGF-β in jaw tumor fluids induces RANKL expression in stromal fibroblasts

    PubMed Central

    Yamada, Chiaki; Aikawa, Tomonao; Okuno, Emi; Miyagawa, Kazuaki; Amano, Katsuhiko; Takahata, Sosuke; Kimata, Masaaki; Okura, Masaya; Iida, Seiji; Kogo, Mikihiko

    2016-01-01

    Odontogenic tumors and cysts, arising in the jawbones, grow by resorption and destruction of the jawbones. However, mechanisms underlying bone resorption by odontogenic tumors/cysts remain unclear. Odontogenic tumors/cysts comprise odontogenic epithelial cells and stromal fibroblasts, which originate from the developing tooth germ. It has been demonstrated that odontogenic epithelial cells of the developing tooth germ induce osteoclastogenesis to prevent the tooth germ from invading the developing bone to maintain its structure in developing bones. Thus, we hypothesized that odontogenic epithelial cells of odontogenic tumors/cysts induce osteoclast formation, which plays potential roles in tumor/cyst outgrowth into the jawbone. The purpose of this study was to examine osteoclastogenesis by cytokines, focusing on transforming growth factor-β (TGF-β), produced by odontogenic epithelial cells. We observed two pathways for receptor activator of NF-κB ligand (RANKL) induction by keratocystic odontogenic tumor fluid: the cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) pathway through interleukin-1α (IL-1α) signaling and non-COX-2/PGE2 pathway through TGF-β receptor signaling. TGF-β1 and IL-1α produced by odontogenic tumors/cysts induced osteoclastogenesis directly in the osteoclast precursor cells and indirectly via increased RANKL induction in the stroma. PMID:27279422

  4. The role of stromal cancer-associated fibroblasts in pancreatic cancer.

    PubMed

    von Ahrens, Dagny; Bhagat, Tushar D; Nagrath, Deepak; Maitra, Anirban; Verma, Amit

    2017-03-28

    Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer generally refractory to conventional treatments. Cancer-associated fibroblasts (CAFs) are cellular components of the desmoplastic stroma characteristic to the tumor that contributes to this treatment resistance. Various markers for CAFs have been explored including palladin and CD146 that have prognostic and functional roles in the pathobiology of PDAC. Mechanisms of CAF-tumor cell interaction have been described including exosomal transfer and paracrine signaling mediated by cytokines such as GM-CSF and IL-6. The role of downstream signaling pathways including JAK/STAT, mTOR, sonic hedge hog (SHH), and NFkB have also been shown to play an important function in PDAC-CAF cross talk. The role of autophagy and other metabolic effects on each cell type within the tumor have also been proposed to play roles in facilitating CAF secretory function and enhancing tumor growth in a low-glucose microenvironment. Targeting the stroma has gained interest with multiple preclinical and clinical trials targeting SHH, JAK2, and methods of either exploiting the secretory capability of CAFs to enhance drug delivery or inhibiting it to prevent its influence on cancer cell chemoresistance. This review summarizes the most recent progress made in understanding stromal formation; its contribution to tumor proliferation, invasion, and metastasis; its role in chemoresistance; and potential therapeutic strategies on the horizon.

  5. 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

  6. 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.

  7. Differences of cell surface marker expression between bone marrow- and kidney-derived murine mesenchymal stromal cells and fibroblasts.

    PubMed

    Cakiroglu, F; Osbahr, J W; Kramer, J; Rohwedel, J

    2016-10-31

    Mesenchymal stromal cells (MSC) are undifferentiated, multipotent adult cells with regenerative properties. They are particularly relevant for therapeutic approaches due to the simplicity of their isolation and cultivation. Since MSC show an expression pattern of cell surface marker, which is almost identical to fibroblasts, many attempts have been made to address the similarities and differences between MSC and fibroblasts. In this study we aimed to isolate murine MSC from bone marrow (BM) and kidney to characterize them in comparison to fibroblasts. Cells were isolated from murine kidney, BM and abdominal skin by plastic adherence and subsequently characterized by analysing their capability to build colony-forming unit-fibroblasts (CFU-F), their morphology, their proliferation, expression of telomerase activity and cell surface antigens as well as their differentiation capacity. Plastic adherent cells from the 3 mouse tissues showed similar morphology, proliferation profiles and CFU-F building capacities. However, while MSC from BM and kidney differentiated into the adipogenic, chondrogenic and osteogenic direction, fibroblasts were not able to do so efficiently. In addition, a tendency for lower expression of telomerase was found in the fibroblast population. Proliferating cells from kidney and BM expressed the MSC-specific cell surface markers CD105 and Sca-1 on a significantly higher and CD117 on a significantly lower level compared to fibroblasts and were thereby distinguishable from fibroblasts. Furthermore, we found that certain CD markers were specifically expressed on a higher level, either in BM-derived cells or fibroblasts. This study demonstrates that murine MSC isolated from different organs express certain specific markers, which enable their discrimination.

  8. The Transcriptomic Evolution of Mammalian Pregnancy: Gene Expression Innovations in Endometrial Stromal Fibroblasts

    PubMed Central

    Kin, Koryu; Maziarz, Jamie; Chavan, Arun R.; Kamat, Manasi; Vasudevan, Sreelakshmi; Birt, Alyssa; Emera, Deena; Lynch, Vincent J.; Ott, Troy L.; Pavlicev, Mihaela; Wagner, Günter P.

    2016-01-01

    The endometrial stromal fibroblast (ESF) is a cell type present in the uterine lining of therian mammals. In the stem lineage of eutherian mammals, ESF acquired the ability to differentiate into decidual cells in order to allow embryo implantation. We call the latter cell type “neo-ESF” in contrast to “paleo-ESF” which is homologous to eutherian ESF but is not able to decidualize. In this study, we compare the transcriptomes of ESF from six therian species: Opossum (Monodelphis domestica; paleo-ESF), mink, rat, rabbit, human (all neo-ESF), and cow (secondarily nondecidualizing neo-ESF). We find evidence for strong stabilizing selection on transcriptome composition suggesting that the expression of approximately 5,600 genes is maintained by natural selection. The evolution of neo-ESF from paleo-ESF involved the following gene expression changes: Loss of expression of genes related to inflammation and immune response, lower expression of genes opposing tissue invasion, increased markers for proliferation as well as the recruitment of FOXM1, a key gene transiently expressed during decidualization. Signaling pathways also evolve rapidly and continue to evolve within eutherian lineages. In the bovine lineage, where invasiveness and decidualization were secondarily lost, we see a re-expression of genes found in opossum, most prominently WISP2, and a loss of gene expression related to angiogenesis. The data from this and previous studies support a scenario, where the proinflammatory paleo-ESF was reprogrammed to express anti-inflammatory genes in response to the inflammatory stimulus coming from the implanting conceptus and thus paving the way for extended, trans-cyclic gestation. PMID:27401177

  9. CD34+ stromal cells/fibroblasts/fibrocytes/telocytes as a tissue reserve and a principal source of mesenchymal cells. Location, morphology, function and role in pathology.

    PubMed

    Díaz-Flores, L; Gutiérrez, R; García, M P; Sáez, F J; Díaz-Flores, L; Valladares, F; Madrid, J F

    2014-07-01

    We review the morphofunctional characteristics of CD34+ stromal fibroblastic/fibrocytic cells (CD34+ SFCs) and report our observations. We consider the following aspects of CD34+ SFCs: A) The confusing terms applied to this cell type, often combining the prefix CD34 with numerous names, including fibroblasts, fibrocytes, dendrocytes, keratocytes, telocytes and stromal, dendritic, adventitial, supraadventitial, perivascular, paravascular and delimiting cells; B) Changes in their immunophenotype, e.g., loss of CD34 expression and gain of other markers, such as those defining mesenchymal and derivate cells (myofibroblasts, osteoblasts, chondroblasts, adipocytes); C) Morphology (elongated or triangular cell body and thin, moniliform, bipolar or multipolar cytoplasmic processes), immunohistochemistry (co-expression of and changes in molecular expression) and structure (characteristics of nucleus and cytoplasmic organelles, and points of contact and junctions in quiescent and activated stages by light and electron microscopy); D) Location and distribution in the vessels (adventitia or external layer), in the tissues (connective, adipose, blood, muscle and nervous) and in the organs and systems (skin, oral cavity and oropharynx, respiratory, digestive, urinary, male, female, endocrine and lymphoid systems, serosal and synovial membranes, heart, eye and meninges); E) Origin from the mesoderm and cranial neural crest in the embryo, and from stem cells (themselves or other cells) and/or peripheral blood pluripotent stem cells (circulating progenitor cells) in post-natal life; F) Functions, such as synthesis of different molecules, progenitor of mesenchymal cells, immunomodulation, parenchymal regulation (growth, maturation and differentiation of adjacent cells), induction of angiogenesis, scaffolding support of other cells and phagocytic properties. Since CD34+ SFCs are the main reservoir of tissue mesenchymal cells (great mesenchymal potential, probably higher than that

  10. The stromal cell-surface protease fibroblast activation protein-α localizes to lipid rafts and is recruited to invadopodia.

    PubMed

    Knopf, Julia D; Tholen, Stefan; Koczorowska, Maria M; De Wever, Olivier; Biniossek, Martin L; Schilling, Oliver

    2015-10-01

    Fibroblast activation protein alpha (FAPα) is a cell surface protease expressed by cancer-associated fibroblasts in the microenvironment of most solid tumors. As there is increasing evidence for proteases having non-catalytic functions, we determined the FAPα interactome in cancer-associated fibroblasts using the quantitative immunoprecipitation combined with knockdown (QUICK) method. Complex formation with adenosin deaminase, erlin-2, stomatin, prohibitin, Thy-1 membrane glycoprotein, and caveolin-1 was further validated by immunoblotting. Co-immunoprecipitation (co-IP) of the known stoichiometric FAPα binding partner dipeptidyl-peptidase IV (DPPIV) corroborated the proteomic strategy. Reverse co-IPs validated the FAPα interaction with caveolin-1, erlin-2, and stomatin while co-IP upon RNA-interference mediated knock-down of DPPIV excluded adenosin deaminase as a direct FAPα interaction partner. Many newly identified FAPα interaction partners localize to lipid rafts, including caveolin-1, a widely-used marker for lipid raft localization. We hypothesized that this indicates a recruitment of FAPα to lipid raft structures. In density gradient centrifugation, FAPα co-fractionates with caveolin-1. Immunofluorescence optical sectioning microscopy of FAPα and lipid raft markers further corroborates recruitment of FAPα to lipid rafts and invadopodia. FAPα is therefore an integral component of stromal lipid rafts in solid tumors. In essence, we provide one of the first interactome analyses of a cell surface protease and translate these results into novel biological aspects of a marker protein for cancer-associated fibroblasts.

  11. Coculturing human endometrial epithelial cells and stromal fibroblasts alters cell-specific gene expression and cytokine production

    PubMed Central

    Chen, Joseph C.; Erikson, David W.; Piltonen, Terhi T.; Meyer, Michelle R.; Barragan, Fatima; McIntire, Ramsey H.; Tamaresis, John S.; Vo, Kim Chi; Giudice, Linda C.; Irwin, Juan C.

    2013-01-01

    Objective To determine the effects of coculturing endometrial epithelial cells (eEC) with paired endometrial stromal fibroblasts (eSF) on cell-specific gene expression and cytokine secretion patterns. Design In vitro study. Setting University research laboratory. Patient(s) Endometrial biopsies were obtained from premenopausal women. Intervention(s) Polarized eEC and subject-paired eSF were cultured for 12.5 hours alone (monoculture) or combined in a two-chamber coculture system without cell-cell contact. Cells and conditioned media were analyzed for global gene expression and cytokine secretion, respectively. Purified, endometrial tissue-derived eEC and eSF isolated by fluorescent activated cell sorting (FACS) were used as noncultured controls. Main Outcome Measure(s) Cell-specific global gene expression profiling and analysis of secreted cytokines in eEC/eSF cocultures and respective monocultures. Result(s) Transepithelial resistance, diffusible tracer exclusion, expression of tight junction proteins, and apical/basolateral vectorial secretion confirmed eEC structural and functional polarization. Distinct transcriptomes of eEC and eSF were consistent with their respective lineages and their endometrial origin. Coculture of eEC with eSF resulted in altered cell-specific gene expression and cytokine secretion. Conclusion(s) This coculture model provides evidence that interactions between endometrial functionally polarized epithelium and stromal fibroblasts affect cell-specific gene expression and cytokine secretion underscoring their relevance when modeling endometrium in vitro. PMID:23849844

  12. Regulation of IL-6 and IL-8 production by reciprocal cell-to-cell interactions between tumor cells and stromal fibroblasts through IL-1α in ameloblastoma

    SciTech Connect

    Fuchigami, Takao; Kibe, Toshiro; Koyama, Hirofumi; Kishida, Shosei; Iijima, Mikio; Nishizawa, Yoshiaki; Hijioka, Hiroshi; Fujii, Tomomi; Ueda, Masahiro; Nakamura, Norifumi; Kiyono, Tohru; Kishida, Michiko

    2014-09-05

    Highlights: • We studied the interaction between tumor cells and fibroblasts in ameloblastoma. • AM-3 ameloblastoma cells secreted significantly high IL-1α levels. • IL-1α derived from AM-3 cells promoted IL-6 and IL-8 secretion of fibroblasts. • IL-6 and IL-8 activated the cellular motility and proliferation of AM-3 cells. - Abstract: Ameloblastoma is an odontogenic benign tumor that occurs in the jawbone, which invades bone and reoccurs locally. This tumor is treated by wide surgical excision and causes various problems, including changes in facial countenance and mastication disorders. Ameloblastomas have abundant tumor stroma, including fibroblasts and immune cells. Although cell-to-cell interactions are considered to be involved in the pathogenesis of many diseases, intercellular communications in ameloblastoma have not been fully investigated. In this study, we examined interactions between tumor cells and stromal fibroblasts via soluble factors in ameloblastoma. We used a human ameloblastoma cell line (AM-3 ameloblastoma cells), human fibroblasts (HFF-2 fibroblasts), and primary-cultured fibroblasts from human ameloblastoma tissues, and analyzed the effect of ameloblastoma-associated cell-to-cell communications on gene expression, cytokine secretion, cellular motility and proliferation. AM-3 ameloblastoma cells secreted higher levels of interleukin (IL)-1α than HFF-2 fibroblasts. Treatment with conditioned medium from AM-3 ameloblastoma cells upregulated gene expression and secretion of IL-6 and IL-8 of HFF-2 fibroblasts and primary-cultured fibroblast cells from ameloblastoma tissues. The AM3-stimulated production of IL-6 and IL-8 in fibroblasts was neutralized by pretreatment of AM-3 cells with anti-IL-1α antibody and IL-1 receptor antagonist. Reciprocally, cellular motility of AM-3 ameloblastoma cells was stimulated by HFF-2 fibroblasts in IL-6 and IL-8 dependent manner. In conclusion, ameloblastoma cells and stromal fibroblasts behave

  13. Stromal-epithelial interactions in aging and cancer: Senescent fibroblasts alter epithelial cell differentiation

    SciTech Connect

    Parrinello, Simona; Coppe, Jean-Philippe; Krtolica, Ana; Campisi, Judith

    2004-07-14

    Cellular senescence suppresses cancer by arresting cells at risk for malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation, and branching morphogenesis. Further, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts--the ability to alter epithelial differentiation--that might also explain the loss of tissue function and organization that is a hallmark of aging.

  14. Regulation of IL-6 and IL-8 production by reciprocal cell-to-cell interactions between tumor cells and stromal fibroblasts through IL-1α in ameloblastoma.

    PubMed

    Fuchigami, Takao; Kibe, Toshiro; Koyama, Hirofumi; Kishida, Shosei; Iijima, Mikio; Nishizawa, Yoshiaki; Hijioka, Hiroshi; Fujii, Tomomi; Ueda, Masahiro; Nakamura, Norifumi; Kiyono, Tohru; Kishida, Michiko

    2014-09-05

    Ameloblastoma is an odontogenic benign tumor that occurs in the jawbone, which invades bone and reoccurs locally. This tumor is treated by wide surgical excision and causes various problems, including changes in facial countenance and mastication disorders. Ameloblastomas have abundant tumor stroma, including fibroblasts and immune cells. Although cell-to-cell interactions are considered to be involved in the pathogenesis of many diseases, intercellular communications in ameloblastoma have not been fully investigated. In this study, we examined interactions between tumor cells and stromal fibroblasts via soluble factors in ameloblastoma. We used a human ameloblastoma cell line (AM-3 ameloblastoma cells), human fibroblasts (HFF-2 fibroblasts), and primary-cultured fibroblasts from human ameloblastoma tissues, and analyzed the effect of ameloblastoma-associated cell-to-cell communications on gene expression, cytokine secretion, cellular motility and proliferation. AM-3 ameloblastoma cells secreted higher levels of interleukin (IL)-1α than HFF-2 fibroblasts. Treatment with conditioned medium from AM-3 ameloblastoma cells upregulated gene expression and secretion of IL-6 and IL-8 of HFF-2 fibroblasts and primary-cultured fibroblast cells from ameloblastoma tissues. The AM3-stimulated production of IL-6 and IL-8 in fibroblasts was neutralized by pretreatment of AM-3 cells with anti-IL-1α antibody and IL-1 receptor antagonist. Reciprocally, cellular motility of AM-3 ameloblastoma cells was stimulated by HFF-2 fibroblasts in IL-6 and IL-8 dependent manner. In conclusion, ameloblastoma cells and stromal fibroblasts behave interactively via these cytokines to create a microenvironment that leads to the extension of ameloblastomas.

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

    SciTech Connect

    Rattigan, Yanique I.; Patel, Brijesh B.; Ackerstaff, Ellen; Sukenick, George; Koutcher, Jason A.; Glod, John W.; and others

    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, 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.

  16. Stromal fibroblast activation and their potential association with uterine fibroids (Review)

    PubMed Central

    ZHENG, LI-HUA; CAI, FENG-FENG; GE, ISABELL; BISKUP, EWELINA; CHENG, ZHONG-PING

    2014-01-01

    Uterine fibroids are the most common type of benign, gynecologic neoplasm and are the primary indication for performance of a hysterectomy, accounting for >200,000 hysterectomies annually in the USA. At present, females are younger and exhibit larger leiomyomas at the time of diagnosis. Cancer-associated fibroblasts in tumor microenvironments have emerged as an important target for cancer therapy. Repeated stimulation by infectious or non-infectious agents in the uterine tissues, including inflammation, mechanical forces or hypoxia, stimulate the resident fibroblasts to undergo specific activation and, thus, are significant in tumorigenesis. Furthermore, complex signaling pathways regulate the mechanisms of fibroblastic activation. The current review focuses on the molecular mechanisms of fibroblastic activation and the potential association with uterine leiomyoma pathogenesis, enabling an integrated pathogenic analysis for review of the therapeutic options. PMID:25013460

  17. Tracing CD34+ Stromal Fibroblasts in Palatal Mucosa and Periodontal Granulation Tissue as a Possible Cell Reservoir for Periodontal Regeneration.

    PubMed

    Roman, Alexandra; Páll, Emőke; Mihu, Carmen M; Petruţiu, Adrian S; Barbu-Tudoran, Lucian; Câmpian, Radu S; Florea, Adrian; Georgiu, Carmen

    2015-08-01

    The aim of the present research was to trace CD34+ stromal fibroblastic cells (CD34+ SFCs) in the palatal connective tissue harvested for muco-gingival surgical procedures and in granulation tissues from periodontal pockets using immunohistochemical and transmission electron microscopy. Immunohistochemical analysis targeted the presence of three antigens: CD31, α-smooth muscle actin (α-SMA), and CD34. In the palate, CD31 staining revealed a colored inner ring of the vessels representing the endothelium, α-SMA+ was located in the medial layer of the vasculature, and CD34 was intensely expressed by endothelial cells and artery adventitial cells (considered to be CD34+ SFCs). Granulation tissue showed the same pattern for CD31+ and α-SMA, but a different staining pattern for CD34. Ultrastructural examination of the palatal tissue highlighted perivascular cells with fibroblast-like characteristics and pericytes in close spatial relationship to endothelial cells. The ultrastructural evaluation of granulation tissue sections confirmed the presence of neovasculature and the inflammatory nature of this tissue. The present study traced the presence of CD34+ SFCs and of pericytes in the palatal connective tissue thus highlighting once more its intrinsic regenerative capabilities. The clinical and systemic factors triggering mobilization and influencing the fate of local CD34+SCFs and other progenitors are issues to be further investigated.

  18. Stromal cells from human long-term marrow cultures, but not cultured marrow fibroblasts, phagocytose horse serum constituents: studies with a monoclonal antibody that reacts with a species-specific epitope common to multiple horse serum proteins.

    PubMed

    Charbord, P; Tippens, D; Wight, T S; Gown, A M; Singer, J W

    1987-01-01

    This report describes an IgG1 mouse monoclonal antibody derived after immunization of mice with washed stromal cells from human, long-term bone marrow cultures. The antigen recognized by the antibody (BMS-1) is a carbohydrate-containing prosthetic group that is common to and specific for multiple horse serum proteins. These proteins are avidly ingested by stromal cells and concentrated in endocytic vesicles. Cultured smooth muscle cells took up the horse proteins in a similar manner to marrow stromal cells while cultured marrow fibroblasts, endothelial cells, and hepatoma cells did not. These data indicate that marrow stromal cells specifically accumulate horse serum proteins which might partially explain the horse serum requirement for long-term marrow culture maintenance. The data also suggest further similarities between marrow stromal and smooth muscle cells and additional differences between marrow fibroblasts and marrow stromal cells.

  19. Stromal fibroblasts and the immune microenvironment: partners in mammary gland biology and pathology?

    PubMed

    Unsworth, Ashleigh; Anderson, Robin; Britt, Kara

    2014-07-01

    The microenvironment of a tumor has emerged recently as a critical contributor to the development of cancer. Within this environment, fibroblasts and immune cells are the cell lineages that seem to be active mediators of tumour development. The activated fibroblasts that are also present during wound healing and chronic inflammation have been studied extensively. Their activation leads to altered gene expression profiles that markedly increase growth factor and cytokine secretion, leading to major alterations in the immune cell microenvironment. To better understand normal tissue development, wound healing and the chronic inflammation that leads to cancer, we review here information available on the role of fibroblasts and immune cells in normal breast development and in cancer. We also discuss the immunogenicity of breast cancer compared to other cancers and the contribution of the immune microenvironment to the initiation, progression and metastasis of tumors. Also reviewed is the limited knowledge on the role of immune cells and fibroblasts in normal development and whether the risk of cancer increases when their control is not tightly regulated.

  20. The Action of Discoidin Domain Receptor 2 in Basal Tumor Cells and Stromal Cancer-Associated Fibroblasts Is Critical for Breast Cancer Metastasis.

    PubMed

    Corsa, Callie A S; Brenot, Audrey; Grither, Whitney R; Van Hove, Samantha; Loza, Andrew J; Zhang, Kun; Ponik, Suzanne M; Liu, Yuming; DeNardo, David G; Eliceiri, Kevin W; Keely, Patricia J; Longmore, Gregory D

    2016-06-14

    High levels of collagen deposition in human and mouse breast tumors are associated with poor outcome due to increased local invasion and distant metastases. Using a genetic approach, we show that, in mice, the action of the fibrillar collagen receptor discoidin domain receptor 2 (DDR2) in both tumor and tumor-stromal cells is critical for breast cancer metastasis yet does not affect primary tumor growth. In tumor cells, DDR2 in basal epithelial cells regulates the collective invasion of tumor organoids. In stromal cancer-associated fibroblasts (CAFs), DDR2 is critical for extracellular matrix production and the organization of collagen fibers. The action of DDR2 in CAFs also enhances tumor cell collective invasion through a pathway distinct from the tumor-cell-intrinsic function of DDR2. This work identifies DDR2 as a potential therapeutic target that controls breast cancer metastases through its action in both tumor cells and tumor-stromal cells at the primary tumor site.

  1. Dermal fibroblast expression of stromal cell-derived factor-1 (SDF-1) promotes epidermal keratinocyte proliferation in normal and diseased skin.

    PubMed

    Quan, Chunji; Cho, Moon Kyun; Shao, Yuan; Mianecki, Laurel E; Liao, Eric; Perry, Daniel; Quan, Taihao

    2015-12-01

    Stromal cells provide a crucial microenvironment for overlying epithelium. Here we investigated the expression and function of a stromal cell-specific protein, stromal cell-derived factor-1 (SDF-1), in normal human skin and in the tissues of diseased skin. Immunohistology and laser capture microdissection (LCM)-coupled quantitative real-time RT-PCR revealed that SDF-1 is constitutively and predominantly expressed in dermal stromal cells in normal human skin in vivo. To our surprise, an extremely high level of SDF-1 transcription was observed in the dermis of normal human skin in vivo, evidenced by much higher mRNA expression level than type I collagen, the most abundant and highly expressed protein in human skin. SDF-1 was also upregulated in the tissues of many human skin disorders including psoriasis, basal cell carcinoma (BCC), and squamous cell carcinoma (SCC). Double immunostaining for SDF-1 and HSP47 (heat shock protein 47), a marker of fibroblasts, revealed that fibroblasts were the major source of stroma-cell-derived SDF-1 in both normal and diseased skin. Functionally, SDF-1 activates the ERK (extracellular-signal-regulated kinases) pathway and functions as a mitogen to stimulate epidermal keratinocyte proliferation. Both overexpression of SDF-1 in dermal fibroblasts and treatment with rhSDF-1 to the skin equivalent cultures significantly increased the number of keratinocyte layers and epidermal thickness. Conversely, the stimulative function of SDF-1 on keratinocyte proliferation was nearly completely eliminated by interfering with CXCR4, a specific receptor of SDF-1, or by knock-down of SDF-1 in fibroblasts. Our data reveal that extremely high levels of SDF-1 provide a crucial microenvironment for epidermal keratinocyte proliferation in both physiologic and pathologic skin conditions.

  2. 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 Central

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

    2012-01-01

    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. PMID:23203033

  3. Identification of an extended N-acetylated sequence adjacent to the protein-linkage region of fibroblast heparan sulphate.

    PubMed Central

    Lyon, M; Steward, W P; Hampson, I N; Gallagher, J T

    1987-01-01

    The distribution of N-sulphate groups within fibroblast heparan sulphate chains was investigated. The detergent-extractable heparan sulphate proteoglycan from adult human skin fibroblasts, radiolabelled with [3H]glucosamine and [35S]sulphate, was coupled to CNBr-activated Sepharose 4B. After partial depolymerization of the heparan sulphate with nitrous acid, the remaining Sepharose-bound fragments were removed by treatment with alkali. These fragments, of various sizes, but all containing an intact reducing xylose residue, were fractionated on Sephacryl S-300 and the distribution of the 3H and 35S radiolabels was analysed. A decreased degree of sulphation was observed towards the reducing termini of the chains. After complete nitrous acid hydrolysis of the Sepharose-bound proteoglycan, analysis of the proximity of N-sulphation to the reducing end revealed the existence of an extended N-acetylated sequence directly adjacent to the protein-linkage sequence. The size of this N-acetylated domain was estimated by gel filtration to be approximately eight disaccharide units. This domain appears to be highly conserved, being present in virtually all the chains derived from this proteoglycan, implying the existence of a mechanism capable of generating such a non-random sequence during the post-polymeric modification of heparan sulphate. Comparison with the corresponding situation in heparin suggests that different mechanisms regulate polymer N-sulphation in the vicinity of the protein-linkage region of these chemically related glycosaminoglycans. PMID:2954540

  4. 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.

  5. Comparison of Morphology, Orientation, and Migration of Tendon Derived Fibroblasts and Bone Marrow Stromal Cells on Electrochemically Aligned Collagen Constructs

    PubMed Central

    Gurkan, Umut Atakan; Cheng, Xingguo; Kishore, Vipuil; Uquillas, Jorge Alfredo; Akkus, Ozan

    2010-01-01

    There are approximately 33 million injuries involving musculoskeletal tissues (including tendons and ligaments) every year in the United States. In certain cases the tendons and ligaments are damaged irreversibly and require replacements that possess the natural functional properties of these tissues. As a biomaterial, collagen has been a key ingredient in tissue engineering scaffolds. The application range of collagen in tissue engineering would be greatly broadened if the assembly process could be better controlled to facilitate the synthesis of dense, oriented tissue-like constructs. An electrochemical method has recently been developed in our laboratory to form highly oriented and densely packed collagen bundles with mechanical strength approaching that of tendons. However, there is limited information whether this electrochemically aligned collagen bundle (ELAC) presents advantages over randomly oriented bundles in terms of cell response. Therefore, the current study aimed to assess the biocompatibility of the collagen bundles in vitro, and compare tendon derived fibroblasts (TDFs) and bone marrow stromal cells (MSCs) in terms of their ability to populate and migrate on the single and braided ELAC bundles. The results indicated that the ELAC was not cytotoxic; both cell types were able to populate and migrate on the ELAC bundles more efficiently than that observed for random collagen bundles. The braided ELAC constructs were efficiently populated by both TDFs and MSCs in vitro. Therefore, both TDFs and MSCs can be used with the ELAC bundles for tissue engineering purposes. PMID:20694974

  6. Hypoxia and loss of PHD2 inactivate stromal fibroblasts to decrease tumour stiffness and metastasis.

    PubMed

    Madsen, Chris D; Pedersen, Jesper T; Venning, Freja A; Singh, Lukram Babloo; Moeendarbary, Emad; Charras, Guillaume; Cox, Thomas R; Sahai, Erik; Erler, Janine T

    2015-10-01

    Cancer-associated fibroblasts (CAFs) interact with tumour cells and promote growth and metastasis. Here, we show that CAF activation is reversible: chronic hypoxia deactivates CAFs, resulting in the loss of contractile force, reduced remodelling of the surrounding extracellular matrix and, ultimately, impaired CAF-mediated cancer cell invasion. Hypoxia inhibits prolyl hydroxylase domain protein 2 (PHD2), leading to hypoxia-inducible factor (HIF)-1α stabilisation, reduced expression of αSMA and periostin, and reduced myosin II activity. Loss of PHD2 in CAFs phenocopies the effects of hypoxia, which can be prevented by simultaneous depletion of HIF-1α. Treatment with the PHD inhibitor DMOG in an orthotopic breast cancer model significantly decreases spontaneous metastases to the lungs and liver, associated with decreased tumour stiffness and fibroblast activation. PHD2 depletion in CAFs co-injected with tumour cells similarly prevents CAF-induced metastasis to lungs and liver. Our data argue that reversion of CAFs towards a less active state is possible and could have important clinical implications.

  7. Stromal influences on breast cancer cell growth.

    PubMed Central

    van Roozendaal, C. E.; van Ooijen, B.; Klijn, J. G.; Claassen, C.; Eggermont, A. M.; Henzen-Logmans, S. C.; Foekens, J. A.

    1992-01-01

    Paracrine influences from fibroblasts derived from different sources of breast tissue on epithelial breast cancer cell growth in vitro were investigated. Medium conditioned (CM) by fibroblasts derived from tumours, adjacent normal breast tissue, and normal breast tissue obtained from reduction mammoplasty or from skin tissue significantly stimulated the growth of the steroid-receptor positive cell lines MCF-7 and ZR 75.1. The proliferation index (PI) on MCF-7 cells with CM from fibroblasts derived from breast tumour tissue was significantly higher than that obtained with fibroblasts derived from adjacent normal breast tissue (2p less than 0.05, n = 8). The PI obtained with CM from normal fibroblast cultures from reduction mammoplasty tissue, like normal tissue adjacent to the tumour, fell in the lower range of values. Skin fibroblast, like tumour tissue derived fibroblast, CM caused a high range PI. MDA-MB-231 and Evsa-T, two steroid-receptor negative cell lines, showed only a minor growth stimulatory responses with some of the fibroblast CM's. Evsa-T was occasionally inhibited by CM's. In conclusion, stromal factors play a role in the growth regulation of human breast cancer cells. The effects on cancer cell growth are, however, varying depending on the source of the stroma and the characteristics of the epithelial tumour cells. PMID:1733444

  8. Fibroblasts from the inner granulation tissue of the pseudocapsule in hips at revision arthroplasty induce osteoclast differentiation, as do stromal cells

    PubMed Central

    Sakai, H; Jingushi, S; Shuto, T; Urabe, K; Ikenoue, T; Okazaki, K; Kukita, T; Kukita, A; Iwamoto, Y

    2002-01-01

    Background: It has previously been shown that many osteoclast precursors are included in the granulation tissue within the pseudocapsule obtained at revision arthroplasty from hips with osteolysis. In vitro culture of only cells isolated from the granulation tissue has been previously shown to generate many mature osteoclasts. Objective: To investigate the presence or otherwise of supporting cells, similar to stromal cells, which differentiate osteoclasts within the granulation tissue. Methods: Cells isolated from the granulation tissue were cultured alone, and after four weeks fibroblast-like cells (granulation fibroblasts) remained. Rat non-adherent bone marrow cells (NA-BMCs) were co-cultured with the granulation fibroblasts with or without 1α,25(OH)2D3 (10-8 M) or heat treated ROS 17/2.8 cell conditioned medium (ht ROSCM), or both. Multinucleated cells (MNCs), which formed, were assessed by biochemical and functional characterisation of osteoclasts. Receptor activator of NFκB ligand (RANKL) was investigated by immunohistochemistry. Results: Co-culture of NA-BMCs and granulation fibroblasts caused the formation of tartrate resistant acid phosphatase (TRAP) positive MNCs, which had the calcitonin receptor (CTR), the Kat-1 antigen, which is specific to the surface of rat osteoclasts, and the ability to form pits in the presence of both 1α,25(OH)2D3 and ht ROSCM or in the presence of just ht ROSCM. RANKL was detected in fibroblast-like cells in the granulation tissue. Conclusion: These data suggest that granulation fibroblasts support osteoclast differentiation, as do osteoblasts/stromal cells, and may play a part in aseptic loosening. PMID:11796394

  9. Cell Injury-Induced Release of Fibroblast Growth Factor 2: Relevance to Intracerebral Mesenchymal Stromal Cell Transplantations

    PubMed Central

    Vinodkumar, Deepti; McGrogan, Michael; Bates, Damien

    2015-01-01

    Beneficial effects of intracerebral transplantation of mesenchymal stromal cells (MSC) and their derivatives are believed to be mediated mostly by factors produced by engrafted cells. However, the mesenchymal cell engraftment rate is low, and the majority of grafted cells disappear within a short post-transplantation period. Here, we hypothesize that dying transplanted cells can affect surrounding tissues by releasing their active intracellular components. To elucidate the type, amounts, and potency of these putative intracellular factors, freeze/thaw extracts of MSC or their derivatives were tested in enzyme-linked immunosorbent assays and bioassays. We found that fibroblast growth factor (FGF)2 and FGF1, but not vascular endothelial growth factor and monocyte chemoattractant protein 1 levels were high in extracts despite being low in conditioned media. Extracts induced concentration-dependent proliferation of rat cortical neural progenitor cells and human umbilical vein endothelial cells; these proliferative responses were specifically blocked by FGF2-neutralizing antibody. In the neuropoiesis assay with rat cortical cells, both MSC extracts and killed cells induced expression of nestin, but not astrocyte differentiation. However, suspensions of killed cells strongly potentiated the astrogenic effects of live MSC. In transplantation-relevant MSC injury models (peripheral blood cell-mediated cytotoxicity and high cell density plating), MSC death coincided with the release of intracellular FGF2. The data showed that MSC contain a major depot of active FGF2 that is released upon cell injury and is capable of acutely stimulating neuropoiesis and angiogenesis. We therefore propose that both dying and surviving grafted MSC contribute to tissue regeneration. PMID:25873141

  10. Mouse embryonic fibroblasts (MEF) exhibit a similar but not identical phenotype to bone marrow stromal stem cells (BMSC).

    PubMed

    Saeed, Hamid; Taipaleenmäki, Hanna; Aldahmash, Abdullah M; Abdallah, Basem M; Kassem, Moustapha

    2012-06-01

    Mouse embryonic fibroblasts have been utilized as a surrogate stem cell model for the postnatal bone marrow-derived stromal stem cells (BMSC) to study mesoderm-type cell differentiation e.g. osteoblasts, adipocytes and chondrocytes. However, no formal characterization of MEF phenotype has been reported. Utilizing standard in vitro and in vivo assays we performed a side-by-side comparison of MEF and BMSC to determine their ability to differentiate into mesoderm-type cells. BMSC were isolated from 8-10 weeks old mouse bone marrow by plastic adherence. MEF were established by trypsin/EDTA digestion from E13.5 embryos after removing heads and viscera, followed by plastic adherence. Compared to BMSC, MEF exhibited telomerase activity and improved cell proliferation as assessed by q-PCR based TRAP assay and cell number quantification, respectively. FACS analysis revealed that MEF exhibited surface markers characteristic of the BMSC: Sca-1(+), CD73(+), CD105(+), CD29(+), CD44(+), CD106(+), CD11b(-), and CD45(-). In contrast to BMSC, ex vivo osteoblast (OB) differentiation of MEF exhibited a less mature osteoblastic phenotype (less alkaline phosphatase, collagen type I and osteocalcin) as assessed by real-time PCR analysis. Compared to BMSC, MEF exhibited a more enhanced differentiation into adipocyte and chondrocyte lineages. Interestingly, both MEF and BMSC formed the same amount of heterotopic bone and bone marrow elements upon in vivo subcutaneous implantation with hydroxyapatite/tricalcium phosphate, in immune deficient mice. In conclusion, MEF contain a population of stem cells that behave in ex vivo and in vivo assays, similar but not identical, to BMSC. Due to their enhanced cell growth, they may represent a good alternative for BMSC in studying molecular mechanisms of stem cell commitment and differentiation to osteoblasts, adipocytes and chondrocytes.

  11. 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.

  12. Mitochondria "fuel" breast cancer metabolism: fifteen markers of mitochondrial biogenesis label epithelial cancer cells, but are excluded from adjacent stromal cells.

    PubMed

    Sotgia, Federica; Whitaker-Menezes, Diana; Martinez-Outschoorn, Ubaldo E; Salem, Ahmed F; Tsirigos, Aristotelis; Lamb, Rebecca; Sneddon, Sharon; Hulit, James; Howell, Anthony; Lisanti, Michael P

    2012-12-01

    Here, we present new genetic and morphological evidence that human tumors consist of two distinct metabolic compartments. First, re-analysis of genome-wide transcriptional profiling data revealed that > 95 gene transcripts associated with mitochondrial biogenesis and/or mitochondrial translation were significantly elevated in human breast cancer cells, as compared with adjacent stromal tissue. Remarkably, nearly 40 of these upregulated gene transcripts were mitochondrial ribosomal proteins (MRPs), functionally associated with mitochondrial translation of protein components of the OXPHOS complex. Second, during validation by immunohistochemistry, we observed that antibodies directed against 15 markers of mitochondrial biogenesis and/or mitochondrial translation (AKAP1, GOLPH3, GOLPH3L, MCT1, MRPL40, MRPS7, MRPS15, MRPS22, NRF1, NRF2, PGC1-α, POLRMT, TFAM, TIMM9 and TOMM70A) selectively labeled epithelial breast cancer cells. These same mitochondrial markers were largely absent or excluded from adjacent tumor stromal cells. Finally, markers of mitochondrial lipid synthesis (GOLPH3) and mitochondrial translation (POLRMT) were associated with poor clinical outcome in human breast cancer patients. Thus, we conclude that human breast cancers contain two distinct metabolic compartments-a glycolytic tumor stroma, which surrounds oxidative epithelial cancer cells-that are mitochondria-rich. The co-existence of these two compartments is indicative of metabolic symbiosis between epithelial cancer cells and their surrounding stroma. As such, epithelial breast cancer cells should be viewed as predatory metabolic "parasites," which undergo anabolic reprogramming to amplify their mitochondrial "power." This notion is consistent with the observation that the anti-malarial agent chloroquine may be an effective anticancer agent. New anticancer therapies should be developed to target mitochondrial biogenesis and/or mitochondrial translation in human cancer cells.

  13. Mitochondria “fuel” breast cancer metabolism: Fifteen markers of mitochondrial biogenesis label epithelial cancer cells, but are excluded from adjacent stromal cells

    PubMed Central

    Sotgia, Federica; Whitaker-Menezes, Diana; Martinez-Outschoorn, Ubaldo E.; Salem, Ahmed F.; Tsirigos, Aristotelis; Lamb, Rebecca; Sneddon, Sharon; Hulit, James; Howell, Anthony; Lisanti, Michael P.

    2012-01-01

    Here, we present new genetic and morphological evidence that human tumors consist of two distinct metabolic compartments. First, re-analysis of genome-wide transcriptional profiling data revealed that > 95 gene transcripts associated with mitochondrial biogenesis and/or mitochondrial translation were significantly elevated in human breast cancer cells, as compared with adjacent stromal tissue. Remarkably, nearly 40 of these upregulated gene transcripts were mitochondrial ribosomal proteins (MRPs), functionally associated with mitochondrial translation of protein components of the OXPHOS complex. Second, during validation by immunohistochemistry, we observed that antibodies directed against 15 markers of mitochondrial biogenesis and/or mitochondrial translation (AKAP1, GOLPH3, GOLPH3L, MCT1, MRPL40, MRPS7, MRPS15, MRPS22, NRF1, NRF2, PGC1-α, POLRMT, TFAM, TIMM9 and TOMM70A) selectively labeled epithelial breast cancer cells. These same mitochondrial markers were largely absent or excluded from adjacent tumor stromal cells. Finally, markers of mitochondrial lipid synthesis (GOLPH3) and mitochondrial translation (POLRMT) were associated with poor clinical outcome in human breast cancer patients. Thus, we conclude that human breast cancers contain two distinct metabolic compartments—a glycolytic tumor stroma, which surrounds oxidative epithelial cancer cells—that are mitochondria-rich. The co-existence of these two compartments is indicative of metabolic symbiosis between epithelial cancer cells and their surrounding stroma. As such, epithelial breast cancer cells should be viewed as predatory metabolic “parasites,” which undergo anabolic reprogramming to amplify their mitochondrial “power.” This notion is consistent with the observation that the anti-malarial agent chloroquine may be an effective anticancer agent. New anticancer therapies should be developed to target mitochondrial biogenesis and/or mitochondrial translation in human cancer cells. PMID

  14. Functionally and Phenotypically Distinct Subpopulations of Marrow Stromal Cells Are Fibroblast in Origin and Induce Different Fates in Peripheral Blood Monocytes

    PubMed Central

    Iwata, Mineo; Sandstrom, Richard S.; Delrow, Jeffrey J.; Stamatoyannopoulos, John A.

    2014-01-01

    Marrow stromal cells constitute a heterogeneous population of cells, typically isolated after expansion in culture. In vivo, stromal cells often exist in close proximity or in direct contact with monocyte-derived macrophages, yet their interaction with monocytes is largely unexplored. In this report, isolated CD146+ and CD146− stromal cells, as well as immortalized cell lines representative of each (designated HS27a and HS5, respectively), were shown by global DNase I hypersensitive site mapping and principal coordinate analysis to have a lineage association with marrow fibroblasts. Gene expression profiles generated for the CD146+ and CD146− cell lines indicate significant differences in their respective transcriptomes, which translates into differences in secreted factors. Consequently, the conditioned media (CM) from these two populations induce different fates in peripheral blood monocytes. Monocytes incubated in CD146+ CM acquire a tissue macrophage phenotype, whereas monocytes incubated in CM from CD146− cells express markers associated with pre-dendritic cells. Importantly, when CD14+ monocytes are cultured in contact with the CD146+ cells, the combined cell populations, assayed as a unit, show increased levels of transcripts associated with organismal development and hematopoietic regulation. In contrast, the gene expression profile from cocultures of monocytes and CD146− cells does not differ from that obtained when monocytes are cultured with CD146− CM. These in vitro results show that the CD146+ marrow stromal cells together with monocytes increase the expression of genes relevant to hematopoietic regulation. In vivo relevance of these data is suggested by immunohistochemistry of marrow biopsies showing juxtaposed CD146+ cells and CD68+ cells associated with these upregulated proteins. PMID:24131213

  15. Notch1—WISP-1 axis determines the regulatory role of mesenchymal stem cell-derived stromal fibroblasts in melanoma metastasis

    PubMed Central

    Moller, Mecker; Issac, Biju; Zhang, Leiming; Owyong, Mark; Moscowitz, Anna Elizabeth; Vazquez-Padron, Roberto; Radtke, Freddy; Liu, Zhao-Jun

    2016-01-01

    Mesenchymal stem cells-derived fibroblasts (MSC-DF) constitute a significant portion of stromal fibroblasts in the tumor microenvironment (TME) and are key modulators of tumor progression. However, the molecular mechanisms that determine their tumor-regulatory function are poorly understood. Here, we uncover the Notch1 pathway as a molecular determinant that selectively controls the regulatory role of MSC-DF in melanoma metastasis. We demonstrate that the Notch1 pathway's activity is inversely correlated with the metastasis-regulating function of fibroblasts and can determine the metastasis-promoting or -suppressing phenotype of MSC-DF. When co-grafted with melanoma cells, MSC-DFNotch1−/− selectively promote, while MSC-DFN1IC+/+ preferentially suppress melanoma metastasis, but not growth, in mouse models. Consistently, conditioned media (CM) from MSC-DFNotch1−/− and MSC-DFN1IC+/+ oppositely, yet selectively regulates migration, but not growth of melanoma cells in vitro. Additionally, when co-cultured with metastatic melanoma cells in vitro, MSC-DFNotch1−/− support, while MSC-DFN1IC+/+ inhibit melanoma cells in the formation of spheroids. These findings expand the repertoire of Notch1 signaling as a molecular switch in determining the tumor metastasis-regulating function of MSC-DF. We also identified Wnt-induced secreted protein-1 (WISP-1) as a key downstream secretory mediator of Notch1 signaling to execute the influential role of MSC-DF on melanoma metastasis. These findings reveal the Notch1—WISP-1 axis as a crucial molecular determinant in governing stromal regulation of melanoma metastasis; thus, establishing this axis as a potential therapeutic target for melanoma metastasis. PMID:27813493

  16. Comparison of Gene Expression Profile Between Tumor Tissue and Adjacent Non-tumor Tissue in Patients with Gastric Gastrointestinal Stromal Tumor (GIST).

    PubMed

    Kou, Youwei; Zhao, Ying; Bao, Chenhui; Wang, Qiang

    2015-06-01

    Gastrointestinal stromal tumors (GISTs) are defined as spindle cell and/or epithelioid tumors originated from interstitial Cajal cells or precursors in the digestive tract. This study was conducted to identify genes differing in expression between the gastric tumors and the adjacent non-cancerous mucosas in patients with primary gastric GIST. The gene expression profile was determined by using oligonucleotide-based DNA microarrays and further validated by quantitative real-time PCR. The Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis was performed to predict signaling pathways involved in gastric GIST. Our data showed that the expression levels of 957 genes (RAB39B, member RAS oncogene family; VCAN, versican; etc.) were higher and that of 526 genes (CXCL14, chemokine C-X-C motif ligand 14; MTUS1, microtubule-associated tumor suppressor 1; etc.) were lower in the gastric tumor tissues as compared with normal gastric tissues. Results from KEGG pathway analysis revealed that the differentially expressed genes were enriched into 16 signaling transduction pathways, including Hedeghog and Wnt signaling pathways. Our study may provide basis for identification of novel biomarkers associated with primary gastric GIST pathogenesis and for exploration of underlying mechanisms involved in this gastric sarcoma.

  17. Sonic hedgehog signals to multiple prostate stromal stem cells that replenish distinct stromal subtypes during regeneration

    PubMed Central

    Peng, Yu-Ching; Levine, Charles M.; Zahid, Sarwar; Wilson, E. Lynette; Joyner, Alexandra L.

    2013-01-01

    The adult mouse prostate has a seemingly endless capacity for regeneration, and sonic hedgehog (SHH) signaling has been implicated in this stem cell-driven process. However, it is not clear whether SHH acts on the epithelium or stromal cells that secrete factors required for epithelial expansion. Because little is known about stromal stem cells compared with their epithelial counterparts, we used in vivo mouse genetics tools to characterize four prostate stromal subtypes and their stem cells. Using knockin reporter alleles, we uncovered that SHH signals from prostate basal epithelial cells to adjacent stromal cells. Furthermore, the SHH target gene Gli1 is preferentially expressed in subepithelial fibroblast-like cells, one of four prostate stromal subtypes and the subtype closest to the epithelial source of SHH. Using Genetic Inducible Fate Mapping to mark adult Gli1- or Smooth muscle actin-expressing cells and follow their fate during regeneration, we uncovered that Gli1-expressing cells exhibit long-term self-renewal capacity during multiple rounds of androgen-mediated regeneration after castration-induced involution, and depleted smooth muscle cells are mainly replenished by preexisting smooth muscle cells. Based on our Genetic Inducible Fate Mapping studies, we propose a model where SHH signals to multiple stromal stem cells, which are largely unipotent in vivo. PMID:24218555

  18. Oxidative stress in cancer associated fibroblasts drives tumor-stroma co-evolution

    PubMed Central

    Martinez-Outschoorn, Ubaldo E; Balliet, Renee M; Rivadeneira, Dayana B; Chiavarina, Barbara; Pavlides, Stephanos; Wang, Chenguang; Whitaker-Menezes, Diana; Daumer, Kristin M; Lin, Zhao; Witkiewicz, Agnieszka K; Flomenberg, Neal; Howell, Anthony; Pestell, Richard G; Knudsen, Erik S; Lisanti, Michael P

    2010-01-01

    Loss of stromal fibroblast caveolin-1 (Cav-1) is a powerful single independent predictor of poor prognosis in human breast cancer patients, and is associated with early tumor recurrence, lymph node metastasis and tamoxifen-resistance. We developed a novel co-culture system to understand the mechanism(s) by which a loss of stromal fibroblast Cav-1 induces a “lethal tumor microenvironment.” Here, we propose a new paradigm to explain the powerful prognostic value of stromal Cav-1. In this model, cancer cells induce oxidative stress in cancer-associated fibroblasts, which then acts as a “metabolic” and “mutagenic” motor to drive tumor-stroma co-evolution, DNA damage and aneuploidy in cancer cells. More specifically, we show that an acute loss of Cav-1 expression leads to mitochondrial dysfunction, oxidative stress and aerobic glycolysis in cancer associated fibroblasts. Also, we propose that defective mitochondria are removed from cancer-associated fibroblasts by autophagy/mitophagy that is induced by oxidative stress. As a consequence, cancer associated fibroblasts provide nutrients (such as lactate) to stimulate mitochondrial biogenesis and oxidative metabolism in adjacent cancer cells (the “Reverse Warburg effect”). We provide evidence that oxidative stress in cancer-associated fibroblasts is sufficient to induce genomic instability in adjacent cancer cells, via a bystander effect, potentially increasing their aggressive behavior. Finally, we directly demonstrate that nitric oxide (NO) over-production, secondary to Cav-1 loss, is the root cause for mitochondrial dysfunction in cancer associated fibroblasts. In support of this notion, treatment with anti-oxidants (such as N-acetyl-cysteine, metformin and quercetin) or NO inhibitors (L-NAME) was sufficient to reverse many of the cancer-associated fibroblast phenotypes that we describe. Thus, cancer cells use “oxidative stress” in adjacent fibroblasts (1) as an “engine” to fuel their own

  19. Ionizing radiation-mediated premature senescence and paracrine interactions with cancer cells enhance the expression of syndecan 1 in human breast stromal fibroblasts: the role of TGF-β

    PubMed Central

    Liakou, Eleni; Mavrogonatou, Eleni; Pratsinis, Harris; Rizou, Sophia; Evangelou, Konstantinos; Panagiotou, Petros N.; Karamanos, Nikos K.; Gorgoulis, Vassilis G.; Kletsas, Dimitris

    2016-01-01

    The cell surface proteoglycan syndecan 1 (SDC1) is overexpressed in the malignant breast stromal fibroblasts, creating a favorable milieu for tumor cell growth. In the present study, we found that ionizing radiation, a well-established treatment in human breast cancer, provokes premature senescence of human breast stromal fibroblasts in vitro, as well as in the breast tissue in vivo. These senescent cells were found to overexpress SDC1 both in vitro and in vivo. By using a series of specific inhibitors and siRNA approaches, we showed that this SDC1 overexpression in senescent cells is the result of an autocrine action of Transforming Growth Factor-β (TGF-β) through the Smad pathway and the transcription factor Sp1, while the classical senescence pathways of p53 or p38 MAPK - NF-kB are not involved. In addition, the highly invasive human breast cancer cells MDA-MB-231 (in contrast to the low-invasive MCF-7) can also enhance SDC1 expression, both in early-passage and senescent fibroblasts via a paracrine action of TGF-β. The above suggest that radiation-mediated premature senescence and invasive tumor cells, alone or in combination, enhance SDC1 expression in breast stromal fibroblasts, a poor prognostic factor for cancer growth, and that TGF-β plays a crucial role in this process. PMID:27434331

  20. Oncogenes induce the cancer-associated fibroblast phenotype

    PubMed Central

    Lisanti, Michael P; Martinez-Outschoorn, Ubaldo E; Sotgia, Federica

    2013-01-01

    Metabolic coupling, between mitochondria in cancer cells and catabolism in stromal fibroblasts, promotes tumor growth, recurrence, metastasis, and predicts anticancer drug resistance. Catabolic fibroblasts donate the necessary fuels (such as L-lactate, ketones, glutamine, other amino acids, and fatty acids) to anabolic cancer cells, to metabolize via their TCA cycle and oxidative phosphorylation (OXPHOS). This provides a simple mechanism by which metabolic energy and biomass are transferred from the host microenvironment to cancer cells. Recently, we showed that catabolic metabolism and “glycolytic reprogramming” in the tumor microenvironment are orchestrated by oncogene activation and inflammation, which originates in epithelial cancer cells. Oncogenes drive the onset of the cancer-associated fibroblast phenotype in adjacent normal fibroblasts via paracrine oxidative stress. This oncogene-induced transition to malignancy is “mirrored” by a loss of caveolin-1 (Cav-1) and an increase in MCT4 in adjacent stromal fibroblasts, functionally reflecting catabolic metabolism in the tumor microenvironment. Virtually identical findings were obtained using BRCA1-deficient breast and ovarian cancer cells. Thus, oncogene activation (RAS, NFkB, TGF-β) and/or tumor suppressor loss (BRCA1) have similar functional effects on adjacent stromal fibroblasts, initiating “metabolic symbiosis” and the cancer-associated fibroblast phenotype. New therapeutic strategies that metabolically uncouple oxidative cancer cells from their glycolytic stroma or modulate oxidative stress could be used to target this lethal subtype of cancers. Targeting “fibroblast addiction” in primary and metastatic tumor cells may expose a critical Achilles’ heel, leading to disease regression in both sporadic and familial cancers. PMID:23860382

  1. Oncogenes induce the cancer-associated fibroblast phenotype: metabolic symbiosis and "fibroblast addiction" are new therapeutic targets for drug discovery.

    PubMed

    Lisanti, Michael P; Martinez-Outschoorn, Ubaldo E; Sotgia, Federica

    2013-09-01

    Metabolic coupling, between mitochondria in cancer cells and catabolism in stromal fibroblasts, promotes tumor growth, recurrence, metastasis, and predicts anticancer drug resistance. Catabolic fibroblasts donate the necessary fuels (such as L-lactate, ketones, glutamine, other amino acids, and fatty acids) to anabolic cancer cells, to metabolize via their TCA cycle and oxidative phosphorylation (OXPHOS). This provides a simple mechanism by which metabolic energy and biomass are transferred from the host microenvironment to cancer cells. Recently, we showed that catabolic metabolism and "glycolytic reprogramming" in the tumor microenvironment are orchestrated by oncogene activation and inflammation, which originates in epithelial cancer cells. Oncogenes drive the onset of the cancer-associated fibroblast phenotype in adjacent normal fibroblasts via paracrine oxidative stress. This oncogene-induced transition to malignancy is "mirrored" by a loss of caveolin-1 (Cav-1) and an increase in MCT4 in adjacent stromal fibroblasts, functionally reflecting catabolic metabolism in the tumor microenvironment. Virtually identical findings were obtained using BRCA1-deficient breast and ovarian cancer cells. Thus, oncogene activation (RAS, NFkB, TGF-β) and/or tumor suppressor loss (BRCA1) have similar functional effects on adjacent stromal fibroblasts, initiating "metabolic symbiosis" and the cancer-associated fibroblast phenotype. New therapeutic strategies that metabolically uncouple oxidative cancer cells from their glycolytic stroma or modulate oxidative stress could be used to target this lethal subtype of cancers. Targeting "fibroblast addiction" in primary and metastatic tumor cells may expose a critical Achilles' heel, leading to disease regression in both sporadic and familial cancers.

  2. Autophagy in cancer associated fibroblasts promotes tumor cell survival

    PubMed Central

    Martinez-Outschoorn, Ubaldo E; Trimmer, Casey; Lin, Zhao; Whitaker-Menezes, Diana; Chiavarina, Barbara; Zhou, Jie; Wang, Chengwang; Pavlides, Stephanos; Martinez-Cantarin, Maria P; Capozza, Franco; Witkiewicz, Agnieszka K; Flomenberg, Neal; Howell, Anthony; Pestell, Richard G; Caro, Jaime

    2010-01-01

    Recently, using a co-culture system, we demonstrated that MCF7 epithelial cancer cells induce oxidative stress in adjacent cancer-associated fibroblasts, resulting in the autophagic/lysosomal degradation of stromal caveolin-1 (Cav-1). However, the detailed signaling mechanism(s) underlying this process remain largely unknown. Here, we show that hypoxia is sufficient to induce the autophagic degradation of Cav-1 in stromal fibroblasts, which is blocked by the lysosomal inhibitor chloroquine. Concomitant with the hypoxia-induced degradation of Cav-1, we see the upregulation of a number of well-established autophagy/mitophagy markers, namely LC3, ATG16L, BNIP3, BNIP3L, HIF-1α and NFκB. In addition, pharmacological activation of HIF-1α drives Cav-1 degradation, while pharmacological inactivation of HIF-1 prevents the downregulation of Cav-1. Similarly, pharmacological inactivation of NFκB—another inducer of autophagy—prevents Cav-1 degradation. Moreover, treatment with an inhibitor of glutathione synthase, namely BSO, which induces oxidative stress via depletion of the reduced glutathione pool, is sufficient to induce the autophagic degradation of Cav-1. Thus, it appears that oxidative stress mediated induction of HIF1- and NFκB-activation in fibroblasts drives the autophagic degradation of Cav-1. In direct support of this hypothesis, we show that MCF7 cancer cells activate HIF-1α- and NFκB-driven luciferase reporters in adjacent cancer-associated fibroblasts, via a paracrine mechanism. Consistent with these findings, acute knockdown of Cav-1 in stromal fibroblasts, using an siRNA approach, is indeed sufficient to induce autophagy, with the upregulation of both lysosomal and mitophagy markers. How does the loss of stromal Cav-1 and the induction of stromal autophagy affect cancer cell survival? Interestingly, we show that a loss of Cav-1 in stromal fibroblasts protects adjacent cancer cells against apoptotic cell death. Thus, autophagic cancer

  3. 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.

  4. The functional interplay between EGFR overexpression, hTERT activation, and p53 mutation in esophageal epithelial cells with activation of stromal fibroblasts induces tumor development, invasion, and differentiation.

    PubMed

    Okawa, Takaomi; Michaylira, Carmen Z; Kalabis, Jiri; Stairs, Douglas B; Nakagawa, Hiroshi; Andl, Claudia D; Johnstone, Cameron N; Klein-Szanto, Andres J; El-Deiry, Wafik S; Cukierman, Edna; Herlyn, Meenhard; Rustgi, Anil K

    2007-11-01

    Esophageal cancer is a prototypic squamous cell cancer that carries a poor prognosis, primarily due to presentation at advanced stages. We used human esophageal epithelial cells as a platform to recapitulate esophageal squamous cell cancer, thereby providing insights into the molecular pathogenesis of squamous cell cancers in general. This was achieved through the retroviral-mediated transduction into normal, primary human esophageal epithelial cells of epidermal growth factor receptor (EGFR), the catalytic subunit of human telomerase (hTERT), and p53(R175H), genes that are frequently altered in human esophageal squamous cell cancer. These cells demonstrated increased migration and invasion when compared with control cells. When these genetically altered cells were placed within the in vivo-like context of an organotypic three-dimensional (3D) culture system, the cells formed a high-grade dysplastic epithelium with malignant cells invading into the stromal extracellular matrix (ECM). The invasive phenotype was in part modulated by the activation of matrix metalloproteinase-9 (MMP-9). Using pharmacological and genetic approaches to decrease MMP-9, invasion into the underlying ECM could be suppressed partially. In addition, tumor differentiation was influenced by the type of fibroblasts within the stromal ECM. To that end, fetal esophageal fibroblasts fostered a microenvironment conducive to poorly differentiated invading tumor cells, whereas fetal skin fibroblasts supported a well-differentiated tumor as illustrated by keratin "pearl" formation, a hallmark feature of well-differentiated squamous cell cancers. When inducible AKT was introduced into fetal skin esophageal fibroblasts, a more invasive, less-differentiated esophageal cancer phenotype was achieved. Invasion into the stromal ECM was attenuated by genetic knockdown of AKT1 as well as AKT2. Taken together, alterations in key oncogenes and tumor suppressor genes in esophageal epithelial cells, the composition

  5. Enhancement of osteoblastic differentiation of mesenchymal stromal cells cultured by selective combination of bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-2 (FGF-2).

    PubMed

    Maegawa, Naoki; Kawamura, Kenji; Hirose, Motohiro; Yajima, Hiroshi; Takakura, Yoshinori; Ohgushi, Hajime

    2007-01-01

    It is well known that bone marrow contains mesenchymal stromal cells (MSCs), which can show osteoblastic differentiation when cultured in osteogenic medium containing ascorbic acid, beta-glycerophosphate and dexamethasone. The differentiation results in the appearance of osteoblasts, together with the formation of bone matrix; thus, in vitro cultured bone (osteoblasts/bone matrix) could be fabricated by MSC culture. This type of cultured bone has already been used in clinical cases involving orthopaedic problems. To improve the therapeutic effect of the cultured bone, we investigated the culture conditions that contributed to extensive osteoblastic differentiation. Rat bone marrow was primarily cultured to expand the number of MSCs and further cultured in osteogenic medium for 12 days. The culture was also conducted in a medium supplemented with either bone morphogenetic protein-2 (BMP-2) or fibroblast growth factor (FGF-2), or with sequential combinations of both supplements. Among them, the sequential supplementation of FGF-2 followed by BMP-2 showed high alkaline phosphatase activity, sufficient bone-specific osteocalcein expression and abundant bone matrix formation of the MSC culture. These data implied that the number of responding cells or immature osteoblasts was increased by the supplementation of FGF-2 in the early phase of the culture and that these cells can show osteoblastic differentiation, of which capability was augmented by BMP-2 in the late phase. The sequential supplementation of these cytokines into MSC culture might be suitable for the fabrication of ideal cultured bone for use in bone tissue engineering.

  6. Degenerative Suspensory Ligament Desmitis (DSLD) in Peruvian Paso Horses Is Characterized by Altered Expression of TGFβ Signaling Components in Adipose-Derived Stromal Fibroblasts

    PubMed Central

    Luo, Wei; Sandy, John; Li, Jun; Brounts, Sabrina; Galante, Jorge; Plaas, Anna

    2016-01-01

    Equine degenerative suspensory ligament desmitis (DSLD) in Peruvian Paso horses typically presents at 7–15 years and is characterized by lameness, focal disorganization of collagen fibrils, and chondroid deposition in the body of the ligament. With the aim of developing a test for disease risk (that can be used to screen horses before breeding) we have quantified the expression of 76 TGFβ-signaling target genes in adipose-derived stromal fibroblasts (ADSCs) from six DSLD-affected and five unaffected Paso horses. Remarkably, 35 of the genes showed lower expression (p<0.05) in cells from DSLD-affected animals and this differential was largely eliminated by addition of exogenous TGFβ1. Moreover, TGFβ1-mediated effects on expression were prevented by the TGFβR1/2 inhibitor LY2109761, showing that the signaling was via a TGFβR1/2 complex. The genes affected by the pathology indicate that it is associated with a generalized metabolic disturbance, since some of those most markedly altered in DSLD cells (ATF3, MAPK14, ACVRL1 (ALK1), SMAD6, FOS, CREBBP, NFKBIA, and TGFBR2) represent master-regulators in a wide range of cellular metabolic responses. PMID:27902739

  7. Degenerative Suspensory Ligament Desmitis (DSLD) in Peruvian Paso Horses Is Characterized by Altered Expression of TGFβ Signaling Components in Adipose-Derived Stromal Fibroblasts.

    PubMed

    Luo, Wei; Sandy, John; Trella, Katie; Gorski, Daniel; Gao, Shuguang; Li, Jun; Brounts, Sabrina; Galante, Jorge; Plaas, Anna

    2016-01-01

    Equine degenerative suspensory ligament desmitis (DSLD) in Peruvian Paso horses typically presents at 7-15 years and is characterized by lameness, focal disorganization of collagen fibrils, and chondroid deposition in the body of the ligament. With the aim of developing a test for disease risk (that can be used to screen horses before breeding) we have quantified the expression of 76 TGFβ-signaling target genes in adipose-derived stromal fibroblasts (ADSCs) from six DSLD-affected and five unaffected Paso horses. Remarkably, 35 of the genes showed lower expression (p<0.05) in cells from DSLD-affected animals and this differential was largely eliminated by addition of exogenous TGFβ1. Moreover, TGFβ1-mediated effects on expression were prevented by the TGFβR1/2 inhibitor LY2109761, showing that the signaling was via a TGFβR1/2 complex. The genes affected by the pathology indicate that it is associated with a generalized metabolic disturbance, since some of those most markedly altered in DSLD cells (ATF3, MAPK14, ACVRL1 (ALK1), SMAD6, FOS, CREBBP, NFKBIA, and TGFBR2) represent master-regulators in a wide range of cellular metabolic responses.

  8. Basic Fibroblast Growth Factor Inhibits Apoptosis and Promotes Proliferation of Adipose-Derived Mesenchymal Stromal Cells Isolated from Patients with Type 2 Diabetes by Reducing Cellular Oxidative Stress

    PubMed Central

    2017-01-01

    Type 2 diabetes (T2D) is a chronic metabolic disorder affecting increasing number of people in developed countries. Therefore new strategies for treatment of T2D and its complications are of special interest. Nowadays, cellular therapies involving mesenchymal stromal cells that reside in adipose tissue (ASCs) constitute a promising approach; however, there are still many obstacles concerning safety and effectiveness that need to be overcome before ASCs could be engaged for the treatment of diabetes mellitus. One of the challenges is preventing ASCs from deterioration caused by elevated oxidative stress present in diabetes milieu. In the current study we investigated the effect of basic fibroblast growth factor (bFGF) treatment on ASCs isolated from patients with diagnosed T2D. We demonstrate here that cell exposition to bFGF in 5 and 10 ng/mL dosages results in improved morphology, increased proliferative activity, reduced cellular senescence and apoptosis, and decreased oxidative stress, indicating recovery of ASCs' function impaired by T2D. Therefore our results provide a support for bFGF as a potential therapeutic agent for improving stem cell-based approaches for the treatment of diabetes mellitus and its complications. PMID:28168007

  9. Increased liposome-mediated gene transfer into haematopoietic cells grown in adhesion to stromal or fibroblast cell line monolayers.

    PubMed

    Marit, G; Cao, Y; Froussard, P; Ripoche, J; Dupouy, M; Elandaloussi, A; Lacombe, F; Mahon, F X; Keller, H; Pla, M; Reiffers, J; Theze, J

    2000-01-01

    We investigated transfection rates of CD34+ haematopoietic progenitor cells (HPC) or haematopoietic cell lines (TF-1, KG1a and K562) using the LacZ gene as a reporter and cationic liposomes. The transfection efficiency of CD34+ haematopoietic progenitor cells (HPC) or TF-1, KG1a and K562 grown in suspension is very low (average percentage of 0.013 for HPC and 0.03 for cell lines). Adhesion of HPC or cell lines to plates by immunological or physical methods significantly enhances transfection efficiency; however, the percentage of transfected cells still remained low. We found that adhesion of TF-1, KG1a and K562 HC to MS-5 stroma cells or NIH-3T3 fibroblast cells increased transfection efficiency. Under these conditions transfection is achieved in 11.2-25% (mean 18.30%) for the cell lines and 13.6% (range 8.2-24.2%) for CD34+ HPC. These results indicate that liposome-mediated transfection of HC is significantly increased when cells are grown in adherence to stroma or fibroblast monolayers.

  10. Human tumors instigate granulin-expressing hematopoietic cells that promote malignancy by activating stromal fibroblasts in mice

    PubMed Central

    Elkabets, Moshe; Gifford, Ann M.; Scheel, Christina; Nilsson, Bjorn; Reinhardt, Ferenc; Bray, Mark-Anthony; Carpenter, Anne E.; Jirström, Karin; Magnusson, Kristina; Ebert, Benjamin L.; Pontén, Fredrik; Weinberg, Robert A.; McAllister, Sandra S.

    2011-01-01

    Systemic instigation is a process by which endocrine signals sent from certain tumors (instigators) stimulate BM cells (BMCs), which are mobilized into the circulation and subsequently foster the growth of otherwise indolent carcinoma cells (responders) residing at distant anatomical sites. The identity of the BMCs and their specific contribution or contributions to responder tumor growth have been elusive. Here, we have demonstrated that Sca1+cKit– hematopoietic BMCs of mouse hosts bearing instigating tumors promote the growth of responding tumors that form with a myofibroblast-rich, desmoplastic stroma. Such stroma is almost always observed in malignant human adenocarcinomas and is an indicator of poor prognosis. We then identified granulin (GRN) as the most upregulated gene in instigating Sca1+cKit– BMCs relative to counterpart control cells. The GRN+ BMCs that were recruited to the responding tumors induced resident tissue fibroblasts to express genes that promoted malignant tumor progression; indeed, treatment with recombinant GRN alone was sufficient to promote desmoplastic responding tumor growth. Further, analysis of tumor tissues from a cohort of breast cancer patients revealed that high GRN expression correlated with the most aggressive triple-negative, basal-like tumor subtype and reduced patient survival. Our data suggest that GRN and the unique hematopoietic BMCs that produce it might serve as novel therapeutic targets. PMID:21266779

  11. Matrix remodeling stimulates stromal autophagy, “fueling” cancer cell mitochondrial metabolism and metastasis

    PubMed Central

    Bonuccelli, Gloria; Molchansky, Alex; Capozza, Franco; Witkiewicz, Agnieszka K; Birbe, Ruth C; Howell, Anthony; Pestell, Richard G; Whitaker-Menezes, Diana; Sotgia, Federica

    2011-01-01

    We have previously demonstrated that loss of stromal caveolin-1 (Cav-1) in cancer-associated fibroblasts is a strong and independent predictor of poor clinical outcome in human breast cancer patients. However, the signaling mechanism(s) by which Cav-1 downregulation leads to this tumor-promoting microenvironment are not well understood. To address this issue, we performed an unbiased comparative proteomic analysis of wild-type (WT) and Cav-1-/- null mammary stromal fibroblasts (MSFs). Our results show that plasminogen activator inhibitor type 1 and type 2 (PAI-1 and PAI-2) expression is significantly increased in Cav-1-/- MSFs. To establish a direct cause-effect relationship, we next generated immortalized human fibroblast lines stably overexpressing either PAI-1 or PAI-2. Importantly, PAI-1/2(+) fibroblasts promote the growth of MDA-MB-231 tumors (a human breast cancer cell line) in a murine xenograft model, without any increases in angiogenesis. Similarly, PAI-1/2(+) fibroblasts stimulate experimental metastasis of MDA-MB-231 cells using an in vivo lung colonization assay. Further mechanistic studies revealed that fibroblasts overexpressing PAI-1 or PAI-2 display increased autophagy (“self-eating”) and are sufficient to induce mitochondrial biogenesis/activity in adjacent cancer cells, in co-culture experiments. In xenografts, PAI-1/2(+) fibroblasts significantly reduce the apoptosis of MDA-MB-231 tumor cells. The current study provides further support for the “Autophagic Tumor Stroma Model of Cancer” and identifies a novel “extracellular matrix”-based signaling mechanism, by which a loss of stromal Cav-1 generates a metastatic phenotype. Thus, the secretion and remodeling of extracellular matrix components (such as PAI-1/2) can directly regulate both (1) autophagy in stromal fibroblasts and (2) epithelial tumor cell mitochondrial metabolism. PMID:21646868

  12. 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 Central

    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. PMID:27732616

  13. Smooth muscle differentiation in scleroderma fibroblastic cells.

    PubMed Central

    Sappino, A. P.; Masouyé, I.; Saurat, J. H.; Gabbiani, G.

    1990-01-01

    Using antibodies to alpha-smooth muscle actin and desmin on paraffin-embedded formalin-fixed tissue sections, the authors demonstrate that fibroblastic cells of localized and systemic scleroderma lesions express features of smooth muscle differentiation. Eleven of eleven skin specimens of systemic sclerosis patients and two of four skin specimens of localized scleroderma displayed the presence of fibroblasts expressing alpha-smooth muscle actin, a cell population that predominated in areas of prominent collagen deposition. A similar fibroblastic phenotype was found in the esophagus, the liver, and the lung specimens obtained from four patients who died of progressive systemic sclerosis. Immunostaining for desmin, performed on adjacent tissue sections, demonstrated that a minority of these fibroblastic cells present in skin and visceral lesions contained this protein. The authors' observations indicate that scleroderma fibroblasts are phenotypically related to the stromal cells previously identified in hypertrophic scars, fibromatoses, and desmoplasia; they might provide novel criteria for the characterization of scleroderma lesions and help to identify the factors responsible for phenotypic modulations in fibroblastic cells. Images Figure 1 Figure 2 Figure 3 PMID:1698026

  14. Endometrial stromal fibroblasts from women with polycystic ovary syndrome have impaired progesterone-mediated decidualization, aberrant cytokine profiles and promote enhanced immune cell migration in vitro

    PubMed Central

    Piltonen, T.T.; Chen, J.C.; Khatun, M.; Kangasniemi, M.; Liakka, A.; Spitzer, T.; Tran, N.; Huddleston, H.; Irwin, J.C.; Giudice, L.C.

    2015-01-01

    STUDY QUESTION Do endometrial stromal fibroblasts (eSF) in women with polycystic ovary syndrome (PCOS) (eSFpcos) exhibit altered estrogen and/or progesterone (P4) responses, which may explain some of the adverse reproductive outcomes and endometrial pathologies in these women? SUMMARY ANSWER In vitro, eSF from women with PCOS exhibit an aberrant decidualization response and concomitant changes in pro-inflammatory cytokine, chemokine and matrix metalloproteinase (MMP) release and immune cell chemoattraction. In vivo these aberrations may result in suboptimal implantation and predisposition to endometrial cancer. WHAT IS KNOWN ALREADY The endometrium in women with PCOS has several abnormalities including progesterone (P4) resistance at the gene expression level, likely contributing to subfertility, pregnancy complications and increased endometrial cancer risk in PCOS women. STUDY DESIGN, SIZE, DURATION Prospective, university-based, case–control, in vitro study. PARTICIPANTS/MATERIALS, SETTING, METHODS Cultures of eSFPCOS (n = 12, Rotterdam and NIH criteria) and eSFControl (Ctrl) (n = 6, regular cycle length, no signs of hyperandrogenism) were treated with vehicle, estradiol (E2, 10 nM) or E2P4 (10 nM/1 μM) for 14 days. Progesterone receptor (PGR) mRNA was assessed with quantitative real-time PCR (qRT–PCR) and eSF decidualization was confirmed by insulin-like growth factor-binding protein-1 (IGFBP-1) transcript and protein expression. Fractalkine (CX3CL1), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL) 6, 8 and 11, macrophage chemoattractant protein (MCP) 1 and 3, CCL5 (RANTES) and MMPs (MMP1, 2, 3, 7, 9, 10 and 12) were measured in conditioned media by Luminex multiplex assays, and chemotactic activity of the conditioned media was tested in a migration assay using CD14+ monocyte and CD4+ T-cell migration assay. Effects of IL-6 (0.02, 0.2, 2 or 20 ng/ml) or IL-8 (0.04, 0.4, 4, or 40 ng/ml) or combination (0.2 ng/ml IL-6 and 4.0 ng

  15. Metabolic reprogramming of cancer-associated fibroblasts by TGF-β drives tumor growth

    PubMed Central

    Guido, Carmela; Whitaker-Menezes, Diana; Capparelli, Claudia; Balliet, Renee; Lin, Zhao; Pestell, Richard G.; Howell, Anthony; Aquila, Saveria; Andò, Sebastiano; Martinez-Outschoorn, Ubaldo; Sotgia, Federica; Lisanti, Michael P.

    2012-01-01

    We have previously shown that a loss of stromal Cav-1 is a biomarker of poor prognosis in breast cancers. Mechanistically, a loss of Cav-1 induces the metabolic reprogramming of stromal cells, with increased autophagy/mitophagy, mitochondrial dysfunction and aerobic glycolysis. As a consequence, Cav-1-low CAFs generate nutrients (such as L-lactate) and chemical building blocks that fuel mitochondrial metabolism and the anabolic growth of adjacent breast cancer cells. It is also known that a loss of Cav-1 is associated with hyperactive TGF-β signaling. However, it remains unknown whether hyperactivation of the TGF-β signaling pathway contributes to the metabolic reprogramming of Cav-1-low CAFs. To address these issues, we overexpressed TGF-β ligands and the TGF-β receptor I (TGFβ-RI) in stromal fibroblasts and breast cancer cells. Here, we show that the role of TGF-β in tumorigenesis is compartment-specific, and that TGF-β promotes tumorigenesis by shifting cancer-associated fibroblasts toward catabolic metabolism. Importantly, the tumor-promoting effects of TGF-β are independent of the cell type generating TGF-β. Thus, stromal-derived TGF-β activates signaling in stromal cells in an autocrine fashion, leading to fibroblast activation, as judged by increased expression of myofibroblast markers, and metabolic reprogramming, with a shift toward catabolic metabolism and oxidative stress. We also show that TGF-β-activated fibroblasts promote the mitochondrial activity of adjacent cancer cells, and in a xenograft model, enhancing the growth of breast cancer cells, independently of angiogenesis. Conversely, activation of the TGF-β pathway in cancer cells does not influence tumor growth, but cancer cell-derived-TGF-β ligands affect stromal cells in a paracrine fashion, leading to fibroblast activation and enhanced tumor growth. In conclusion, ligand-dependent or cell-autonomous activation of the TGF-β pathway in stromal cells induces their metabolic

  16. Cancer-Associated Fibroblasts and Their Putative Role in Potentiating the Initiation and Development of Epithelial Ovarian Cancer1

    PubMed Central

    Schauer, Isaiah G; Sood, Anil K; Mok, Samuel; Liu, Jinsong

    2011-01-01

    The progression of ovarian cancer, from cell transformation through invasion of normal tissue, relies on communication between tumor cells and their adjacent stromal microenvironment. Through a natural selection process, an autocrine-paracrine communication loop establishes reciprocal reinforcement of growth and migration signals. Thus, the cancer-activated stromal response is similar to an off-switch-defective form of the normal, universal response needed to survive insult or injury. It is becoming clearer within the cancer literature base that tumor stroma plays a bimodal role in cancer development: it impedes neoplastic growth in normal tissue while encouraging migration and tumor growth in a co-opted desmoplastic response during tumor progression. In this review, we discuss this reciprocal influence that ovarian cancer epithelial cells may have on ovarian stromal cell-reactive phenotype, stromal cell behavior, disrupted signaling networks, and tumor suppressor status in the stroma, within the context of cancer fibroblast studies from alternate cancer tissue settings. We focus on the exchange of secreted factors, in particular interleukin 1β and SDF-1α, between activated fibroblasts and cancer cells as a key area for future investigation and therapeutic development. A better understanding of the bidirectional reliance of early epithelial cancer cells on activated stromal cells could lead to the identification of novel diagnostic stromal markers and targets for therapy. PMID:21532880

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

    PubMed

    Hsu, Hsiang-Chi; Liu, Liang-Chih; 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.

  18. 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

  19. 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

  20. Mitochondrial oxidative stress in cancer-associated fibroblasts drives lactate production, promoting breast cancer tumor growth: understanding the aging and cancer connection.

    PubMed

    Balliet, Renee M; Capparelli, Claudia; Guido, Carmela; Pestell, Timothy G; Martinez-Outschoorn, Ubaldo E; Lin, Zhao; Whitaker-Menezes, Diana; Chiavarina, Barbara; Pestell, Richard G; Howell, Anthony; Sotgia, Federica; Lisanti, Michael P

    2011-12-01

    Increasing chronological age is the most significant risk factor for cancer. Recently, we proposed a new paradigm for understanding the role of the aging and the tumor microenvironment in cancer onset. In this model, cancer cells induce oxidative stress in adjacent stromal fibroblasts. This, in turn, causes several changes in the phenotype of the fibroblast including mitochondrial dysfunction, hydrogen peroxide production, and aerobic glycolysis, resulting in high levels of L-lactate production. L-lactate is then transferred from these glycolytic fibroblasts to adjacent epithelial cancer cells and used as "fuel" for oxidative mitochondrial metabolism.  Here, we created a new pre-clinical model system to directly test this hypothesis experimentally. To synthetically generate glycolytic fibroblasts, we genetically-induced mitochondrial dysfunction by knocking down TFAM using an sh-RNA approach.  TFAM is mitochondrial transcription factor A, which is important in functionally maintaining the mitochondrial respiratory chain. Interestingly, TFAM-deficient fibroblasts showed evidence of mitochondrial dysfunction and oxidative stress, with the loss of certain mitochondrial respiratory chain components, and the over-production of hydrogen peroxide and L-lactate. Thus, TFAM-deficient fibroblasts underwent metabolic reprogramming towards aerobic glycolysis.  Most importantly, TFAM-deficient fibroblasts significantly promoted tumor growth, as assayed using a human breast cancer (MDA-MB-231) xenograft model. These increases in glycolytic fibroblast driven tumor growth were independent of tumor angiogenesis. Mechanistically, TFAM-deficient fibroblasts increased the mitochondrial activity of adjacent epithelial cancer cells in a co-culture system, as seen using MitoTracker. Finally, TFAM-deficient fibroblasts also showed a loss of caveolin-1 (Cav-1), a known breast cancer stromal biomarker. Loss of stromal fibroblast Cav-1 is associated with early tumor recurrence, metastasis

  1. Human corneal fibroblast migration and ECM synthesis during stromal repair: Role played by PDGF-BB, bFGF, and TGFβ1.

    PubMed

    Gallego-Muñoz, Patricia; Ibares-Frías, Lucía; Garrote, José A; Valsero-Blanco, María Cruz; Cantalapiedra-Rodríguez, Roberto; Merayo-Lloves, Jesús; Martínez-García, M Carmen

    2016-11-15

    The development of treatments that modulate corneal wound healing to avoid fibrosis during tissue repair is important for the restoration of corneal transparency after an injury. To date, few studies have studied the influence of growth factors (GFs) on human corneal fibroblast (HCF) expression of extracellular matrix (ECM) proteins such as collagen types I and III, proteoglycans such as perlecan, or proteins implicated in cellular migration such as α5β1-integrin and syndecan-4. Using in vitro HCFs, we developed a mechanical wound model to study the influence of the GFs basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF-BB), and transforming growth factor beta 1 (TGFβ1) on ECM protein production and cellular migration. Our results show that mechanical wounding provokes the autocrine release of bFGF and TGFβ1 at different time points during the wound closure. The HCF response to PDGF-BB was a rapid closure due to fast cellular migration associated with a high focal adhesion replacement and a high expression of collagen and proteoglycans, producing a non-fibrotic healing. bFGF stimulated non-fibrotic ECM production and limited the migration process. Finally, TGFβ1 induced expression of the fibrotic markers collagen type III and α5β1 integrin, and it inhibited cellular migration due to the formation of focal adhesions with a low turnover rate. The novel in vitro HCF mechanical wound model can be used to understand the role played by GFs in human corneal repair. The model can also be used to test the effects of different treatments aimed at improving the healing process.

  2. Evidence for a stromal-epithelial “lactate shuttle” in human tumors

    PubMed Central

    Lin, Zhao; Ertel, Adam; Flomenberg, Neal; Witkiewicz, Agnieszka K; Birbe, Ruth C; Howell, Anthony; Pavlides, Stephanos; Gandara, Ricardo; Pestell, Richard G; Sotgia, Federica

    2011-01-01

    Recently, we proposed a new mechanism for understanding the Warburg effect in cancer metabolism. In this new paradigm, cancer-associated fibroblasts undergo aerobic glycolysis, and extrude lactate to “feed” adjacent cancer cells, which then drives mitochondrial biogenesis and oxidative mitochondrial metabolism in cancer cells. Thus, there is vectorial transport of energy-rich substrates from the fibroblastic tumor stroma to anabolic cancer cells. A prediction of this hypothesis is that cancer-associated fibroblasts should express MCT4, a mono-carboxylate transporter that has been implicated in lactate efflux from glycolytic muscle fibers and astrocytes in the brain. To address this issue, we co-cultured MCF7 breast cancer cells with normal fibroblasts. Interestingly, our results directly show that breast cancer cells specifically induce the expression of MCT4 in cancer-associated fibroblasts; MCF7 cells alone and fibroblasts alone, both failed to express MCT4. We also show that the expression of MCT4 in cancer-associated fibroblasts is due to oxidative stress, and can be prevented by pre-treatment with the anti-oxidant N-acetyl-cysteine. In contrast to our results with MCT4, we see that MCT1, a transporter involved in lactate uptake, is specifically upregulated in MCF7 breast cancer cells when co-cultured with fibroblasts. Virtually identical results were also obtained with primary human breast cancer samples. In human breast cancers, MCT4 selectively labels the tumor stroma, e.g., the cancer-associated fibroblast compartment. Conversely, MCT1 was selectively expressed in the epithelial cancer cells within the same tumors. Functionally, we show that overexpression of MCT4 in fibroblasts protects both MCF7 cancer cells and fibroblasts against cell death, under co-culture conditions. Thus, we provide the first evidence for the existence of a stromal-epithelial lactate shuttle in human tumors, analogous to the lactate shuttles that are essential for the normal

  3. Metabolic reprogramming of cancer-associated fibroblasts by TGF-β drives tumor growth: connecting TGF-β signaling with "Warburg-like" cancer metabolism and L-lactate production.

    PubMed

    Guido, Carmela; Whitaker-Menezes, Diana; Capparelli, Claudia; Balliet, Renee; Lin, Zhao; Pestell, Richard G; Howell, Anthony; Aquila, Saveria; Andò, Sebastiano; Martinez-Outschoorn, Ubaldo; Sotgia, Federica; Lisanti, Michael P

    2012-08-15

    We have previously shown that a loss of stromal Cav-1 is a biomarker of poor prognosis in breast cancers. Mechanistically, a loss of Cav-1 induces the metabolic reprogramming of stromal cells, with increased autophagy/mitophagy, mitochondrial dysfunction and aerobic glycolysis. As a consequence, Cav-1-low CAFs generate nutrients (such as L-lactate) and chemical building blocks that fuel mitochondrial metabolism and the anabolic growth of adjacent breast cancer cells. It is also known that a loss of Cav-1 is associated with hyperactive TGF-β signaling. However, it remains unknown whether hyperactivation of the TGF-β signaling pathway contributes to the metabolic reprogramming of Cav-1-low CAFs. To address these issues, we overexpressed TGF-β ligands and the TGF-β receptor I (TGFβ-RI) in stromal fibroblasts and breast cancer cells. Here, we show that the role of TGF-β in tumorigenesis is compartment-specific, and that TGF-β promotes tumorigenesis by shifting cancer-associated fibroblasts toward catabolic metabolism. Importantly, the tumor-promoting effects of TGF-β are independent of the cell type generating TGF-β. Thus, stromal-derived TGF-β activates signaling in stromal cells in an autocrine fashion, leading to fibroblast activation, as judged by increased expression of myofibroblast markers, and metabolic reprogramming, with a shift toward catabolic metabolism and oxidative stress. We also show that TGF-β-activated fibroblasts promote the mitochondrial activity of adjacent cancer cells, and in a xenograft model, enhancing the growth of breast cancer cells, independently of angiogenesis. Conversely, activation of the TGF-β pathway in cancer cells does not influence tumor growth, but cancer cell-derived-TGF-β ligands affect stromal cells in a paracrine fashion, leading to fibroblast activation and enhanced tumor growth. In conclusion, ligand-dependent or cell-autonomous activation of the TGF-β pathway in stromal cells induces their metabolic

  4. 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

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

    SciTech Connect

    Kim, Sun-Ah; Lee, Eun Kyung; Kuh, Hyo-Jeong

    2015-07-15

    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. Active 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.

  6. Expression of smooth muscle-specific proteins in myoepithelium and stromal myofibroblasts of normal and malignant human breast tissue.

    PubMed Central

    Lazard, D; Sastre, X; Frid, M G; Glukhova, M A; Thiery, J P; Koteliansky, V E

    1993-01-01

    The expression of several differentiation markers in normal human mammary gland myoepithelium and in certain stromal fibroblasts ("myofibroblasts") associated with breast carcinomas was studied by immunofluorescence microscopy of frozen sections. Several antibodies to smooth muscle-specific proteins (smooth muscle alpha-actin, smooth muscle myosin heavy chains, calponin, alpha 1-integrin, and high molecular weight caldesmon) and to epithelial-specific proteins (cytokeratins, E-cadherin, and desmoplakin) were used to show that myoepithelial cells concomitantly express epithelial and smooth muscle markers whereas adjacent luminal cells express only epithelial markers. The same antibodies were used to establish that stromal myofibroblasts exhibit smooth muscle phenotypic properties characterized by the expression of all the smooth muscle markers examined except for high molecular weight caldesmon. In addition, both myoepithelium and myofibroblasts show a significant degree of heterogeneity in smooth muscle protein expression. Thus, myoepithelial cells and stromal myofibroblasts are epithelial and mesenchymal cells, respectively, which coordinately express a set of smooth muscle markers while maintaining their specific original features. The dual nature of myoepithelial cells and the phenotypic transition of fibroblasts to myofibroblasts are examples of the plasticity of the differentiated cell phenotype. Images PMID:8430113

  7. Caveolin-1 and mitochondrial SOD2 (MnSOD) function as tumor suppressors in the stromal microenvironment

    PubMed Central

    Trimmer, Casey; Sotgia, Federica; Whitaker-Menezes, Diana; Balliet, Renee M; Eaton, Gregory; Martinez-Outschoorn, Ubaldo E; Pavlides, Stephanos; Howell, Anthony; Iozzo, Renato V; Pestell, Richard G; Scherer, Philipp E

    2011-01-01

    We have recently proposed a new model for understanding tumor metabolism, termed: “The Autophagic Tumor Stroma Model of Cancer Metabolism”. In this new paradigm, catabolism (autophagy) in the tumor stroma fuels the anabolic growth of aggressive cancer cells. Mechanistically, tumor cells induce autophagy in adjacent cancer-associated fibroblasts via the loss of caveolin-1 (Cav-1), which is sufficient to promote oxidative stress in stromal fibroblasts. To further test this hypothesis, here we created human Cav-1 deficient immortalized fibroblasts using a targeted sh-RNA knock-down approach. Relative to control fibroblasts, Cav-1 deficient fibroblasts dramatically promoted tumor growth in xenograft assays employing an aggressive human breast cancer cell line, namely MDA-MB-231 cells. Co-injection of Cav-1 deficient fibroblasts, with MDA-MB-231 cells, increased both tumor mass and tumor volume by ∼4-fold. Immuno-staining with CD31 indicated that this paracrine tumor promoting effect was clearly independent of angiogenesis. Mechanistically, proteomic analysis of these human Cav-1 deficient fibroblasts identified >40 protein biomarkers that were upregulated, most of which were associated with (i) myofibroblast differentiation or (ii) oxidative stress/hypoxia. In direct support of these findings, the tumor promoting effects of Cav-1 deficient fibroblasts could be functionally suppressed (nearly 2-fold) by the recombinant overexpression of SOD2 (superoxide dismutase 2), a known mitochondrial enzyme that de-activates superoxide, thereby reducing mitochondrial oxidative stress. In contrast, cytoplasmic soluble SOD1 had no effect, further highlighting a specific role for mitochondrial oxidative stress in this process. In summary, here we provide new evidence directly supporting a key role for a loss of stromal Cav-1 expression and oxidative stress in cancerassociated fibroblasts, in promoting tumor growth, which is consistent with “The Autophagic Tumor Stroma Model of

  8. Senescent fibroblasts in melanoma initiation and progression: an integrated theoretical, experimental, and clinical approach.

    PubMed

    Kim, Eunjung; Rebecca, Vito; Fedorenko, Inna V; Messina, Jane L; Mathew, Rahel; Maria-Engler, Silvya S; Basanta, David; Smalley, Keiran S M; Anderson, Alexander R A

    2013-12-01

    We present an integrated study to understand the key role of senescent fibroblasts in driving melanoma progression. Based on the hybrid cellular automata paradigm, we developed an in silico model of normal skin. The model focuses on key cellular and microenvironmental variables that regulate interactions among keratinocytes, melanocytes, and fibroblasts, key components of the skin. The model recapitulates normal skin structure and is robust enough to withstand physical as well as biochemical perturbations. Furthermore, the model predicted the important role of the skin microenvironment in melanoma initiation and progression. Our in vitro experiments showed that dermal fibroblasts, which are an important source of growth factors in the skin, adopt a secretory phenotype that facilitates cancer cell growth and invasion when they become senescent. Our coculture experiments showed that the senescent fibroblasts promoted the growth of nontumorigenic melanoma cells and enhanced the invasion of advanced melanoma cells. Motivated by these experimental results, we incorporated senescent fibroblasts into our model and showed that senescent fibroblasts transform the skin microenvironment and subsequently change the skin architecture by enhancing the growth and invasion of normal melanocytes. The interaction between senescent fibroblasts and the early-stage melanoma cells leads to melanoma initiation and progression. Of microenvironmental factors that senescent fibroblasts produce, proteases are shown to be one of the key contributing factors that promoted melanoma development from our simulations. Although not a direct validation, we also observed increased proteolytic activity in stromal fields adjacent to melanoma lesions in human histology. This leads us to the conclusion that senescent fibroblasts may create a prooncogenic skin microenvironment that cooperates with mutant melanocytes to drive melanoma initiation and progression and should therefore be considered as a

  9. 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

  10. Analysis of stromal cell secretomes reveals a critical role for stromal cell-derived HGF and fibronectin in angiogenesis

    PubMed Central

    Newman, Andrew C.; Chou, Wayne; Welch-Reardon, Katrina M.; Fong, Ashley H.; Popson, Stephanie A.; Phan, Duc Thien; Sandoval, Daniel R.; Nguyen, Dananh P.; Gershon, Paul D.; Hughes, Christopher C. W.

    2013-01-01

    Objective Angiogenesis requires tightly coordinated cross-talk between endothelial cells and stromal cells such as fibroblasts and smooth muscle cells. The specific molecular mechanisms moderating this process are still poorly understood. Method and Results Stromal cell-derived factors are essential for endothelial cell sprouting and lumen formation. We therefore compared the abilities of two primary fibroblast isolates and a primary smooth muscle cell isolate to promote in vitro angiogenesis and analyzed their secretomes using a combination of nanoLC-MS/MS, qPCR and ELISA. Each isolate exhibited a different level of angiogenic ability. Using quantitative MS, we then compared the secretomes of a fibroblast isolate exhibiting low angiogenic activity, a fibroblast isolate exhibiting high angiogenic activity and human umbilical vein endothelial cells. High angiogenic fibroblast supernatants exhibited an over-abundance of proteins associated with extracellular matrix constituents compared to low angiogenic fibroblasts or endothelial cells. Finally, siRNA technology and purified protein were used to confirm a role for stromal cell-derived hepatocyte growth factor and fibronectin in inducing endothelial cell sprouting. Conclusion Differences in stromal cell ability to induce angiogenesis are due to differences in the secreted proteomes of both extracellular matrix proteins and pro-angiogenic growth factors. PMID:23288153

  11. Molecular transitions from papillomavirus infection to cervical precancer and cancer: Role of stromal estrogen receptor signaling

    PubMed Central

    den Boon, Johan A.; Pyeon, Dohun; Wang, Sophia S.; Horswill, Mark; Schiffman, Mark; Sherman, Mark; Zuna, Rosemary E.; Wang, Zhishi; Hewitt, Stephen M.; Pearson, Rachel; Schott, Meghan; Chung, Lisa; He, Qiuling; Lambert, Paul; Walker, Joan; Newton, Michael A.; Wentzensen, Nicolas; Ahlquist, Paul

    2015-01-01

    To study the multistep process of cervical cancer development, we analyzed 128 frozen cervical samples spanning normalcy, increasingly severe cervical intraepithelial neoplasia (CIN1– CIN3), and cervical cancer (CxCa) from multiple perspectives, revealing a cascade of progressive changes. Compared with normal tissue, expression of many DNA replication/repair and cell proliferation genes was increased in CIN1/CIN2 lesions and further sustained in CIN3, consistent with high-risk human papillomavirus (HPV)-induced tumor suppressor inactivation. The CIN3-to-CxCa transition showed metabolic shifts, including decreased expression of mitochondrial electron transport complex components and ribosomal protein genes. Significantly, despite clinical, epidemiological, and animal model results linking estrogen and estrogen receptor alpha (ERα) to CxCa, ERα expression declined >15-fold from normalcy to cancer, showing the strongest inverse correlation of any gene with the increasing expression of p16, a marker for HPV-linked cancers. This drop in ERα in CIN and tumor cells was confirmed at the protein level. However, ERα expression in stromal cells continued throughout CxCa development. Our further studies localized stromal ERα to FSP1+, CD34+, SMA− precursor fibrocytes adjacent to normal and precancerous CIN epithelium, and FSP1−, CD34−, SMA+ activated fibroblasts in CxCas. Moreover, rank correlations with ERα mRNA identified IL-8, CXCL12, CXCL14, their receptors, and other angiogenesis and immune cell infiltration and inflammatory factors as candidates for ERα-induced stroma–tumor signaling pathways. The results indicate that estrogen signaling in cervical cancer has dramatic differences from ERα+ breast cancers, and imply that estrogen signaling increasingly proceeds indirectly through ERα in tumor-associated stromal fibroblasts. PMID:26056290

  12. Pancreatic fibroblasts smoothen their activities via AKT–GLI2–TGFα

    PubMed Central

    Rustgi, Anil K.

    2016-01-01

    Pancreatic stromal fibroblasts provide structural support. Activated fibroblasts are critical in the tumor microenvironment. In this issue of Genes & Development, Liu and colleagues (pp. 1943–1955) unravel the finding that depletion of Smoothened (Smo) in pancreatic stromal fibroblasts results in AKT activation and noncanonical GLI2 activation with subsequent TGFα secretion, activation of EGFR in pancreatic epithelial cells, and augmentation of acinar–ductal metaplasia. Additionally, Smo-mediated signaling has proproliferative effects on pancreatic tumor cells. PMID:27664234

  13. Differential regulation of extracellular matrix protein expression in carcinoma-associated fibroblasts by TGF-β1 regulates cancer cell spreading but not adhesion

    PubMed Central

    Van Bockstal, Mieke; Lambein, Kathleen; Van Gele, Mireille; De Vlieghere, Elly; Limame, Ridha; Braems, Geert; Van den Broecke, Rudy; Cocquyt, Veronique; Denys, Hannelore; Bracke, Marc; Libbrecht, Louis; De Wever, Olivier

    2014-01-01

    Cancer progression is characterized by a complex reciprocity between neoplastic epithelium and adjacent stromal cells. In ductal carcinoma in situ (DCIS) of the breast, both reduced stromal decorin expression and myxoid stroma are correlated with increased recurrence risk. In this study, we aimed to investigate paracrine regulation of expression of decorin and related extracellular matrix (ECM) proteins in cancer-associated fibroblasts (CAFs). Transforming growth factor-β1 (TGF-β1) was identified as a competent ECM modulator, as it reduced decorin and strongly enhanced versican, biglycan and type I collagen expression. Similar but less pronounced effects were observed when fibroblasts were treated with basic fibroblast growth factor (bFGF). Despite this concerted ECM modulation, TGF-β1 and bFGF differentially regulated alpha-smooth muscle actin (α-SMA) expression, which is often proposed as a CAF-marker. Cancer cell-derived secretomes induced versican and biglycan expression in fibroblasts. Immunohistochemistry on twenty DCIS specimens showed a trend toward periductal versican overexpression in DCIS with myxoid stroma. Cancer cell adhesion was inhibited by decorin, but not by CAF-derived matrices. Cancer cells presented significantly enhanced spreading when seeded on matrices derived from TGF-β1-treated CAF. Altogether these data indicate that preinvasive cancerous lesions might modulate the composition of surrounding stroma through TGF-β1 release to obtain an invasion-permissive microenvironment. PMID:25593993

  14. The Interaction Between Human Papillomaviruses and the Stromal Microenvironment.

    PubMed

    Woodby, B; Scott, M; Bodily, J

    2016-01-01

    Human papillomaviruses (HPVs) 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, focusing on stromal fibroblasts, immune cells, and endothelial cells. 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.

  15. 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

  16. Gastric stromal tumor.

    PubMed

    Ovali, Gülgün Yilmaz; Tarhan, Serdar; Serter, Selim; Pabuşçu, Yüksel

    2005-06-01

    Gastric stromal tumors are rare neoplasms of the stomach. In this report we present a gastric stromal tumor with an exophytic growth pattern, and describe magnetic resonance imaging and endoscopic ultrasonography findings.

  17. The Role of Stromally Produced Cathepsin D in Promoting Prostate Tumorigenesis

    DTIC Science & Technology

    2014-11-01

    beta (TGF-β) and stromal cell derived factor-1 ( SDF -1) as being overexpressed in CAF cells. These factors were 11 Figure 7. Stromal specific...expressed in cancer associated fibroblasts (CAFs) (including cyclin D1 [CD1], TGFß and SDF -1) induce tumorigenesis and malignant transformation in tissue

  18. 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.

  19. 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

  20. Melanoma Cells Block PEDF Production in Fibroblasts to Induce the Tumor-Promoting Phenotype of Cancer-Associated Fibroblasts.

    PubMed

    Nwani, Nkechiyere G; Deguiz, Maria L; Jimenez, Benilde; Vinokour, Elena; Dubrovskyi, Oleksii; Ugolkov, Andrey; Mazar, Andrew P; Volpert, Olga V

    2016-04-15

    Loss of pigment epithelium-derived factor (PEDF, SERPINF1) in cancer cells is associated with poor prognosis and metastasis, but the contribution of stromal PEDF to cancer evolution is poorly understood. Therefore, we investigated the role of fibroblast-derived PEDF in melanoma progression. We demonstrate that normal dermal fibroblasts expressing high PEDF levels attenuated melanoma growth and angiogenesis in vivo, whereas PEDF-depleted fibroblasts exerted tumor-promoting effects. Accordingly, mice with global PEDF knockout were more susceptible to melanoma metastasis. We also demonstrate that normal fibroblasts in close contact with PEDF-null melanoma cells lost PEDF expression and tumor-suppressive properties. Further mechanistic investigations underlying the crosstalk between tumor and stromal cells revealed that melanoma cells produced PDGF-BB and TGFβ, which blocked PEDF production in fibroblasts. Notably, cancer-associated fibroblasts (CAF) isolated from patient-derived tumors expressed markedly low levels of PEDF. Treatment of patient CAF and TGFβ-treated normal fibroblasts with exogenous PEDF decreased the expression of CAF markers and restored PEDF expression. Finally, expression profiling of PEDF-depleted fibroblasts revealed induction of IL8, SERPINB2, hyaluronan synthase-2, and other genes associated with tumor promotion and metastasis. Collectively, our results demonstrate that PEDF maintains tumor-suppressive functions in fibroblasts to prevent CAF conversion and illustrate the mechanisms by which melanoma cells silence stromal PEDF to promote malignancy. Cancer Res; 76(8); 2265-76. ©2016 AACR.

  1. 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.

  2. [Frequent allelic losses in tumor-associated stromal cells and tumor epitelium of prostate cancer].

    PubMed

    Kekeeva, T V; Popova, O P; Shegaĭ, P V; Zavalishina, L E; Andreeva, Iu Iu; Zaletaev, D V; Nemtsova, M V

    2008-01-01

    It has become increasingly clear that tumor microenvironment plays a critical role in carcinogenesis. Accumulation of genetic alterations is typical not only for cancer epithelial cells but tumor-associated fibroblasts as well. Tumor epithelia, tumor-associated stroma from prostatectomy specimens of patients with prostate cancer and cells from prostatic intraepithelial neoplasia (PIN) and adjacent stroma from males with PIN were isolated by using laser capture microdissection. Microsatellite allelotyping was evaluated using 4 highly polymorphic markers for chromosomal regions 8p22, 16q23-24 and 13q14. Incidences of alterations (loss of heterozygosity or allelic imbalance) were 48% for region 8p22, 72% for 16q23 and 37% for 13q14. The LOH frequencies in tumor-associated stroma cells were very similar. Alterations at chromosome 13q were significantly associated with advanced tumor stage, whereas AI at 16q was also associated with high Gleason score and lymph node metastasis. We find some incidences of allelic imbalance in premalignant lesions in epithelial (16-27%) and stromal (7-22%) components. Our results show that the frequencies of genetic aberrations are as high in stromal cells as in tumor cells.

  3. Correlation of vascular endothelial growth factor expression with fibroblast growth factor-8 expression and clinico-pathologic parameters in human prostate cancer

    PubMed Central

    West, A F; O'Donnell, M; Charlton, R G; Neal, D E; Leung, H Y

    2001-01-01

    Vascular endothelial growth factor (VEGF) mediates neo-angiogenesis during tumour progression and is known to cooperate with the fibroblast growth factor (FGF) system to facilitate angiogenesis in a synergistic manner. In view of this, we have investigated VEGF expression in 67 cases of prostate cancer previously characterized for fibroblast growth factor-8 (FGF-8) expression. Cytoplasmic VEGF staining was detected in malignant cells in 45 out of 67 cases. Cytoplasmic staining was found in adjacent stromal cells in 32 cases, being particularly strong around nests of invasive tumour. Positive VEGF immunoreactivity in benign glands was restricted to basal epithelium. A significant association was observed between tumour VEGF and FGF-8 expression (P = 0.004). We identified increased VEGF immunoreactivity in both malignant epithelium and adjacent stroma and both were found to be significantly associated with high tumour stage (P = 0.0047 and P = 0.0002, respectively). VEGF expression also correlated with increased serum PSA levels (P = 0.01). Among positively stained tumours, VEGF expression showed a significant association with Gleason score (P = 0.04). Cases showing positive VEGF immunoreactivity in the stroma had a significantly reduced survival rate compared to those with negative staining (P = 0.037). Cases with tumours expressing both FGF-8 in the malignant epithelium and VEGF in the adjacent stroma had a significantly worse survival rate than those with tumours negative for both, or only expressing one of the two growth factors (P = 0.029). Cox multivariate regression analysis of survival demonstrated that stromal VEGF and tumour stage were the most significant independent predictors of survival. In conclusion, we report for the first time a correlation of both tumour and stromal VEGF expression in prostate cancer with clinical parameters as well as its correlation to FGF-8 expression. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11506499

  4. 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

  5. Cellular and molecular phenotypes of proliferating stromal cells from human carcinomas

    PubMed Central

    Kopantzev, E P; Vayshlya, N A; Kopantseva, M R; Egorov, V I; Pikunov, M; Zinovyeva, M V; Vinogradova, T V; Zborovskaya, I B; Sverdlov, E D

    2010-01-01

    Background: Stromal cells are a functionally important component of human carcinomas. The aim of this study was to obtain and characterise primary cultures of stromal cells from human carcinomas and the corresponding surrounding normal tissue. Methods: Primary stromal cell cultures from tumours of lung, oesophagus and pancreas were obtained using a mild tissue dissociation method and a medium for culturing mesenchymal cells. Immunofluorescence staining and western blotting were used to analyse the expression of differentiation markers and selected known oncoproteins in the cell cultures obtained. Results: A panel of stromal primary cultures was prepared from different human tumours and from matched normal cancer-free tissues. The in vitro proliferative potential of tumour-associated fibroblasts was shown to be higher than that of matched normal stromal cells. A mutational analysis of the TP53 and KRAS2 genes in a number of stromal cultures did not reveal known mutations in most cells of the cultures studied. Western blot analysis showed that stromal cells of lung tumours were characterised by a statistically significantly lower expression level of the p16 protein as compared with that in normal lung stromal cells. An important finding of our study was that, according to immunofluorescence assay, a fraction of fibroblast-like vimentin-positive cells in some tumour and normal stromal cell cultures expressed an epithelial marker – cytokeratins. Conclusions: Proliferating stromal cells from the carcinomas studied proved to be genetically normal cells with altered expression profiles of some genes involved in carcinogenesis, as compared with normal stromal cells. Epithelial-mesenchymal transition may lead to the emergence of transdifferentiated fibroblast-like cells in tumour stroma and in the tumour-surrounding tissue. PMID:20407446

  6. Decellularization of human stromal refractive lenticules for corneal tissue engineering

    PubMed Central

    Yam, Gary Hin-Fai; Yusoff, Nur Zahirah Binte M.; Goh, Tze-Wei; Setiawan, Melina; Lee, Xiao-Wen; Liu, Yu-Chi; Mehta, Jodhbir S.

    2016-01-01

    Small incision lenticule extraction (SMILE) becomes a procedure to correct myopia. The extracted lenticule can be used for other clinical scenarios. To prepare for allogeneic implantation, lenticule decellularization with preserved optical property, stromal architecture and chemistry would be necessary. We evaluated different methods to decellularize thin human corneal stromal lenticules created by femtosecond laser. Treatment with 0.1% sodium dodecylsulfate (SDS) followed by extensive washes was the most efficient protocol to remove cellular and nuclear materials. Empty cell space was found inside the stroma, which displayed aligned collagen fibril architecture similar to native stroma. The SDS-based method was superior to other treatments with hyperosmotic 1.5 M sodium chloride, 0.1% Triton X-100 and nucleases (from 2 to 10 U/ml DNase and RNase) in preserving extracellular matrix content (collagens, glycoproteins and glycosaminoglycans). The stromal transparency and light transmittance was indifferent to untreated lenticules. In vitro recellularization showed that the SDS-treated lenticules supported corneal stromal fibroblast growth. In vivo re-implantation into a rabbit stromal pocket further revealed the safety and biocompatibility of SDS-decellularized lenticules without short- and long-term rejection risk. Our results concluded that femtosecond laser-derived human stromal lenticules decellularized by 0.1% SDS could generate a transplantable bioscaffold with native-like stromal architecture and chemistry. PMID:27210519

  7. 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

  8. Senescent stromal-derived osteopontin promotes preneoplastic cell growth.

    PubMed

    Pazolli, Ermira; Luo, Xianmin; Brehm, Sarah; Carbery, Kelly; Chung, Jun-Jae; Prior, Julie L; Doherty, Jason; Demehri, Shadmehr; Salavaggione, Lorena; Piwnica-Worms, David; Stewart, Sheila A

    2009-02-01

    Alterations in the tissue microenvironment collaborate with cell autonomous genetic changes to contribute to neoplastic progression. The importance of the microenvironment in neoplastic progression is underscored by studies showing that fibroblasts isolated from a tumor stimulate the growth of preneoplastic and neoplastic cells in xenograft models. Similarly, senescent fibroblasts promote preneoplastic cell growth in vitro and in vivo. Because senescent cells accumulate with age, their presence is hypothesized to facilitate preneoplastic cell growth and tumor formation in older individuals. To identify senescent stromal factors directly responsible for stimulating preneoplastic cell growth, we carried out whole-genome transcriptional profiling and compared senescent fibroblasts with their younger counterparts. We identified osteopontin (OPN) as one of the most highly elevated transcripts in senescent fibroblasts. Importantly, reduction of OPN protein levels by RNA interference did not affect senescence induction in fibroblasts; however, it dramatically reduced the growth-promoting activities of senescent fibroblasts in vitro and in vivo, showing that OPN is necessary for paracrine stimulation of preneoplastic cell growth. In addition, we found that recombinant OPN was sufficient to stimulate preneoplastic cell growth. Finally, we show that OPN is expressed in senescent stroma within preneoplastic lesions that arise following 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate treatment of mice, suggesting that stromal-derived OPN-mediated signaling events affect neoplastic progression.

  9. Senescent Stromal-Derived Osteopontin Promotes Preneoplastic Cell Growth

    PubMed Central

    Pazolli, Ermira; Luo, Xianmin; Brehm, Sarah; Carbery, Kelly; Chung, Jun-Jae; Prior, Julie L.; Doherty, Jason; Demehri, Shadmehr; Salavaggione, Lorena; Piwnica-Worms, David; Stewart, Sheila A.

    2008-01-01

    Alterations in the tissue microenvironment collaborate with cell autonomous genetic changes to contribute to neoplastic progression. The importance of the microenvironment in neoplastic progression is underscored by studies demonstrating that fibroblasts isolated from a tumor stimulate the growth of preneoplastic and neoplastic cells in xenograft models. Similarly, senescent fibroblasts promote preneoplastic cell growth in vitro and in vivo. Because senescent cells accumulate with age, their presence is hypothesized to facilitate preneoplastic cell growth and tumor formation in older individuals. To identify senescent stromal factors directly responsible for stimulating preneoplastic cell growth, we carried out whole genome transcriptional profiling and compared senescent fibroblasts to their younger counterparts. We identified osteopontin (OPN) as one of the most highly elevated transcripts in senescent fibroblasts. Importantly, reduction of OPN protein levels by RNAi did not impact senescence induction in fibroblasts; however, it dramatically reduced the growth-promoting activities of senescent fibroblasts in vitro and in vivo, demonstrating that OPN is necessary for paracrine stimulation of preneoplastic cell growth. In addition, we found that recombinant OPN was sufficient to stimulate preneoplastic cell growth. Finally, we demonstrate that OPN is expressed in senescent stroma within preneoplastic lesions that arise following DMBA/TPA treatment of mice, suggesting that stromal-derived OPN-mediated signaling events impact neoplastic progression. PMID:19155301

  10. Fibroblast cell interactions with human melanoma cells affect tumor cell growth as a function of tumor progression.

    PubMed Central

    Cornil, I; Theodorescu, D; Man, S; Herlyn, M; Jambrosic, J; Kerbel, R S

    1991-01-01

    It is known from a variety of experimental systems that the ability of tumor cells to grow locally and metastasize can be affected by the presence of adjacent normal tissues and cells, particularly mesenchymally derived stromal cells such as fibroblasts. However, the comparative influence of such normal cell-tumor cell interactions on tumor behavior has not been thoroughly investigated from the perspective of different stages of tumor progression. To address this question we assessed the influence of normal dermal fibroblasts on the growth of human melanoma cells obtained from different stages of tumor progression. We found that the in vitro growth of most (4 out of 5) melanoma cell lines derived from early-stage radial growth phase or vertical growth phase metastatically incompetent primary lesions is repressed by coculture with normal dermal fibroblasts, suggesting that negative homeostatic growth controls are still operative on melanoma cells from early stages of disease. On the other hand, 9 out of 11 melanoma cell lines derived from advanced metastatically competent vertical growth phase primary lesions, or from distant metastases, were found to be consistently stimulated to grow in the presence of dermal fibroblasts. Evidence was obtained to show that this discriminatory fibroblastic influence is mediated by soluble inhibitory and stimulatory growth factor(s). Taken together, these results indicate that fibroblast-derived signals can have antithetical growth effects on metastatic versus metastatically incompetent tumor subpopulations. This resultant conversion in responsiveness to host tissue environmental factors may confer upon small numbers of metastatically competent cells a growth advantage, allowing them to escape local growth constraints both in the primary tumor site and at distant ectopic tissue sites. PMID:2068080

  11. Transplantation of a mammary stromal cell line into a mammary fat pad: development of the site-specific in vivo analysis system for mammary stromal cells.

    PubMed

    Nakatani, Hajime; Aoki, Naohito; Nadano, Daita; Matsuda, Tsukasa

    2011-01-01

    The interaction between mammary epithelial and stromal tissue is considered to be important in breast tissue development. In this study, we developed a transplantation procedure for the mammary stromal fibroblastic cell line (MSF) to examine its life in vivo. First we established MSF cells which stably expressed lacZ (lacZ/MSF) and had characteristics of mammary stromal cells. The lacZ/MSF cells were then transplanted into a cleared mammary fat pad of syngenic mice with and without mammary primary epithelial organoids. Whole mount X-gal and carmine staining of the transplants revealed that a number of undifferentiated lacZ/MSF cells survived around the mammary epithelial tissue when transplanted with organoids. These results indicate that transplantation of MSF cells into mammary fat pad was accomplished by co-transplantation with primary mammary organoids. Finally, we discuss the application of transplantation procedure for in vivo studies of the mammary stromal tissue development and stromal-epithelial interactions.

  12. Stromal-cell and cancer-cell exosomes leading the metastatic exodus for the promised niche.

    PubMed

    Hoffman, Robert M

    2013-06-18

    Exosomes are thought to play an important role in metastasis. Luga and colleagues have described the production of exosomes by stromal cells such as cancer-associated fibroblasts that are taken up by breast cancer cells and are then loaded with Wnt 11, which is associated with stimulation of the invasiveness and metastasis of the breast cancer cells. Previous studies have shown that exosomes produced by breast cancer cells are taken up by stromal fibroblasts and other stromal cells, suggesting that exosomes are agents of cross-talk between cancer and stromal cells to stimulate metastasis. Imaging of exosomes by labeling with fluorescent proteins will enlighten the process by which exosomes enhance metastasis, including premetastatic niche formation.

  13. 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

  14. Targeting stromal androgen receptor suppresses prolactin-driven benign prostatic hyperplasia (BPH).

    PubMed

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

    2013-10-01

    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.

  15. [Fibroblast growth factor-2].

    PubMed

    Faitová, J

    2004-01-01

    Fibroblast growth factor-2 is a member of a large family of proteins that bind heparin and heparan sulfate and modulate the function of a wide range of cell types. FGF-2 occurs in several isoforms resulting from alternative initiations of traslation: an 18 kDa cytoplasmic isoform and four larger molecular weight nuclear isoforms (22, 22.5, 24 and 34 kDa). It acts mainly through a paracrine/autocrine mechanism involving high affinity transmembrane receptors and heparan sulfate proteoglycan low affinity receptors. It is expressed mostly in tissues of mesoderm and neuroectoderm origin, and plays an important role in mesoderm induction, stimulates the growth and development of the new blood vessels (angiogenesis), normal wound healing and tissue development. FGF-2 positively regulates hematopoiesis by acting on various cellular targets: stromal cells, early and committed hematopoietic progenitors and possibly some mature blood cells. FGF-2 is a potent hematopoietic growth factor that is likely to play an important role in physiological and pathological hematopoiesis.

  16. Stromal response to Hedgehog signaling restrains pancreatic cancer progression.

    PubMed

    Lee, John J; Perera, Rushika M; Wang, Huaijun; Wu, Dai-Chen; Liu, X Shawn; Han, Shiwei; Fitamant, Julien; Jones, Phillip D; Ghanta, Krishna S; Kawano, Sally; Nagle, Julia M; Deshpande, Vikram; Boucher, Yves; Kato, Tomoyo; Chen, James K; Willmann, Jürgen K; Bardeesy, Nabeel; Beachy, Philip A

    2014-07-29

    Pancreatic ductal adenocarcinoma (PDA) is the most lethal of common human malignancies, with no truly effective therapies for advanced disease. Preclinical studies have suggested a therapeutic benefit of targeting the Hedgehog (Hh) signaling pathway, which is activated throughout the course of PDA progression by expression of Hh ligands in the neoplastic epithelium and paracrine response in the stromal fibroblasts. Clinical trials to test this possibility, however, have yielded disappointing results. To further investigate the role of Hh signaling in the formation of PDA and its precursor lesion, pancreatic intraepithelial neoplasia (PanIN), we examined the effects of genetic or pharmacologic inhibition of Hh pathway activity in three distinct genetically engineered mouse models and found that Hh pathway inhibition accelerates rather than delays progression of oncogenic Kras-driven disease. Notably, pharmacologic inhibition of Hh pathway activity affected the balance between epithelial and stromal elements, suppressing stromal desmoplasia but also causing accelerated growth of the PanIN epithelium. In striking contrast, pathway activation using a small molecule agonist caused stromal hyperplasia and reduced epithelial proliferation. These results indicate that stromal response to Hh signaling is protective against PDA and that pharmacologic activation of pathway response can slow tumorigenesis. Our results provide evidence for a restraining role of stroma in PDA progression, suggesting an explanation for the failure of Hh inhibitors in clinical trials and pointing to the possibility of a novel type of therapeutic intervention.

  17. Colon cancer cell-derived 12(S)-HETE induces the retraction of cancer-associated fibroblast via MLC2, RHO/ROCK and Ca(2+) signalling.

    PubMed

    Stadler, Serena; Nguyen, Chi Huu; Schachner, Helga; Milovanovic, Daniela; Holzner, Silvio; Brenner, Stefan; Eichsteininger, Julia; Stadler, Mira; Senfter, Daniel; Krenn, Liselotte; Schmidt, Wolfgang M; Huttary, Nicole; Krieger, Sigurd; Koperek, Oskar; Bago-Horvath, Zsuzsanna; Brendel, Konstantin Alexander; Marian, Brigitte; de Wever, Oliver; Mader, Robert M; Giessrigl, Benedikt; Jäger, Walter; Dolznig, Helmut; Krupitza, Georg

    2016-12-24

    Retraction of mesenchymal stromal cells supports the invasion of colorectal cancer cells (CRC) into the adjacent compartment. CRC-secreted 12(S)-HETE enhances the retraction of cancer-associated fibroblasts (CAFs) and therefore, 12(S)-HETE may enforce invasivity of CRC. Understanding the mechanisms of metastatic CRC is crucial for successful intervention. Therefore, we studied pro-invasive contributions of stromal cells in physiologically relevant three-dimensional in vitro assays consisting of CRC spheroids, CAFs, extracellular matrix and endothelial cells, as well as in reductionist models. In order to elucidate how CAFs support CRC invasion, tumour spheroid-induced CAF retraction and free intracellular Ca(2+) levels were measured and pharmacological- or siRNA-based inhibition of selected signalling cascades was performed. CRC spheroids caused the retraction of CAFs, generating entry gates in the adjacent surrogate stroma. The responsible trigger factor 12(S)-HETE provoked a signal, which was transduced by PLC, IP3, free intracellular Ca(2+), Ca(2+)-calmodulin-kinase-II, RHO/ROCK and MYLK which led to the activation of myosin light chain 2, and subsequent CAF mobility. RHO activity was observed downstream as well as upstream of Ca(2+) release. Thus, Ca(2+) signalling served as central signal amplifier. Treatment with the FDA-approved drugs carbamazepine, cinnarizine, nifedipine and bepridil HCl, which reportedly interfere with cellular calcium availability, inhibited CAF-retraction. The elucidation of signalling pathways and identification of approved inhibitory drugs warrant development of intervention strategies targeting tumour-stroma interaction.

  18. Distinct effects of SIRT1 in cancer and stromal cells on tumor promotion.

    PubMed

    Shin, Dong Hoon; Choi, Yong-Joon; Jin, Peng; Yoon, Haejin; Chun, Yang-Sook; Shin, Hyun-Woo; Kim, Ja-Eun; Park, Jong-Wan

    2016-04-26

    The lysyl deacetylase SIRT1 acts as a metabolic sensor in adjusting metabolic imbalance. To explore the role of SIRT1 in tumor-stroma interplay, we designed an in vivo tumor model using SIRT1-transgenic mice. B16F10 mouse melanoma grew more quickly in SIRT1-transgenic mice than in wild-type mice, whereas SIRT1-overexpressing one grew slowly in both mice. Of human tumors, SIRT1 expression in stromal fibroblasts was found to correlate with poor prognosis in ovarian cancer. B16F10 and human ovarian cancer (SKOV3 and SNU840) cells were more proliferative in co-culture with SIRT1-overexpressiong fibroblasts. In contrast, SIRT1 within cancer cells has a negative effect on cell proliferation. In conditioned media from SIRT1-overexpressing fibroblasts, matrix metalloproteinase-3 (MMP3) was identified in cytokine arrays to be secreted from fibroblasts SIRT1-dependently. Fibroblast-derived MMP3 stimulated cancer cell proliferation, and such a role of MMP3 was also demonstrated in cancer/fibroblast co-grafts. In conclusion, SIRT1 plays differential roles in cancer and stromal cells. SIRT1 in stromal cells promotes cancer growth by producing MMP3, whereas SIRT1 in cancer cells inhibits growth via an intracellular event. The present study provides a basis for setting new anticancer strategies targeting SIRT1.

  19. 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

  20. Antitumor effects in gastrointestinal stromal tumors using photodynamic therapy with a novel glucose-conjugated chlorin.

    PubMed

    Tanaka, Mamoru; Kataoka, Hiromi; Yano, Shigenobu; Ohi, Hiromi; Moriwaki, Kazuhiro; Akashi, Haruo; Taguchi, Takahiro; Hayashi, Noriyuki; Hamano, Shingo; Mori, Yoshinori; Kubota, Eiji; Tanida, Satoshi; Joh, Takashi

    2014-04-01

    Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the gastrointestinal tract. Except for surgical resection, no effective treatment strategies have been established. Photodynamic therapy (PDT) consists of intravenous administration of a photosensitizer, activated by a specific wavelength of light, which produces reactive oxygen species that directly kill tumor cells. We analyzed the efficacy of PDT using a newly developed photosensitizer, 5,10,15,20-tetrakis [4-[β-d-glucopyranosylthio-2,3,5,6-tetrafluorophenyl]-2,3,[methano[N-methyl] iminomethano] chlorin (H(2)TFPC-SGlc), for the GIST treatment. Various photosensitizers were administered in vitro to GIST (GIST-T1) and fibroblast (WI-38) cells, followed by irradiation, after which cell death was compared. We additionally established xenograft mouse models with GIST-T1 tumors and examined the accumulation and antitumor effects of these photosensitizers in vivo. In vitro, the expression of the glucose transporters GLUT1, GLUT3, and GLUT4, the cellular uptake of H(2)TFPC-SGlc, and apoptosis mediated by PDT with H(2)TFPC-SGlc were significantly higher in GIST-T1 than in WI-38 cells. In vivo, H(2)TFPC-SGlc accumulation was higher in xenograft tumors of GIST-T1 cells than in the adjacent normal tissue, and tumor growth was significantly suppressed following PDT. PDT with novel H(2)TFPC-SGlc is potentially useful for clinical applications about the treatment of GIST.

  1. Inhibition of tumor-stromal interaction through HGF/Met signaling by valproic acid

    SciTech Connect

    Matsumoto, Yohsuke; Motoki, Takahiro; Kubota, Satoshi; Takigawa, Masaharu; Tsubouchi, Hirohito; Gohda, Eiichi

    2008-02-01

    Hepatocyte growth factor (HGF), which is produced by surrounding stromal cells, including fibroblasts and endothelial cells, has been shown to be a significant factor responsible for cancer cell invasion mediated by tumor-stromal interactions. We found in this study that the anti-tumor agent valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, strongly inhibited tumor-stromal interaction. VPA inhibited HGF production in fibroblasts induced by epidermal growth factor (EGF), platelet-derived growth factor, basic fibroblast growth factor, phorbol 12-myristate 13-acetate (PMA) and prostaglandin E{sub 2} without any appreciable cytotoxic effect. Other HDAC inhibitors, including butyric acid and trichostatin A (TSA), showed similar inhibitory effects on HGF production stimulated by various inducers. Up-regulations of HGF gene expression induced by PMA and EGF were also suppressed by VPA and TSA. Furthermore, VPA significantly inhibited HGF-induced invasion of HepG2 hepatocellular carcinoma cells. VPA, however, did not affect the increases in phosphorylation of MAPK and Akt in HGF-treated HepG2 cells. These results demonstrated that VPA inhibited two critical processes of tumor-stromal interaction, induction of fibroblastic HGF production and HGF-induced invasion of HepG2 cells, and suggest that those activities serve for other anti-tumor mechanisms of VPA besides causing proliferation arrest, differentiation, and/or apoptosis of tumor cells.

  2. 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

  3. Fibroblast nemosis induces angiogenic responses of endothelial cells

    SciTech Connect

    Enzerink, Anna; Rantanen, Ville; Vaheri, Antti

    2010-03-10

    Increasing evidence points to a central link between inflammation and activation of the stroma, especially of fibroblasts therein. However, the mechanisms leading to such activation mostly remain undescribed. We have previously characterized a novel type of fibroblast activation (nemosis) where clustered fibroblasts upregulated the production of cyclooxygenase-2, secretion of prostaglandins, proteinases, chemotactic cytokines, and hepatocyte growth factor (HGF), and displayed activated nuclear factor-{kappa}B. Now we show that nemosis drives angiogenic responses of endothelial cells. In addition to HGF, nemotic fibroblasts secreted vascular endothelial growth factor (VEGF), and conditioned medium from spheroids promoted sprouting and networking of human umbilical venous endothelial cells (HUVEC). The response was partly inhibited by function-blocking antibodies against HGF and VEGF. Conditioned nemotic fibroblast medium promoted closure of HUVEC and human dermal microvascular endothelial cell monolayer wounds, by increasing the motility of the endothelial cells. Wound closure in HUVEC cells was partly inhibited by the antibodies against HGF. The stromal microenvironment regulates wound healing responses and often promotes tumorigenesis. Nemosis offers clues to the activation process of stromal fibroblasts and provides a model to study the part they play in angiogenesis-related conditions, as well as possibilities for therapeutical approaches desiring angiogenesis in tissue.

  4. Infection Programs Sustained Lymphoid Stromal Cell Responses and Shapes Lymph Node Remodeling upon Secondary Challenge.

    PubMed

    Gregory, Julia L; Walter, Anne; Alexandre, Yannick O; Hor, Jyh Liang; Liu, Ruijie; Ma, Joel Z; Devi, Sapna; Tokuda, Nobuko; Owada, Yuji; Mackay, Laura K; Smyth, Gordon K; Heath, William R; Mueller, Scott N

    2017-01-10

    Lymph nodes (LNs) are constructed of intricate networks of endothelial and mesenchymal stromal cells. How these lymphoid stromal cells (LSCs) regulate lymphoid tissue remodeling and contribute to immune responses remains poorly understood. We performed a comprehensive functional and transcriptional analysis of LSC responses to skin viral infection and found that LSC subsets responded robustly, with different kinetics for distinct pathogens. Recruitment of cells to inflamed LNs induced LSC expansion, while B cells sustained stromal responses in an antigen-independent manner. Infection induced rapid transcriptional responses in LSCs. This transcriptional program was transient, returning to homeostasis within 1 month of infection, yet expanded fibroblastic reticular cell networks persisted for more than 3 months after infection, and this altered LN composition reduced the magnitude of LSC responses to subsequent heterologous infection. Our results reveal the complexity of LSC responses during infection and suggest that amplified networks of LN stromal cells support successive immune responses.

  5. Atypical presentation of gastrointestinal stromal tumours-a case report.

    PubMed

    Raja, Kalpana; Dev, Bhawna; Santosham, Roy; Santhosh, Joseph

    2013-06-01

    Gastrointestinal stromal tumors (GISTs) are benign mesenchymal tumors of the gastrointestinal tract (GIT). Their clinical presentations are variable. We report a case of a 31-year-old man who presented with pain in the abdomen and vomiting. CT abdomen revealed a large exophytic mass in the epigastrium with enhancement pattern similar to hemangioma. No relationship of the mass could be made out with the adjacent structures on CT, histopathology proved it to be a GIST.

  6. What's New in Gastrointestinal Stromal Tumor Research and Treatment?

    MedlinePlus

    ... Stromal Tumor (GIST) About Gastrointestinal Stromal Tumor What’s New in Gastrointestinal Stromal Tumor Research and Treatment? There ... the Key Statistics About Gastrointestinal Stromal Tumors? What’s New in Gastrointestinal Stromal Tumor Research and Treatment? More ...

  7. 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

  8. Tumor-associated fibroblasts predominantly come from local and not circulating precursors

    PubMed Central

    Arina, Ainhoa; Idel, Christian; Hyjek, Elizabeth M.; Alegre, Maria-Luisa; Wang, Ying; Bindokas, Vytautas P.; Weichselbaum, Ralph R.; Schreiber, Hans

    2016-01-01

    Fibroblasts are common cell types in cancer stroma and lay down collagen required for survival and growth of cancer cells. Although some cancer therapy strategies target tumor fibroblasts, their origin remains controversial. Multiple publications suggest circulating mesenchymal precursors as a source of tumor-associated fibroblasts. However, we show by three independent approaches that tumor fibroblasts derive primarily from local, sessile precursors. First, transplantable tumors developing in a mouse expressing green fluorescent reporter protein (EGFP) under control of the type I collagen (Col-I) promoter (COL-EGFP) had green stroma, whereas we could not find COL-EGFP+ cells in tumors developing in the parabiotic partner lacking the fluorescent reporter. Lack of incorporation of COL-EGFP+ cells from the circulation into tumors was confirmed in parabiotic pairs of COL-EGFP mice and transgenic mice developing autochthonous intestinal adenomas. Second, transplantable tumors developing in chimeric mice reconstituted with bone marrow cells from COL-EGFP mice very rarely showed stromal fibroblasts expressing EGFP. Finally, cancer cells injected under full-thickness COL-EGFP skin grafts transplanted in nonreporter mice developed into tumors containing green stromal cells. Using multicolor in vivo confocal microscopy, we found that Col-I–expressing fibroblasts constituted approximately one-third of the stromal mass and formed a continuous sheet wrapping the tumor vessels. In summary, tumors form their fibroblastic stroma predominantly from precursors present in the local tumor microenvironment, whereas the contribution of bone marrow-derived circulating precursors is rare. PMID:27317748

  9. Epigenetic Classification of Human Mesenchymal Stromal Cells

    PubMed Central

    de Almeida, Danilo Candido; Ferreira, Marcelo R.P.; Franzen, Julia; Weidner, Carola I.; Frobel, Joana; Zenke, Martin; Costa, Ivan G.; Wagner, Wolfgang

    2016-01-01

    Summary Standardization of mesenchymal stromal cells (MSCs) is hampered by the lack of a precise definition for these cell preparations; for example, there are no molecular markers to discern MSCs and fibroblasts. In this study, we followed the hypothesis that specific DNA methylation (DNAm) patterns can assist classification of MSCs. We utilized 190 DNAm profiles to address the impact of tissue of origin, donor age, replicative senescence, and serum supplements on the epigenetic makeup. Based on this, we elaborated a simple epigenetic signature based on two CpG sites to classify MSCs and fibroblasts, referred to as the Epi-MSC-Score. Another two-CpG signature can distinguish between MSCs from bone marrow and adipose tissue, referred to as the Epi-Tissue-Score. These assays were validated by site-specific pyrosequencing analysis in 34 primary cell preparations. Furthermore, even individual subclones of MSCs were correctly classified by our epigenetic signatures. In summary, we propose an alternative concept to use DNAm patterns for molecular definition of cell preparations, and our epigenetic scores facilitate robust and cost-effective quality control of MSC cultures. PMID:26862701

  10. Catalytically inactive human cathepsin D triggers fibroblast invasive growth

    PubMed Central

    Laurent-Matha, Valérie; Maruani-Herrmann, Sharon; Prébois, Christine; Beaujouin, Mélanie; Glondu, Murielle; Noël, Agnès; Alvarez-Gonzalez, Marie Luz; Blacher, Sylvia; Coopman, Peter; Baghdiguian, Stephen; Gilles, Christine; Loncarek, Jadranka; Freiss, Gilles; Vignon, Françoise; Liaudet-Coopman, Emmanuelle

    2005-01-01

    The aspartyl-protease cathepsin D (cath-D) is overexpressed and hypersecreted by epithelial breast cancer cells and stimulates their proliferation. As tumor epithelial–fibroblast cell interactions are important events in cancer progression, we investigated whether cath-D overexpression affects also fibroblast behavior. We demonstrate a requirement of cath-D for fibroblast invasive growth using a three-dimensional (3D) coculture assay with cancer cells secreting or not pro-cath-D. Ectopic expression of cath-D in cath-D–deficient fibroblasts stimulates 3D outgrowth that is associated with a significant increase in fibroblast proliferation, survival, motility, and invasive capacity, accompanied by activation of the ras–MAPK pathway. Interestingly, all these stimulatory effects on fibroblasts are independent of cath-D proteolytic activity. Finally, we show that pro-cath-D secreted by cancer cells is captured by fibroblasts and partially mimics effects of transfected cath-D. We conclude that cath-D is crucial for fibroblast invasive outgrowth and could act as a key paracrine communicator between cancer and stromal cells, independently of its catalytic activity. PMID:15668295

  11. Stromal hedgehog signaling maintains smooth muscle and hampers micro-invasive prostate cancer

    PubMed Central

    Yang, Zhaohui; Peng, Yu-Ching; Gopalan, Anuradha; Gao, Dong; Chen, Yu

    2017-01-01

    ABSTRACT It is widely appreciated that reactive stroma or carcinoma-associated fibroblasts can influence epithelial tumor progression. In prostate cancer (PCa), the second most common male malignancy worldwide, the amount of reactive stroma is variable and has predictive value for tumor recurrence. By analyzing human PCa protein and RNA expression databases, we found smooth muscle cells (SMCs) are decreased in advanced tumors, whereas fibroblasts are maintained. In three mouse models of PCa, PB-MYC, ERG/PTEN and TRAMP, we found the composition of the stroma is distinct. SMCs are greatly depleted in advanced PB-MYC tumors and locally reduced in ERG/PTEN prostates, whereas in TRAMP tumors the SMC layers are increased. In addition, interductal fibroblast-like cells expand in PB-MYC and ERG/PTEN tumors, whereas in TRAMP PCa they expand little and stromal cells invade into intraductal adenomas. Fate mapping of SMCs showed that in PB-MYC tumors the cells are depleted, whereas they expand in TRAMP tumors and interestingly contribute to the stromal cells in intraductal adenomas. Hedgehog (HH) ligands secreted by epithelial cells are known to regulate prostate mesenchyme expansion differentially during development and regeneration. Any possible role of HH signaling in stromal cells during PCa progression is poorly understood. We found that HH signaling is high in SMCs and fibroblasts near tumor cells in all models, and epithelial Shh expression is decreased whereas Ihh and Dhh are increased. In human primary PCa, expression of IHH is the highest of the three HH genes, and elevated HH signaling correlates with high stromal gene expression. Moreover, increasing HH signaling in the stroma of PB-MYC PCa resulted in more intact SMC layers and decreased tumor progression (micro-invasive carcinoma). Thus, we propose HH signaling restrains tumor progression by maintaining the smooth muscle and preventing invasion by tumor cells. Our studies highlight the importance of understanding

  12. A Novel Bacterial Artificial Chromosome-Transgenic Podoplanin–Cre Mouse Targets Lymphoid Organ Stromal Cells in vivo

    PubMed Central

    Onder, Lucas; Scandella, Elke; Chai, Qian; Firner, Sonja; Mayer, Christian T.; Sparwasser, Tim; Thiel, Volker; Rülicke, Thomas; Ludewig, Burkhard

    2011-01-01

    Stromal cells provide the structural foundation of secondary lymphoid organs (SLOs), and regulate leukocyte access and cell migration within the different compartments of spleen and lymph nodes (LNs). Furthermore, several stromal cell subsets have been implied in shaping of T cell responses through direct presentation of antigen. Despite significant gain of knowledge on the biology of different SLO-resident stromal cell subsets, their molecular and functional characterization has remained incomplete. To address this need, we have generated a bacterial artificial chromosome-transgenic mouse model that utilizes the podoplanin (pdpn) promoter to express the Cre-recombinase exclusively in stromal cells of SLOs. The characterization of the Pdpn–Cre mouse revealed transgene expression in subsets of fibroblastic reticular cells and lymphatic endothelial cells in LNs. Furthermore, the transgene facilitated the identification of a novel splenic perivascular stromal cell subpopulation that forms web-like structures around central arterioles. Assessment of the in vivo antigen expression in the genetically tagged stromal cells in Pdpn–Cre mice revealed activation of both MHC I and II-restricted TCR transgenic T cells. Taken together, stromal pdpn–Cre expression is well-suited to characterize the phenotype and to dissect the function of lymphoid organ stromal cells. PMID:22566840

  13. 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

  14. Altered expression profile of micrornas in gastric stromal tumor.

    PubMed

    Xiao, Jun; Wang, Qi-xian; Zhu, You-qing

    2015-12-01

    MicroRNAs (miRNAs) play important roles in carcinogenesis, but the global miRNA expression profile in gastric stromal tumor tissues remains unclear. This study was to examine the miRNA expression profile in gastric stromal tumor tissues and explore the function of dysregulated miRNAs by performing gene ontology (GO) and pathway enrichment analysis. Total RNA was extracted and purified from 3 pairs of frozen gastric stromal tumor tissues and the adjacent non-tumor tissues by using mirVana™ miRNA isolation kit. The miRNA expression was analyzed with Affymetrix microarrays (version 4.0) containing 2578 human mature microRNA probes. The dysregulated microRNAs were validated by quantitative RT-PCR in 30 pairs of gastric stromal tumor tissues. The target gene of the dysregulated microRNAs was predicted by miRanda, TargetScan and PicTar. GO and pathway enrichment analysis was conducted to examine the potential function of miR-3178 and miR-193a-5p. The results showed that there were 12 differently expressed microRNAs in gastric stromal tumor tissues, among which 10 miRNAs were down-regulated, and 2 were up-regulated (P<0.05). The validation results by RT-PCR were in accordance with those by microRNA microarry. GO analysis found that the target genes of miR-3178 were involved in 5 GO terms and those of miR-193a-5p in 7 GO terms in level 2. Pathway enrichment analysis suggested that miR-3178 and miR-193a-5p were related to 57 and 122 signaling pathways, respectively. It was concluded that gastric stromal tumor displays a unique miRNA signature. This specific expression may become a new diagnostic and prognostic biomarker for gastric stromal tumor. miR-3178 and miR-193a-5p function as suppressive microRNAs, and they may also become diagnosis and treatment targets for gastric stromal tumor.

  15. Controlling herpetic stromal keratitis by modulating lymphotoxin-alpha-mediated inflammatory pathways.

    PubMed

    Veiga-Parga, Tamara; Giménez, Fernanda; Mulik, Sachin; Chiang, Eugene Y; Grogan, Jane L; Rouse, Barry T

    2013-01-01

    Herpes simplex virus 1 infection of the eye can result in stromal keratitis, a chronic immunoinflammatory lesion that is a significant cause of human blindness. A key to controlling the severity of lesions is to identify cellular and molecular events responsible for tissue damage. This report evaluates the role of lymphotoxin-α, a proinflammatory cytokine that could be involved during stromal keratitis. We demonstrate that after infection, both lymphotoxin-α and lymphotoxin-β transcripts are detectable at high levels 48 h postinfection, suggesting roles for the secreted homotrimer lymphotoxin-α3 and the membrane-bound lymphotoxin-α1β2 heterotrimer in stromal keratitis. Using a corneal stromal fibroblast cell line, lymphotoxin-α3 and lymphotoxin-α1β2 were found to have proinflammatory roles by stimulating production of chemokines. Treatment of mice with a depleting anti-lymphotoxin-α mAb during the clinical phase of the disease significantly attenuated stromal keratitis lesions. In treated mice, expression of proinflammatory molecules and chemokines was reduced, as were numbers of cornea-infiltrating proinflammatory cells, particularly Th1 cells. The protective effect of anti-lymphotoxin-α mAb was highly reduced with a mutant version of the mAb that lacks Fc receptor binding activity, indicating that depletion of lymphotoxin-expressing cells was mainly responsible for efficacy, with LT-α3 contributing minimally to inflammation. These data demonstrate that lymphotoxin-expressing cells, such as Th1 cells, mediate stromal keratitis.

  16. CollagenVI-Cre mice: A new tool to target stromal cells in secondary lymphoid organs

    PubMed Central

    Prados, Alejandro; Kollias, George; Koliaraki, Vasiliki

    2016-01-01

    Stromal cells in secondary lymphoid organs (SLOs) are non-hematopoietic cells involved in the regulation of adaptive immune responses. Three major stromal populations have been identified in adult SLOs: fibroblastic reticular cells (FRCs), follicular dendritic cells (FDCs) and marginal reticular cells (MRCs). The properties of these individual populations are not clearly defined, mainly due to the lack of appropriate genetic tools, especially for MRCs. Here, we analyzed stromal cell targeting in SLOs from a transgenic mouse strain that expresses Cre recombinase under the CollagenVI promoter, using lineage tracing approaches. We show that these mice target specifically MRCs and FDCs, but not FRCs in Peyer’s patches and isolated lymphoid follicles in the intestine. In contrast, stromal cells in lymph nodes and the spleen do not express the transgene, which renders ColVI-cre mice ideal for the specific targeting of stromal cells in the gut-associated lymphoid tissue (GALT). This funding further supports the hypothesis of organ-specific stromal precursors in SLOs. Interestingly, in all tissues analyzed, there was also high specificity for perivascular cells, which have been proposed to act as FDC precursors. Taken together, ColVI-Cre mice are a useful new tool for the dissection of MRC- and FDC-specific functions and plasticity in the GALT. PMID:27604178

  17. Tumor-secreted LOXL2 Activates Fibroblasts Through FAK Signaling

    PubMed Central

    Barker, Holly E.; Bird, Demelza; Lang, Georgina; Erler, Janine T.

    2013-01-01

    Cancer-associated fibroblasts enhance cancer progression when activated by tumor cells through mechanisms not yet fully understood. Blocking mammary tumor cell-derived lysyl oxidase-like 2 (LOXL2) significantly inhibited mammary tumor cell invasion and metastasis in transgenic and orthotopic mouse models. Here we discovered that tumor-derived LOXL2 directly activated stromal fibroblasts in the tumor microenvironment. Genetic manipulation or antibody inhibition of LOXL2 in orthotopically grown mammary tumors reduced the expression of α-smooth muscle actin (α-SMA). Using a marker for reticular fibroblasts, it was determined that expression of α-SMA was localized to fibroblasts recruited from the host tissue. This marker also revealed that the matrix present in tumors with reduced levels of LOXL2 was more scattered compared to control tumors which exhibited matrices with dense, parallel alignments. Importantly, in vitro assays revealed that tumor-derived LOXL2 and a recombinant LOXL2 protein induced fibroblast branching on collagen matrices, as well as increased fibroblast-mediated collagen contraction and invasion of fibroblasts through extracellular matrix (ECM). Moreover, LOXL2 induced the expression of α-SMA in fibroblasts grown on collagen matrices. Mechanistically, it was determined that LOXL2 activated fibroblasts through integrin-mediated FAK activation. These results indicate that inhibition of LOXL2 in tumors not only reduces tumor cell invasion but also attenuates the activation of host cells in the tumor microenvironment. Implications: These findings reveal new insight into the mechanisms of fibroblast activation, a novel function of LOXL2, and further highlight the importance of generating LOXL2-targeted therapies for the prevention of tumor progression and metastasis. PMID:24008674

  18. Mesenchymal Stem Cell-Induced DDR2 Mediates Stromal-Breast Cancer Interactions and Metastasis Growth.

    PubMed

    Gonzalez, Maria E; Martin, Emily E; Anwar, Talha; Arellano-Garcia, Caroline; Medhora, Natasha; Lama, Arjun; Chen, Yu-Chih; Tanager, Kevin S; Yoon, Euisik; Kidwell, Kelley M; Ge, Chunxi; Franceschi, Renny T; Kleer, Celina G

    2017-01-31

    Increased collagen deposition by breast cancer (BC)-associated mesenchymal stem/multipotent stromal cells (MSC) promotes metastasis, but the mechanisms are unknown. Here, we report that the collagen receptor discoidin domain receptor 2 (DDR2) is essential for stromal-BC communication. In human BC metastasis, DDR2 is concordantly upregulated in metastatic cancer and multipotent mesenchymal stromal cells. In MSCs isolated from human BC metastasis, DDR2 maintains a fibroblastic phenotype with collagen deposition and induces pathological activation of DDR2 signaling in BC cells. Loss of DDR2 in MSCs impairs their ability to promote DDR2 phosphorylation in BC cells, as well as BC cell alignment, migration, and metastasis. Female ddr2-deficient mice homozygous for the slie mutation show inefficient spontaneous BC metastasis. These results point to a role for mesenchymal stem cell DDR2 in metastasis and suggest a therapeutic approach for metastatic BC.

  19. Stromal-epithelial paracrine interactions in the neoplastic rat and human prostate.

    PubMed

    Djakiew, D; Pflug, B; Onoda, M

    1993-01-01

    Homotypic paracrine interactions in the rat and human prostate have been investigated using prostatic stromal cells and neoplastic epithelial cells (PA-III, rat; TSU-pr1, human). Secretory proteins prepared from each cell type were used to determine the dose dependent regulation of growth (DNA synthesis) of the corresponding homotypic responder cell, as determined by 3H-thymidine incorporation. PA-III secretory protein stimulated rat stromal cell proliferation by 1.8-fold. This stimulatory activity of PA-III protein on stromal cell proliferation was partially reduced (approximately 35%) by treatment with nerve growth factor (NGF) antibody, whereas neither acidic fibroblast growth factor (aFGF) antibody nor basic fibroblast growth factor (bFGF) antibody immunoneutralized the stimulatory activity of PA-III cell protein. In the corresponding opposite interaction, rat stromal cell protein modulated PA-III growth in a biphasic manner. At lower concentrations of stromal cell protein (1.25 micrograms/ml) PA-III cell growth was stimulated by 1.6-fold, whereas at higher concentrations of protein (100 micrograms/ml) PA-III cell growth was inhibited to 60%. Treatment of the stromal cell protein (1.25 micrograms/ml and 100 micrograms/ml) with NGF antibody reduced PA-III cell relative growth to approximately 30% and 5%, respectively. bFGF antibody treatment of stromal cell protein at 1.25 micrograms/ml did not influence relative growth, whereas bFGF antibody treatment of 100 micrograms/ml stromal cell protein reduced relative growth by an additional 40%. Treatment of the stromal cell protein (1.25 micrograms/ml and 100 micrograms/ml) with aFGF antibodies reduced relative growth from that observed at these two protein concentrations by approximately 50% in both cases. Human epithelial TSU-pr1 protein stimulated human stromal cell proliferation approximately 1.7-fold. Treatment of TSU-pr1 protein with NGF antibody resulted in stimulation of human stromal cell proliferation (4

  20. Stromal-epithelial interaction study: The effect of corneal epithelial cells on growth factor expression in stromal cells using organotypic culture model.

    PubMed

    Kobayashi, Takeshi; Shiraishi, Atsushi; Hara, Yuko; Kadota, Yuko; Yang, Lujun; Inoue, Tomoyuki; Shirakata, Yuji; Ohashi, Yuichi

    2015-06-01

    Interactions between stromal and epithelial cells play important roles in the development, homeostasis, and pathological conditions of the cornea. Soluble cytokines are critical factors in stromal-epithelial interactions, and growth factors secreted from corneal stromal cells contribute to the regulation of proliferation and differentiation of corneal epithelial cells (CECs). However, the manner in which the expression of growth factors is regulated in stromal cells has not been completely determined. To study stromal-epithelial cell interactions, we used an organotypic culture model. Human or rabbit CECs (HCECs or RCECs) were cultured on amniotic membranes placed on human corneal fibroblasts (HCFs) embedded in a collagen gel. The properties of the organotypic culture were examined by hematoxylin-eosin staining and immunofluorescence. In the organotypic culture, HCECs or RCECs were stratified into two-three layers after five days and five-seven layers after nine days. However, stratification was not observed when the HCECs were seeded on a collagen gel without fibroblasts. K3/K12 were expressed on day 9. The HCF-embedded collagen gels were collected on days 3, 5, or 9 after seeding the RCECs, and mRNA expression of growth factors FGF7, HGF, NGF, EGF, TGF-α, SCF, TGF-β1, TGF-β2, and TGF-β3 were quantified by real-time PCR. mRNA expression of the growth factors in HCFs cultured with RCECs were compared with those cultured without RCECs, as well as in monolayer cultures. mRNA expression of TGF-α was markedly increased in HCFs cultured with RCECs. However, mRNA expression of the TGF-β family was suppressed in HCFs cultured with RCECs. Principal component analysis revealed that mRNA expression of the growth factors in HCFs were generally similar when they were cultured with RCECs. In organotypic cultures, the morphological changes in the CECs and the expression patterns of the growth factors in the stromal cells clearly demonstrated stromal-epithelial cell

  1. Very Late Antigen-5 Facilitates Stromal Progenitor Cell Differentiation Into Myofibroblast

    PubMed Central

    Sen, Namita; Weingarten, Mark

    2014-01-01

    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. PMID:25273539

  2. The Influence of Stromal Transforming Growth Factor-Beta Receptor Signaling on Mouse Mammary Neoplasia

    DTIC Science & Technology

    2004-08-01

    and -P3) are members of a family of peptide growth factors that include inhibins, bone morphogenic proteins (BMPs) and growth and differentiation...DNIIR) in the mammary epithelium and in stromal fibroblasts resulted in precocious lobuloalveolar development and increased lateral branching...necessary for proper ductal development during puberty . It has been suggested that TGF-P regulates pubertal mammary development through the epithelium and

  3. The Role of Cyclin D1 in Altering Stromal-Epithelial Interactions in Prostate Carcinogenesis

    DTIC Science & Technology

    2008-03-01

    adenocarcinomas. One study of cyclin D1 expression in esophageal carcinomas indicated that cyclin D1 is strongly expressed in stromal fibroblasts. In this study...proliferate faster than controls in vivo in our tissue recombination model. Although cyclin D1 can increase BPH-1 cell motility and promote cell...Alarid ET, Turner T, Donjacour AA, Boutin EL, Foster BA. Normal and abnormal development of the male urogenital tract: role of androgens, mesenchymal

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

    PubMed Central

    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

    2016-01-01

    Objectives: 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. Design: Animal study and primary cell culture. Setting: Laboratory investigation. Subjects: BALB/c mice. Interventions: 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. Measurements and Main Results: 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

  5. Inhibition of Stromal PlGF Suppresses the Growth of Prostate Cancer Xenografts

    PubMed Central

    Zins, Karin; Thomas, Anita; Lucas, Trevor; Sioud, Mouldy; Aharinejad, Seyedhossein; Abraham, Dietmar

    2013-01-01

    The growth and vascularization of prostate cancer is dependent on interactions between cancer cells and supporting stromal cells. The primary stromal cell type found in prostate tumors is the carcinoma-associated fibroblast, which produces placental growth factor (PlGF). PlGF is a member of the vascular endothelial growth factor (VEGF) family of angiogenic molecules and PlGF mRNA levels increase after androgen deprivation therapy in prostate cancer. In this study, we show that PlGF has a direct dose-dependent proliferative effect on human PC-3 prostate cancer cells in vitro and fibroblast-derived PlGF increases PC-3 proliferation in co-culture. In xenograft tumor models, intratumoral administration of murine PlGF siRNA reduced stromal-derived PlGF expression, reduced tumor burden and decreased the number of Ki-67 positive proliferating cells associated with reduced vascular density. These data show that targeting stromal PlGF expression may represent a therapeutic target for the treatment of prostate cancer. PMID:24005860

  6. SerpinB2 regulates stromal remodelling and local invasion in pancreatic cancer.

    PubMed

    Harris, N L E; Vennin, C; Conway, J R W; Vine, K L; Pinese, M; Cowley, M J; Shearer, R F; Lucas, M C; Herrmann, D; Allam, A H; Pajic, M; Morton, J P; Biankin, A V; Ranson, M; Timpson, P; Saunders, D N

    2017-03-27

    Pancreatic cancer has a devastating prognosis, with an overall 5-year survival rate of ~8%, restricted treatment options and characteristic molecular heterogeneity. SerpinB2 expression, particularly in the stromal compartment, is associated with reduced metastasis and prolonged survival in pancreatic ductal adenocarcinoma (PDAC) and our genomic analysis revealed that SERPINB2 is frequently deleted in PDAC. We show that SerpinB2 is required by stromal cells for normal collagen remodelling in vitro, regulating fibroblast interaction and engagement with collagen in the contracting matrix. In a pancreatic cancer allograft model, co-injection of PDAC cancer cells and SerpinB2(-/-) mouse embryonic fibroblasts (MEFs) resulted in increased tumour growth, aberrant remodelling of the extracellular matrix (ECM) and increased local invasion from the primary tumour. These tumours also displayed elevated proteolytic activity of the primary biochemical target of SerpinB2-urokinase plasminogen activator (uPA). In a large cohort of patients with resected PDAC, we show that increasing uPA mRNA expression was significantly associated with poorer survival following pancreatectomy. This study establishes a novel role for SerpinB2 in the stromal compartment in PDAC invasion through regulation of stromal remodelling and highlights the SerpinB2/uPA axis for further investigation as a potential therapeutic target in pancreatic cancer.Oncogene advance online publication, 27 March 2017; doi:10.1038/onc.2017.63.

  7. Identification of Meflin as a Potential Marker for Mesenchymal Stromal Cells

    PubMed Central

    Maeda, Keiko; Enomoto, Atsushi; Hara, Akitoshi; Asai, Naoya; Kobayashi, Takeshi; Horinouchi, Asuka; Maruyama, Shoichi; Ishikawa, Yuichi; Nishiyama, Takahiro; Kiyoi, Hitoshi; Kato, Takuya; Ando, Kenju; Weng, Liang; Mii, Shinji; Asai, Masato; Mizutani, Yasuyuki; Watanabe, Osamu; Hirooka, Yoshiki; Goto, Hidemi; Takahashi, Masahide

    2016-01-01

    Bone marrow-derived mesenchymal stromal cells (BM-MSCs) in culture are derived from BM stromal cells or skeletal stem cells. Whereas MSCs have been exploited in clinical medicine, the identification of MSC-specific markers has been limited. Here, we report that a cell surface and secreted protein, Meflin, is expressed in cultured MSCs, fibroblasts and pericytes, but not other types of cells including epithelial, endothelial and smooth muscle cells. In vivo, Meflin is expressed by immature osteoblasts and chondroblasts. In addition, Meflin is found on stromal cells distributed throughout the BM, and on pericytes and perivascular cells in multiple organs. Meflin maintains the undifferentiated state of cultured MSCs and is downregulated upon their differentiation, consistent with the observation that Meflin-deficient mice exhibit increased number of osteoblasts and accelerated bone development. In the bone and BM, Meflin is more highly expressed in primitive stromal cells that express platelet-derived growth factor receptor α and Sca-1 than the Sca-1-negative adipo-osteogenic progenitors, which create a niche for hematopoiesis. Those results are consistent with a decrease in the number of clonogenic colony-forming unit-fibroblasts within the BM of Meflin-deficient mice. These preliminary data suggest that Meflin is a potential marker for cultured MSCs and their source cells in vivo. PMID:26924503

  8. Characteristic Gene Expression Profiles of Human Fibroblasts and Breast Cancer Cells in a Newly Developed Bilateral Coculture System.

    PubMed

    Ueno, Takayuki; Utsumi, Jun; Toi, Masakazu; Shimizu, Kazuharu

    2015-01-01

    The microenvironment of cancer cells has been implicated in cancer development and progression. Cancer-associated fibroblast constitutes a major stromal component of the microenvironment. To analyze interaction between cancer cells and fibroblasts, we have developed a new bilateral coculture system using a two-sided microporous collagen membrane. Human normal skin fibroblasts were cocultured with three different human breast cancer cell lines: MCF-7, SK-BR-3, and HCC1937. After coculture, mRNA was extracted separately from cancer cells and fibroblasts and applied to transcriptomic analysis with microarray. Top 500 commonly up- or downregulated genes were characterized by enrichment functional analysis using MetaCore Functional Analysis. Most of the genes upregulated in cancer cells were downregulated in fibroblasts while most of the genes downregulated in cancer cells were upregulated in fibroblasts, indicating that changing patterns of mRNA expression were reciprocal between cancer cells and fibroblasts. In coculture, breast cancer cells commonly increased genes related to mitotic response and TCA pathway while fibroblasts increased genes related to carbohydrate metabolism including glycolysis, glycogenesis, and glucose transport, indicating that fibroblasts support cancer cell proliferation by supplying energy sources. We propose that the bilateral coculture system using collagen membrane is useful to study interactions between cancer cells and stromal cells by mimicking in vivo tumor microenvironment.

  9. Characteristic Gene Expression Profiles of Human Fibroblasts and Breast Cancer Cells in a Newly Developed Bilateral Coculture System

    PubMed Central

    Ueno, Takayuki; Utsumi, Jun; Toi, Masakazu; Shimizu, Kazuharu

    2015-01-01

    The microenvironment of cancer cells has been implicated in cancer development and progression. Cancer-associated fibroblast constitutes a major stromal component of the microenvironment. To analyze interaction between cancer cells and fibroblasts, we have developed a new bilateral coculture system using a two-sided microporous collagen membrane. Human normal skin fibroblasts were cocultured with three different human breast cancer cell lines: MCF-7, SK-BR-3, and HCC1937. After coculture, mRNA was extracted separately from cancer cells and fibroblasts and applied to transcriptomic analysis with microarray. Top 500 commonly up- or downregulated genes were characterized by enrichment functional analysis using MetaCore Functional Analysis. Most of the genes upregulated in cancer cells were downregulated in fibroblasts while most of the genes downregulated in cancer cells were upregulated in fibroblasts, indicating that changing patterns of mRNA expression were reciprocal between cancer cells and fibroblasts. In coculture, breast cancer cells commonly increased genes related to mitotic response and TCA pathway while fibroblasts increased genes related to carbohydrate metabolism including glycolysis, glycogenesis, and glucose transport, indicating that fibroblasts support cancer cell proliferation by supplying energy sources. We propose that the bilateral coculture system using collagen membrane is useful to study interactions between cancer cells and stromal cells by mimicking in vivo tumor microenvironment. PMID:26171396

  10. 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

  11. Mesenchymal stromal cell cryopreservation.

    PubMed

    Renzi, Sabrina; Lombardo, Tina; Dotti, Silvia; Dessì, Sara S; De Blasio, Pasquale; Ferrari, Maura

    2012-06-01

    The advent of stem cells and stem cell-based therapies for specific diseases requires particular knowledge of laboratory procedures, which not only guarantee the continuous production of cells, but also provide them an identity and integrity as close as possible to their origin. Their cryopreservation at temperatures below -80°C and typically below -140°C is of paramount importance. This target can be achieved by incorporating high molar concentrations of cryoprotectant mixtures that preserve cells from deleterious ice crystal formation. Usually, dimethyl sulfoxide (DMSO) and animal proteins are used as protectant reagents, but unexpected changes in stem cell fate and downstream toxicity effects have been reported, limiting their wide use in clinical settings. In scientific reviews, there are not much data regarding viability of mesenchymal stromal cells (MSCs) after the freezing/thawing process. During our routine analysis, a poor resistance to cryopreservation of these cells was observed, as well as their weak ability to replicate. This is an important point in the study of MSCs; moreover, it represents a limit for preservation and long-term storage. For this reason, MSCs isolated from equine, ovine, and rodent bone marrow and equine adipose tissue were compared using different cryopreservation solutions for this study of vitality. Our findings showed the best results regarding cell viability using a solution of fetal bovine serum with addition of 10% DMSO. In particular, we noted an increase in survival of equine bone marrow MSCs. This parameter has been evaluated by Trypan blue staining at fixed times (0, 24, and 48 hours post-thaw). This result highlights the fact that equine bone marrow MSCs are the frailest we analyzed. Therefore, it could be useful to delve further into this topic in order to improve the storage possibility for these cells and their potential use in cell-based therapies.

  12. Fibroblast TGF-beta Signaling in Breast Development and Cancer

    DTIC Science & Technology

    2010-09-01

    expres- sion in the tumor stroma can activate CXCR4 expression in adjacent epithelial cells allowing the cognate ligand for this receptor, SDF1/CXCL12...thus allowing cells to ignore the growth inhibitory effects of TGF- [37]. CXCR4 is elevated in localizedandmetastatic cancer and is amarker of poor...cancer is the finding that mice with Smad7 deletion in stromal cells develop PanIN accompanied with increased fibrosis around theductal regions [56

  13. Immunohistochemical expression of epithelial and stromal immunomodulatory signalling molecules is a prognostic indicator in breast cancer

    PubMed Central

    2012-01-01

    Background The immune system has paradoxical roles during cancer development and the prognostic significance of immune modulating factors is controversial. The aim of this study was to determine the expression of cyclooxygenase 2 (COX-2), transforming growth factor-beta (TGF- beta), interleukin-10 (IL-10) and their prognostic significance in breast cancers. Ki67 was included as a measure of growth fraction of tumor cells. Methods On immunohistochemical stained slides from 38 breast cancer patients, we performed digital video analysis of tumor cell areas and adjacent tumor stromal areas from the primary tumors and their corresponding lymph node metastases. COX-2 was recorded as graded staining intensity. Results The expression of TGF-beta, IL-10 and Ki67 were recorded in tumor cell areas and adjacent tumor stromal areas. In both primary tumors and metastases, the expression of COX-2 was higher in the tumor stromal areas than in the tumor cell areas (both P < 0.001). High stromal staining intensity in the primary tumors was associated with a 3.9 (95% CI 1.1-14.2) times higher risk of death compared to the low staining group (P = 0.036). The expression of TGF-beta was highest in the tumor cell areas of both primary tumors and metastases (both P < 0.001). High stromal expression of TGF-beta was associated with increased mortality. For IL-10, the stromal expression was highest in the primary tumors (P < 0.001), whereas in the metastases the expression was highest in tumor cell areas (P < 0.001). High IL-10 expression in tumor- and stromal cell areas of primary tumors predicted mortality. Ki67 was higher expressed in tumor stromal areas of the metastases, and in tumor cell areas of the primary tumors (P < 0.001). Ki67 expression in tumor cell areas and stromal areas of the metastases was independently associated with breast cancer mortality. Conclusions Stromal expression of COX-2, TGF-beta and Ki67 may facilitate tumor progression in breast cancer. PMID:22353218

  14. Membrane-bound ICAM-1 contributes to the onset of proinvasive tumor stroma by controlling acto-myosin contractility in carcinoma-associated fibroblasts

    PubMed Central

    Bonan, Stephanie; Albrengues, Jean; Grasset, Eloise; Kuzet, Sanya-Eduarda; Nottet, Nicolas; Bourget, Isabelle; Bertero, Thomas; Mari, Bernard; Meneguzzi, Guerrino; Gaggioli, Cedric

    2017-01-01

    Acto-myosin contractility in carcinoma-associated fibroblasts leads to assembly of the tumor extracellular matrix. The pro-inflammatory cytokine LIF governs fibroblast activation in cancer by regulating the myosin light chain 2 activity. So far, however, how LIF mediates cytoskeleton contractility remains unknown. Using phenotypic screening assays based on knock-down of LIF-dependent genes in fibroblasts, we identified the glycoprotein ICAM-1 as a crucial regulator of stroma fibroblast proinvasive matrix remodeling. We demonstrate that the membrane-bound ICAM-1 isoform is necessary and sufficient to promote inflammation-dependent extracellular matrix contraction, which favors cancer cell invasion. Indeed, ICAM-1 mediates generation of acto-myosin contractility downstream of the Src kinases in stromal fibroblasts. Moreover, acto-myosin contractility regulates ICAM-1 expression by establishing a positive feedback signaling. Thus, targeting stromal ICAM-1 might constitute a possible therapeutic mean to counteract tumor cell invasion and dissemination. PMID:27901489

  15. Different patterns of stromal and cancer cell thymidine phosphorylase reactivity in non-small-cell lung cancer: impact on tumour neoangiogenesis and survival.

    PubMed Central

    Koukourakis, M. I.; Giatromanolaki, A.; Kakolyris, S.; O'Byrne, K. J.; Apostolikas, N.; Skarlatos, J.; Gatter, K. C.; Harris, A. L.

    1998-01-01

    Angiogenesis is recognized as an important step in tumour pathogenesis that is related to invasion and metastatic spread and which consequently results in poor clinical outcome. In this study, we have examined the role of tumour stroma-activated fibroblasts and macrophage infiltration in the development of the angiogenic and metastatic phenotype in non-small-cell lung cancer (NSCLC). A total of 141 cases of early stage I-II NSCLC treated with surgery alone were analysed. The JC-70 (anti-CD31) MAb was used for the assessment of vascular grade. The P-GF.44C MAb was used to assess thymidine phosphorylase (TP) reactivity in cancer cells, stromal fibroblasts and macrophages. Cancer cell TP overexpression related to high vascular grade and to advanced T stage (P = 0.0004 and P = 0.02). Expression of TP in stromal fibroblasts also correlated with high angiogenesis (P = 0.01), but was independent of cancer cell expression. Fibroblast TP overexpression was related to abundant stroma (P = 0.003), suggesting that TP may be a marker of active stroma. Moreover, intense macrophage infiltration was associated with fibroblast TP reactivity, regardless of the amount of stroma, suggesting that macrophages may be a major contributor to TP expression in stroma. Survival analysis showed that cancer cell TP overexpression was related to poor prognosis (P = 0.005). Although stroma TP is related to angiogenesis, in the low vascular grade group it defined a group of patients with better prognosis (P = 0.02). It may be that fibroblast TP reactivity is an indirect marker of tumour infiltration by functional macrophages, which have an antitumour effect. We conclude that stromal macrophage and fibroblast TP reactivity may have an important role in non-small-cell lung cancer behaviour. Understanding the role of stromal fibroblasts and inflammatory cells and their interaction with oncoprotein expression is essential for the elucidation of lung cancer pathogenesis. Images Figure 1 PMID:9635852

  16. PDCD4 is a CSL associated protein with a transcription repressive function in cancer associated fibroblast activation

    PubMed Central

    Jo, Seung-Hee; Kim, Dong Eun; Clocchiatti, Andrea; Dotto, G. Paolo

    2016-01-01

    The Notch/CSL pathway plays an important role in skin homeostasis and carcinogenesis. CSL, the key effector of canonical Notch signaling endowed with an intrinsic transcription repressive function, suppresses stromal fibroblast senescence and Cancer Associated Fibroblast (CAF) activation through direct down-modulation of key effector genes. Interacting proteins that participate with CSL in this context are as yet to be identified. We report here that Programmed Cell Death 4 (PDCD4), a nuclear/cytoplasmic shuttling protein with multiple functions, associates with CSL and plays a similar role in suppressing dermal fibroblast senescence and CAF activation. Like CSL, PDCD4 is down-regulated in stromal fibroblasts of premalignant skin actinic keratosis (AKs) lesions and squamous cell carcinoma (SCC). While devoid of intrinsic DNA binding capability, PDCD4 is present at CSL binding sites of CAF marker genes as well as canonical Notch/CSL targets and suppresses expression of these genes in a fibroblast-specific manner. Thus, we propose that PDCD4 is part of the CSL repressive complex involved in negative control of stromal fibroblasts conversion into CAFs. PMID:27542230

  17. Gastrointestinal pacemaker cell tumor (GIPACT): gastrointestinal stromal tumors show phenotypic characteristics of the interstitial cells of Cajal.

    PubMed Central

    Kindblom, L. G.; Remotti, H. E.; Aldenborg, F.; Meis-Kindblom, J. M.

    1998-01-01

    The interstitial cells of Cajal (ICC) form a complex cell network within the gastrointestinal tract wall where they function as a pacemaker system. Expression of the kit proto-oncogene is essential for the development of this system. The aim of our study was to examine the hypothesis that gastrointestinal stromal tumors differentiate toward cells with an ICC phenotype. Ultrastructurally, 58 stromal tumors were characterized and found to share many features with ICC. Seventy-eight stromal tumors were immunophenotyped, particularly with regard to the kit receptor. All 78 tumors revealed strong, homogeneous immunoreactivity for the kit receptor as did ICC of adjacent and control gastrointestinal walls. Focal hyperplasia and hypertrophy of kit receptor positive cells were also observed in the gastrointestinal wall adjacent to the tumors. CD34 immunoreactivity observed in interstitial cells surrounding Auerbach's ganglia suggests that a subpopulation of ICC is CD34 positive and may explain why 56 of 78 stromal tumors were CD34 positive. Thirty control tumors, including gastrointestinal leiomyomas and leiomyosarcomas, were all negative for the kit receptor. We conclude that gastrointestinal stromal tumors show striking morphological and immunophenotypic similarities with ICC and that they may originate from stem cells that differentiate toward a pacemaker cell phenotype. We propose that the noncommittal name "gastrointestinal stromal tumor" be replaced by gastrointestinal pacemaker cell tumor. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:9588894

  18. 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

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

    SciTech Connect

    Bao, Ci-Hang; Wang, Xin-Tong; Ma, Wei; Wang, Na-Na; Nesa, Effat un; Wang, Jian-Bo; Wang, Cong; Jia, Yi-Bin; Wang, Kai; Tian, Hui; Cheng, Yu-Feng

    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 irradiated 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.

  20. CDCP1 Identifies a CD146 Negative Subset of Marrow Fibroblasts Involved with Cytokine Production

    PubMed Central

    Iwata, Mineo; Torok-Storb, Beverly; Wayner, Elizabeth A.; Carter, William G.

    2014-01-01

    In vitro expanded bone marrow stromal cells contain at least two populations of fibroblasts, a CD146/MCAM positive population, previously reported to be critical for establishing the stem cell niche and a CD146-negative population that expresses CUB domain-containing protein 1 (CDCP1)/CD318. Immunohistochemistry of marrow biopsies shows that clusters of CDCP1+ cells are present in discrete areas distinct from areas of fibroblasts expressing CD146. Using a stromal cell line, HS5, which approximates primary CDCP1+ stromal cells, we show that binding of an activating antibody against CDCP1 results in tyrosine-phosphorylation of CDCP1, paralleled by phosphorylation of Src Family Kinases (SFKs) Protein Kinase C delta (PKC-δ). When CDCP1 expression is knocked-down by siRNA, the expression and secretion of myelopoietic cytokines is increased. These data suggest CDCP1 expression can be used to identify a subset of marrow fibroblasts functionally distinct from CD146+ fibroblasts. Furthermore the CDCP1 protein may contribute to the defining function of these cells by regulating cytokine expression. PMID:25275584

  1. Attenuated proliferation and trans-differentiation of prostatic stromal cells indicate suitability of phosphodiesterase type 5 inhibitors for prevention and treatment of benign prostatic hyperplasia.

    PubMed

    Zenzmaier, Christoph; Sampson, Natalie; Pernkopf, Dominik; Plas, Eugen; Untergasser, Gerold; Berger, Peter

    2010-08-01

    Benign prostatic hyperplasia (BPH) is characterized by tissue overgrowth and stromal reorganization primarily due to cellular proliferation and fibroblast-to-myofibroblast trans-differentiation. To evaluate the potential of phosphodiesterase type 5 (PDE5) inhibitors like tadalafil for prevention and treatment of BPH, we analyzed the role of the nitric oxide/cyclic GMP (cGMP)/PDE5 pathway for cellular proliferation and TGFbeta1-induced fibroblast-to-myofibroblast trans-differentiation in primary prostate stromal cells. Inhibition by tadalafil of PDE5, which is mainly expressed in the stromal compartment of the prostate, reduced proliferation of primary prostate stromal cells and to a lesser extent of primary prostate basal epithelial cells. Attenuated proliferation due to elevated intracellular cGMP levels was confirmed by inhibition of the cGMP-dependent protein kinase G by its inhibitor KT2358. Moreover, tadalafil strongly attenuated TGFbeta1-induced fibroblast-to-myofibroblast trans-differentiation. The inhibitory effect on trans-differentiation was also observed after small interfering RNA-mediated PDE5 knockdown. As confirmed by the MAPK kinase 1 inhibitor PD98059, this effect was mediated via MAPK kinase 1 signaling. We conclude that BPH patients might benefit from adjuvant therapies with PDE5 inhibitors that inhibit stromal enlargement due to cell proliferation, as well as TGFbeta1-induced trans-differentiation processes.

  2. Supernatant of Bone Marrow Mesenchymal Stromal Cells Induces Peripheral Blood Mononuclear Cells Possessing Mesenchymal Features

    PubMed Central

    Hu, Gang; Xu, Jun-jun; Deng, Zhi-hong; Feng, Jie; Jin, Yan

    2011-01-01

    Increasing evidence shows that some cells from peripheral blood fibroblast-like mononuclear cells have the capacity to differentiate into mesenchymal lineages. However, the insufficiency of these cells in the circulation challenges the cell isolation and subsequently limits the clinical application of these cells. In the present study, the peripheral blood mononuclear cells (pbMNCs) were isolated from wound animals and treated with the supernatant of bone marrow mesenchymal stromal cells (bmMSCs). Results showed these pbMNCs were fibroblast-like, had stromal morphology, were negative for CD34 and CD45, but positive for Vimentin and Collagen I, and had the multipotency to differentiate into adipocytes and osteoblasts. We named these induced peripheral blood-derived mesenchymal stromal cells (ipbMSCs). Skin grafts in combination with ipbMSCs and collagen I were applied for wound healing, and results revealed ipbMSC exhibited similar potency and effectiveness in the promotion of wound healing to the bmMSCs. Hereafter, we speculate that the mixture of growth factors and chemokines secreted by bmMSCs may play an important roles in the induction of the proliferation and mesenchymal differentiation of mononuclear cells. Our results are clinically relevant because it provide a new method for the acquisition of MSCs which can be used as a candidate for the wound repair. PMID:21494428

  3. Breast tumor and stromal cell responses to TGF-β and hypoxia in matrix deposition

    PubMed Central

    Curran, Colleen S.; Keely, Patricia J

    2012-01-01

    The components that comprise the extracellular matrix (ECM) are integral to normal tissue homeostasis as well as the development and progression of breast tumors. The secretion, construction, and remodeling of the ECM are each regulated by a complex interplay between tumor cells, fibroblasts and macrophages. Transforming growth factor-β (TGF-β) is an essential molecule in regulating the cellular production of ECM molecules and the adhesive interactions of cells with the ECM. Additionally, hypoxic cell signals, initiated by oxygen deprivation, additional metabolic factors or receptor activation, are associated with ECM formation and the progression of breast cancer. Both TGF-β and hypoxic cell signals are implicated in the functional and morphological changes of cancer-associated-fibroblasts and tumor-associated-macrophages. Moreover, the enhanced recruitment of tumor and stromal cells in response to hypoxia-induced chemokines leads to increased ECM deposition and remodeling, increased blood vessel formation, and enhanced tumor migration. Thus, elucidation of the collaborative networks between tumor and stromal cells in response to the combined signals of TGF-β and hypoxia may yield insight into treatment parameters that target both tumor and stromal cells. PMID:23262216

  4. What Are the Risk Factors for Gastrointestinal Stromal Tumors?

    MedlinePlus

    ... Gastrointestinal Stromal Tumors Be Prevented? Gastrointestinal Stromal Tumor (GIST) Causes, Risk Factors, and Prevention What Are the ... few known risk factors for gastrointestinal stromal tumors (GISTs). Being older These tumors can occur in people ...

  5. What Are the Key Statistics about Gastrointestinal Stromal Tumors?

    MedlinePlus

    ... About Gastrointestinal Stromal Tumor What Are the Key Statistics About Gastrointestinal Stromal Tumors? Gastrointestinal stromal tumors (GISTs) ... They are slightly more common in men. Survival statistics for GIST are discussed in “ Survival rates for ...

  6. Interleukin-6 receptor in spindle-shaped stromal cells, a prognostic determinant of early breast cancer.

    PubMed

    Labovsky, Vivian; Martinez, Leandro Marcelo; Calcagno, María de Luján; Davies, Kevin Mauro; García-Rivello, Hernán; Wernicke, Alejandra; Feldman, Leonardo; Giorello, María Belén; Matas, Ayelén; Borzone, Francisco Raúl; Howard, Scott C; Chasseing, Norma Alejandra

    2016-10-01

    Spindle-shaped stromal cells, like carcinoma-associated fibroblasts and mesenchymal stem cells, influence tumor behavior and can serve as parameters in the clinical diagnosis, therapy, and prognosis of early breast cancer. Therefore, the aim of this study is to explore the clinicopathological significance of tumor necrosis factor-related apoptosis-induced ligand (TRAIL) receptors (Rs) 2 and 4 (TRAIL-R2 and R4), and interleukin-6 R (IL-6R) in spindle-shaped stromal cells, not associated with the vasculature, as prognostic determinants of early breast cancer patients. Receptors are able to trigger the migratory activity, among other functions, of these stromal cells. We conducted immunohistochemical analysis for the expression of these receptors in spindle-shaped stromal cells, not associated with the vasculature, of primary tumors from early invasive breast cancer patients, and analyzed their association with clinicopathological characteristics. Here, we demonstrate that the elevated levels of TRAIL-R2, TRAIL-R4, and IL-6R in these stromal cells were significantly associated with a higher risk of metastatic occurrence (p = 0.034, 0.026, and 0.006; respectively). Moreover, high expression of TRAIL-R4 was associated with shorter disease-free survival and metastasis-free survival (p = 0.013 and 0.019; respectively). Also, high expression of IL-6R was associated with shorter disease-free survival, metastasis-free survival, and overall survival (p = 0.003, 0.001, and 0.003; respectively). Multivariate analysis showed that IL-6R expression was an independent prognostic factor for disease-free survival and metastasis-free survival (p = 0.035). This study is the first to demonstrate that high levels of IL-6R expression in spindle-shaped stromal cells, not associated with the vasculature, could be used to identify early breast cancer patients with poor outcomes.

  7. Genetic ablation of Smoothened in pancreatic fibroblasts increases acinar–ductal metaplasia

    PubMed Central

    Liu, Xin; Pitarresi, Jason R.; Cuitiño, Maria C.; Kladney, Raleigh D.; Woelke, Sarah A.; Sizemore, Gina M.; Nayak, Sunayana G.; Egriboz, Onur; Schweickert, Patrick G.; Yu, Lianbo; Trela, Stefan; Schilling, Daniel J.; Halloran, Shannon K.; Li, Maokun; Dutta, Shourik; Fernandez, Soledad A.; Rosol, Thomas J.; Lesinski, Gregory B.; Shakya, Reena; Ludwig, Thomas; Konieczny, Stephen F.; Leone, Gustavo; Wu, Jinghai; Ostrowski, Michael C.

    2016-01-01

    The contribution of the microenvironment to pancreatic acinar-to-ductal metaplasia (ADM), a preneoplastic transition in oncogenic Kras-driven pancreatic cancer progression, is currently unclear. Here we show that disruption of paracrine Hedgehog signaling via genetic ablation of Smoothened (Smo) in stromal fibroblasts in a KrasG12D mouse model increased ADM. Smo-deleted fibroblasts had higher expression of transforming growth factor-α (Tgfa) mRNA and secreted higher levels of TGFα, leading to activation of EGFR signaling in acinar cells and increased ADM. The mechanism involved activation of AKT and noncanonical activation of the GLI family transcription factor GLI2. GLI2 was phosphorylated at Ser230 in an AKT-dependent fashion and directly regulated Tgfa expression in fibroblasts lacking Smo. Additionally, Smo-deleted fibroblasts stimulated the growth of KrasG12D/Tp53R172H pancreatic tumor cells in vivo and in vitro. These results define a non-cell-autonomous mechanism modulating KrasG12D-driven ADM that is balanced by cross-talk between Hedgehog/SMO and AKT/GLI2 pathways in stromal fibroblasts. PMID:27633013

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

    SciTech Connect

    Ishino, Ruri; Minami, Kaori; Tanaka, Satowa; Nagai, Mami; Matsui, Keiji; Hasegawa, Natsumi; Roeder, Robert G.; Asano, Shigetaka; Ito, Mitsuhiro

    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 for 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.

  9. Fibroblastic reticular cells: organization and regulation of the T lymphocyte life cycle.

    PubMed

    Brown, Flavian D; Turley, Shannon J

    2015-02-15

    The connective tissue of any organ in the body is generally referred to as stroma. This complex network is commonly composed of leukocytes, extracellular matrix components, mesenchymal cells, and a collection of nerves, blood, and lymphoid vessels. Once viewed primarily as a structural entity, stromal cells of mesenchymal origin are now being intensely examined for their ability to directly regulate various components of immune cell function. There is particular interest in the ability of stromal cells to influence the homeostasis, activation, and proliferation of T lymphocytes. One example of this regulation occurs in the lymph node, where fibroblastic reticular cells support the maintenance of naive T cells, induce Ag-specific tolerance, and restrict the expansion of newly activated T cells. In an effort to highlight the varied immunoregulatory properties of fibroblastic reticular cells, we reviewed the most recent advances in this field and provide some insights into potential future directions.

  10. Analysis of stromal cells in osteofibrous dysplasia and adamantinoma of long bones.

    PubMed

    Taylor, Richard M; Kashima, Takeshi G; Ferguson, David J; Szuhai, Károly; Hogendoorn, Pancras C; Athanasou, Nicholas A

    2012-01-01

    Adamantinoma of long bones and osteofibrous dysplasia are rare, osteolytic primary bone tumours of uncertain origin containing areas of fibrous and fibro-osseous proliferation. We investigated the nature of the stromal cells in adamantinoma of long bones and osteofibrous dysplasia, and determined cellular and molecular mechanisms of osteolysis in these tumours. Cell culture, molecular (RT-PCR, western blot) and immunohistochemical studies on cases of adamantinoma of long bones and of osteofibrous dysplasia were undertaken to determine the expression of epithelial, osteoblast and osteoclast markers. Ultrastructural and immunophenotypic studies on cultured adamantinoma and osteofibrous dysplasia stromal cells showed that these cells were mainly fibroblast-like with few cells expressing epithelial markers. Osteofibrous dysplasia but not adamantinoma cells expressed alkaline phosphatase. Both osteofibrous dysplasia and adamantinoma cells expressed the ostoclastogenic factors M-CSF and RANKL. Adamantinoma and osteofibrous dysplasia cells also expressed messenger RNA for osteocalcin, osteonectin, osteopontin, osterix and collagen type 1. Adamantinoma and osteofibrous dysplasia cells cultured alone on dentine slices were not capable of lacunar resorption, but in co-cultures with monocytes induced formation of osteoclast-like cells was observered. Cultured osteofibrous dysplasia and adamantinoma stromal cells show similar ultrastructural and immunophenotypic characteristics, and differentially express osteoblast markers. Promotion of osteoclastogenesis by stromal cells may contribute to osteolysis in adamantinoma of long bones and osteofibrous dysplasia.

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

    PubMed Central

    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. PMID:25374926

  12. 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.

  13. A molecular classification of human mesenchymal stromal cells

    PubMed Central

    Rohart, Florian; Mason, Elizabeth A.; Matigian, Nicholas; Mosbergen, Rowland; Korn, Othmar; Chen, Tyrone; Butcher, Suzanne; Patel, Jatin; Atkinson, Kerry; Khosrotehrani, Kiarash; Fisk, Nicholas M.; Lê Cao, Kim-Anh

    2016-01-01

    Mesenchymal stromal cells (MSC) are widely used for the study of mesenchymal tissue repair, and increasingly adopted for cell therapy, despite the lack of consensus on the identity of these cells. In part this is due to the lack of specificity of MSC markers. Distinguishing MSC from other stromal cells such as fibroblasts is particularly difficult using standard analysis of surface proteins, and there is an urgent need for improved classification approaches. Transcriptome profiling is commonly used to describe and compare different cell types; however, efforts to identify specific markers of rare cellular subsets may be confounded by the small sample sizes of most studies. Consequently, it is difficult to derive reproducible, and therefore useful markers. We addressed the question of MSC classification with a large integrative analysis of many public MSC datasets. We derived a sparse classifier (The Rohart MSC test) that accurately distinguished MSC from non-MSC samples with >97% accuracy on an internal training set of 635 samples from 41 studies derived on 10 different microarray platforms. The classifier was validated on an external test set of 1,291 samples from 65 studies derived on 15 different platforms, with >95% accuracy. The genes that contribute to the MSC classifier formed a protein-interaction network that included known MSC markers. Further evidence of the relevance of this new MSC panel came from the high number of Mendelian disorders associated with mutations in more than 65% of the network. These result in mesenchymal defects, particularly impacting on skeletal growth and function. The Rohart MSC test is a simple in silico test that accurately discriminates MSC from fibroblasts, other adult stem/progenitor cell types or differentiated stromal cells. It has been implemented in the www.stemformatics.org resource, to assist researchers wishing to benchmark their own MSC datasets or data from the public domain. The code is available from the CRAN

  14. Dupuytren's Contracture: Fibroblast Contraction?

    PubMed Central

    Gabbiani, Giulio; Majno, Guido

    1972-01-01

    In 6 cases of Dupuytren's disease and 1 of Ledderhose's disease, the nodules of the palmar and plantar aponeurosis were examined by light and electron microscopy. The cells composing these nodules, presumably fibroblasts, showed three significant ultrastructural features: (1) a fibrillar system similar to that of smooth muscle cells; (2) nuclear deformations such as are found in contracted cells, the severest being recognizable by light microscopy (cross-banded nuclei); (3) cell-to-cell and cell-to-stroma attachments. Based on these data and on recent information about the biology of the fibroblasts, it is suggested that these cells are fibroblasts that have modulated into contractile cells (myofibroblasts), and that their contraction plays a role in the pathogenesis of the contracture observed clinically. ImagesFig 10Fig 5Fig 11Fig 6 and 7Fig 8Fig 1Fig 2Fig 9Fig 3Fig 4 PMID:5009249

  15. Proteomic identification of prognostic tumour biomarkers, using chemotherapy-induced cancer-associated fibroblasts

    PubMed Central

    Peiris-Pagès, Maria; Smith, Duncan L.; Győrffy, Balázs; Sotgia, Federica; Lisanti, Michael P.

    2015-01-01

    Cancer cells grow in highly complex stromal microenvironments, which through metabolic remodelling, catabolism, autophagy and inflammation nurture them and are able to facilitate metastasis and resistance to therapy. However, these changes in the metabolic profile of stromal cancer-associated fibroblasts and their impact on cancer initiation, progression and metastasis are not well-known. This is the first study to provide a comprehensive proteomic portrait of the azathioprine and taxol-induced catabolic state on human stromal fibroblasts, which comprises changes in the expression of metabolic enzymes, myofibroblastic differentiation markers, antioxidants, proteins involved in autophagy, senescence, vesicle trafficking and protein degradation, and inducers of inflammation. Interestingly, many of these features are major contributors to the aging process. A catabolic stroma signature, generated with proteins found differentially up-regulated in taxol-treated fibroblasts, strikingly correlates with recurrence, metastasis and poor patient survival in several solid malignancies. We therefore suggest the inhibition of the catabolic state in healthy cells as a novel approach to improve current chemotherapy efficacies and possibly avoid future carcinogenic processes. PMID:26539730

  16. Stromal Cell Subsets Directing Neonatal Spleen Regeneration

    PubMed Central

    Tan, Jonathan K. H.; Watanabe, Takeshi

    2017-01-01

    Development of lymphoid tissue is determined by interactions between stromal lymphoid tissue organiser (LTo) and hematopoietic lymphoid tissue inducer (LTi) cells. A failure for LTo to receive appropriate activating signals during embryogenesis through lymphotoxin engagement leads to a complete cessation of lymph node (LN) and Peyer’s patch development, identifying LTo as a key stromal population for lymphoid tissue organogenesis. However, little is known about the equivalent stromal cells that induce spleen development. Here, by dissociating neonatal murine spleen stromal tissue for re-aggregation and transplant into adult mouse recipients, we have identified a MAdCAM-1+CD31+CD201+ spleen stromal organizer cell-type critical for new tissue formation. This finding provides an insight into the regulation of post-natal spleen tissue organogenesis, and could be exploited in the development of spleen regenerative therapies. PMID:28067323

  17. A Reproducible Method for Isolation and In Vitro Culture of Functional Human Lymphoid Stromal Cells from Tonsils

    PubMed Central

    Bar-Ephraim, Yotam E.; Konijn, Tanja; Gönültas, Mehmet; Mebius, Reina E.

    2016-01-01

    The stromal compartment of secondary lymphoid organs is classicaly known for providing a mechanical scaffold for the complex interactions between hematopoietic cells during immune activation as well as for providing a niche which is favorable for survival of lymphocytes. In recent years, it became increasingly clear that these cells also play an active role during such a response. Currently, knowledge of the interactions between human lymphoid stroma and hematopoietic cells is still lacking and most insight is based on murine systems. Although methods to isolate stromal cells from tonsils have been reported, data on stability in culture, characterization, and functional properties are lacking. Here, we describe a reproducible and easy method for isolation and in vitro culture of functional human lymphoid stromal cells from palatine tonsils. The cells isolated express markers and characteristics of T cell zone fibroblastic reticular cells (FRCs) and react to inflammatory stimuli by upregulating inflammatory cytokines and chemokines as well as adhesion molecules, as previously described for mouse lymphoid stroma. Also, cultured tonsil stromal cells support survival of human innate lymphoid cells, showing that these stromal cells can function as bone fide FRCs, providing a favorable microenvironment for hematopoietic cells. PMID:27907202

  18. Implication of Tumor Microenvironment in Chemoresistance: Tumor-Associated Stromal Cells Protect Tumor Cells from Cell Death

    PubMed Central

    Castells, Magali; Thibault, Benoît; Delord, Jean-Pierre; Couderc, Bettina

    2012-01-01

    Tumor development principally occurs following the accumulation of genetic and epigenetic alterations in tumor cells. These changes pave the way for the transformation of chemosensitive cells to chemoresistant ones by influencing the uptake, metabolism, or export of drugs at the cellular level. Numerous reports have revealed the complexity of tumors and their microenvironment with tumor cells located within a heterogeneous population of stromal cells. These stromal cells (fibroblasts, endothelial or mesothelial cells, adipocytes or adipose tissue-derived stromal cells, immune cells and bone marrow-derived stem cells) could be involved in the chemoresistance that is acquired by tumor cells via several mechanisms: (i) cell–cell and cell–matrix interactions influencing the cancer cell sensitivity to apoptosis; (ii) local release of soluble factors promoting survival and tumor growth (crosstalk between stromal and tumor cells); (iii) direct cell-cell interactions with tumor cells (crosstalk or oncologic trogocytosis); (iv) generation of specific niches within the tumor microenvironment that facilitate the acquisition of drug resistance; or (v) conversion of the cancer cells to cancer-initiating cells or cancer stem cells. This review will focus on the implication of each member of the heterogeneous population of stromal cells in conferring resistance to cytotoxins and physiological mediators of cell death. PMID:22949815

  19. 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

  20. Stromal uptake and transmission of acid is a pathway for venting cancer cell-generated acid

    PubMed Central

    Hulikova, Alzbeta; Black, Nicholas; Hsia, Lin-Ting; Wilding, Jennifer; Bodmer, Walter F.; Swietach, Pawel

    2016-01-01

    Proliferation and invasion of cancer cells require favorable pH, yet potentially toxic quantities of acid are produced metabolically. Membrane-bound transporters extrude acid from cancer cells, but little is known about the mechanisms that handle acid once it is released into the poorly perfused extracellular space. Here, we studied acid handling by myofibroblasts (colon cancer-derived Hs675.T, intestinal InMyoFib, embryonic colon-derived CCD-112-CoN), skin fibroblasts (NHDF-Ad), and colorectal cancer (CRC) cells (HCT116, HT29) grown in monoculture or coculture. Expression of the acid-loading transporter anion exchanger 2 (AE2) (SLC4A2 product) was detected in myofibroblasts and fibroblasts, but not in CRC cells. Compared with CRC cells, Hs675.T and InMyoFib myofibroblasts had very high capacity to absorb extracellular acid. Acid uptake into CCD-112-CoN and NHDF-Ad cells was slower and comparable to levels in CRC cells, but increased alongside SLC4A2 expression under stimulation with transforming growth factor β1 (TGFβ1), a cytokine involved in cancer–stroma interplay. Myofibroblasts and fibroblasts are connected by gap junctions formed by proteins such as connexin-43, which allows the absorbed acid load to be transmitted across the stromal syncytium. To match the stimulatory effect on acid uptake, cell-to-cell coupling in NHDF-Ad and CCD-112-CoN cells was strengthened with TGFβ1. In contrast, acid transmission was absent between CRC cells, even after treatment with TGFβ1. Thus, stromal cells have the necessary molecular apparatus for assembling an acid-venting route that can improve the flow of metabolic acid through tumors. Importantly, the activities of stromal AE2 and connexin-43 do not place an energetic burden on cancer cells, allowing resources to be diverted for other activities. PMID:27543333

  1. Contribution of Fibroblast and Mast Cell (Afferent) and Tumor (Efferent) IL-6 Effects within the Tumor Microenvironment.

    PubMed

    Hugo, Honor J; Lebret, Stephanie; Tomaskovic-Crook, Eva; Ahmed, Nuzhat; Blick, Tony; Newgreen, Donald F; Thompson, Erik W; Ackland, M Leigh

    2012-04-01

    Hyperactive inflammatory responses following cancer initiation have led to cancer being described as a 'wound that never heals'. These inflammatory responses elicit signals via NFκB leading to IL-6 production, and IL-6 in turn has been shown to induce epithelial to mesenchymal transition in breast cancer cells in vitro, implicating a role for this cytokine in cancer cell invasion. We previously have shown that conditioned medium derived from cancer-associated fibroblasts induced an Epithelial to Mesenchymal transition (EMT) in PMC42-LA breast cancer cells and we have now identify IL-6 as present in this medium. We further show that IL-6 is expressed approximately 100 fold higher in a cancer-associated fibroblast line compared to normal fibroblasts. Comparison of mouse-specific (stroma) and human-specific (tumor) IL-6 mRNA expression from MCF-7, MDA MB 468 and MDA MB 231 xenografts also indicated the stroma rather than tumor as a significantly higher source of IL-6 expression. Mast cells (MCs) feature in inflammatory cancer-associated stroma, and activated MCs secrete IL-6. We observed a higher MC index (average number of mast cells per xenograft section/average tumor size) in MDA MB 468 compared to MDA MB 231 xenografts, where all MC were observed to be active (degranulating). This higher MC index correlated with greater mouse-specific IL-6 expression in the MDA MB 468 xenografts, implicating MC as an important source of stromal IL-6. Furthermore, immunohistochemistry on these xenografts for pSTAT3, which lies downstream of the IL-6 receptor indicated frequent correlations between pSTAT3 and mast cell positive cells. Analysis of publically available databases for IL-6 expression in patient tissue revealed higher IL-6 in laser capture microdissected stroma compared to adjacent tissue epithelium from patients with inflammatory breast cancer (IBC) and invasive non-inflammatory breast cancer (non-IBC) and we show that IL-6 expression was significantly higher in Basal

  2. Wnt-3a-activated human fibroblasts promote human keratinocyte proliferation and matrix destruction.

    PubMed

    Sobel, Katrin; Tham, Marius; Stark, Hans-Jürgen; Stammer, Hermann; Prätzel-Wunder, Silke; Bickenbach, Jackie R; Boukamp, Petra

    2015-06-15

    Aberrant Wnt regulation, detectable by nuclear translocation of beta-catenin, is a hallmark of many cancers including skin squamous cell carcinomas (SCCs). By analyzing primary human skin SCCs, we demonstrate that nuclear beta-catenin is not restricted to SCC cells but also detected in stromal fibroblasts, suggesting an important role for aberrant Wnt regulation also in the tumor microenvironment. When human keratinocytes and fibroblasts were treated with Wnt-3a, fibroblasts proved to be more responsive. Accordingly, Wnt-3a did not alter HaCaT cell functions in a cell-autonomous manner. However, when organotypic cultures (OTCs) were treated with Wnt-3a, HaCaT keratinocytes responded with increased proliferation. As nuclear beta-catenin was induced only in the fibroblasts, this argued for a Wnt-dependent, paracrine keratinocyte stimulation. Global gene expression analysis of Wnt-3a-stimulated fibroblasts identified genes encoding interleukin-8 (IL-8) and C-C motif chemokine 2 (CCL-2) as well as matrix metalloproteinase-1 (MMP-1) as Wnt-3a targets. In agreement, we show that IL-8 and CCL-2 were secreted in high amounts by Wnt-3a-stimulated fibroblasts also in OTCs. The functional role of IL-8 and CCL-2 as keratinocyte growth regulators was confirmed by directly stimulating HaCaT cell proliferation in conventional cultures. Most important, neutralizing antibodies against IL-8 and CCL-2 abolished the Wnt-dependent HaCaT cell hyperproliferation in OTCs. Additionally, MMP-1 was expressed in high amounts in Wnt-3a-stimulated OTCs and degraded the stromal matrix. Thus, our data show that Wnt-3a stimulates fibroblasts to secrete both keratinocyte proliferation-inducing cytokines and stroma-degrading metalloproteinases, thereby providing evidence for a novel Wnt deregulation in the tumor-stroma directly contributing to skin cancer progression.

  3. Inflammatory stromal keratopathies: medical management of stromal keratomalacia, stromal abscesses, eosinophilic keratitis, and band keratopathy in the horse.

    PubMed

    Brooks, Dennis E

    2004-08-01

    This article discusses the diagnosis and medical treatment of stromal keratomalacia or "melting ulcers," stromal abscesses, eosinophilic keratitis (EK), and calcific band keratopathy. These are common and important inflammatory keratopathies of the equine corneal stroma. Keratomalacia and stromal abscesses are associated with infection, leukocytic invasion of the stroma, and loss of tissue and tear film proteinase homeostasis. Eosinophils infiltrate the stroma in response to unknown stimuli in EK. Calcium is deposited in the stroma and epithelium secondary to chronic equine recurrent uveitis in calcific band keratopathy. They are all associated with varying degrees of iridocyclitis.

  4. Assessment of the stromal contribution to Sonic Hedgehog-dependent pancreatic adenocarcinoma.

    PubMed

    Damhofer, Helene; Medema, Jan Paul; Veenstra, Veronique L; Badea, Liviu; Popescu, Irinel; Roelink, Henk; Bijlsma, Maarten F

    2013-12-01

    Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. It is typically detected at an advanced stage, at which the therapeutic options are very limited. One remarkable feature of PDAC that contributes to its resilience to treatment is the extreme stromal activation seen in these tumors. Often, the vast majority of tumor bulk consists of non-tumor cells that together provide a tumor-promoting environment. One of the signals that maintains and activates the stroma is the developmental protein Sonic Hedgehog (SHH). As the disease progresses, tumor cells produce increasing amounts of SHH, which activates the surrounding stroma to aid in tumor progression. To better understand this response and identify targets for inhibition, we aimed to elucidate the proteins that mediate the SHH-driven stromal response in PDAC. For this a novel mixed-species coculture model was set up in which the cancer cells are human, and the stroma is modeled by mouse fibroblasts. In conjunction with next-generation sequencing we were able to use the sequence difference between these species to genetically distinguish between the epithelial and stromal responses to SHH. The stromal SHH-dependent genes from this analysis were validated and their relevance for human disease was subsequently determined in two independent patient cohorts. In non-microdissected tissue from PDAC patients, in which a large amount of stroma is present, the targets were confirmed to associate with tumor stroma versus normal pancreatic tissue. Patient survival analysis and immunohistochemistry identified CDA, EDIL3, ITGB4, PLAUR and SPOCK1 as SHH-dependent stromal factors that are associated with poor prognosis in PDAC patients. Summarizing, the presented data provide insight into the role of the activated stroma in PDAC, and how SHH acts to mediate this response. In addition, the study has yielded several candidates that are interesting therapeutic targets for a disease for which treatment

  5. Pancreatic cancer-secreted miR-155 implicates in the conversion from normal fibroblasts to cancer-associated fibroblasts

    PubMed Central

    Pang, Wenjing; Su, Jiaojiao; Wang, Yalei; Feng, Hui; Dai, Xin; Yuan, Yaozong; Chen, Xi; Yao, Weiyan

    2015-01-01

    Cancer-associated fibroblasts (CAF) are a major constituent of the pancreatic cancer microenvironment and that the meaning is as intended. Pancreatic cancer cells can induce normal fibroblasts to convert into CAF and, reciprocally, CAF promote tumor invasions and proliferations. The mechanism of the conversion from normal fibroblasts (NF) to CAF remains unclear. MicroRNA are short non-coding RNA involved in the post-transcription gene regulation, which have been defined as an imperative controller in tumor invasions, proliferations and colony formations. Microvesicles (MV) have been proved to be an important mediator of intercellular communication and can selectively transport secreted microRNA from a donor cell into a recipient cell. In this study, we isolated primary pancreatic fibroblasts from wild type C57 mice and co-cultured them with pancreatic cancer cell lines, BxPC-3 and SW1990, and observed the conversion from NF to CAF, or at least CAF-like cells. This phenomenon could also be replicated in primary fibroblasts treated with MV separated from a cancer cell media. We identified that miR-155 was upregulated in PaC-derived MV and we confirmed that normal fibroblasts could convert into CAF after MV containing miR-155 had been taken up. TP53INP1 is a target of miR-155 in fibroblasts and a downregulation of TP53INP1 protein levels could contribute to the fibroblasts’ activation. These results indicated that pancreatic cancer cells might reprogram normal adjacent fibroblasts into CAF by means of secreted MV containing miR-155. Targeting the circulating microRNA might be a potential therapy for malignant tumors. PMID:26195069

  6. Fibroblastic activities post implantation of cobalt chromium alloy and pure germanium in rabbits.

    PubMed

    Carter, J M; Natiella, J R; Baier, R E; Natiella, R R

    1984-02-01

    Different preimplantation surface finishes were applied to surgical vitallium discs and germanium prisms implanted for 20 days within the back muscles of adult rabbits. Histopathologic analysis of the numbers of nuclei of active fibroblasts immediately adjacent to the implants was carried out. The mean apparent volume fractions (MAVF) for the subdermal implant sites were found to depend on the surface cleanliness of the implant, the cleanest or highest-surface-energy surfaces giving the highest MAVF values for active fibroblasts.

  7. Cigarette smoke metabolically promotes cancer, via autophagy and premature aging in the host stromal microenvironment

    PubMed Central

    Salem, Ahmed F.; Al-Zoubi, Mazhar Salim; Whitaker-Menezes, Diana; Martinez-Outschoorn, Ubaldo E.; Lamb, Rebecca; Hulit, James; Howell, Anthony; Gandara, Ricardo; Sartini, Marina; Galbiati, Ferruccio; Bevilacqua, Generoso; Sotgia, Federica; Lisanti, Michael P.

    2013-01-01

    Cigarette smoke has been directly implicated in the disease pathogenesis of a plethora of different human cancer subtypes, including breast cancers. The prevailing view is that cigarette smoke acts as a mutagen and DNA damaging agent in normal epithelial cells, driving tumor initiation. However, its potential negative metabolic effects on the normal stromal microenvironment have been largely ignored. Here, we propose a new mechanism by which carcinogen-rich cigarette smoke may promote cancer growth, by metabolically “fertilizing” the host microenvironment. More specifically, we show that cigarette smoke exposure is indeed sufficient to drive the onset of the cancer-associated fibroblast phenotype via the induction of DNA damage, autophagy and mitophagy in the tumor stroma. In turn, cigarette smoke exposure induces premature aging and mitochondrial dysfunction in stromal fibroblasts, leading to the secretion of high-energy mitochondrial fuels, such as L-lactate and ketone bodies. Hence, cigarette smoke induces catabolism in the local microenvironment, directly fueling oxidative mitochondrial metabolism (OXPHOS) in neighboring epithelial cancer cells, actively promoting anabolic tumor growth. Remarkably, these autophagic-senescent fibroblasts increased breast cancer tumor growth in vivo by up to 4-fold. Importantly, we show that cigarette smoke-induced metabolic reprogramming of the fibroblastic stroma occurs independently of tumor neo-angiogenesis. We discuss the possible implications of our current findings for the prevention of aging-associated human diseases and, especially, common epithelial cancers, as we show that cigarette smoke can systemically accelerate aging in the host microenvironment. Finally, our current findings are consistent with the idea that cigarette smoke induces the “reverse Warburg effect,” thereby fueling “two-compartment tumor metabolism” and oxidative mitochondrial metabolism in epithelial cancer cells. PMID:23388463

  8. PDGFRβ Is a Novel Marker of Stromal Activation in Oral Squamous Cell Carcinomas

    PubMed Central

    Han, Rong; Haines, Paul; Gallagher, George; Noonan, Vikki; Kukuruzinska, Maria; Monti, Stefano; Trojanowska, Maria

    2016-01-01

    Carcinoma associated fibroblasts (CAFs) form the main constituents of tumor stroma and play an important role in tumor growth and invasion. The presence of CAFs is a strong predictor of poor prognosis of head and neck squamous cell carcinoma. Despite significant progress in determining the role of CAFs in tumor progression, the mechanisms contributing to their activation remain poorly characterized, in part due to fibroblast heterogeneity and the scarcity of reliable fibroblast surface markers. To search for such markers in oral squamous cell carcinoma (OSCC), we applied a novel approach that uses RNA-sequencing data derived from the cancer genome atlas (TCGA). Specifically, our strategy allowed for an unbiased identification of genes whose expression was closely associated with a set of bona fide stroma-specific transcripts, namely the interstitial collagens COL1A1, COL1A2, and COL3A1. Among the top hits were genes involved in cellular matrix remodeling and tumor invasion and migration, including platelet-derived growth factor receptor beta (PDGFRβ), which was found to be the highest-ranking receptor protein genome-wide. Similar analyses performed on ten additional TCGA cancer datasets revealed that other tumor types shared CAF markers with OSCC, including PDGFRβ, which was found to significantly correlate with the reference collagen expression in ten of the 11 cancer types tested. Subsequent immunostaining of OSCC specimens demonstrated that PDGFRβ was abundantly expressed in stromal fibroblasts of all tested cases (12/12), while it was absent in tumor cells, with greater specificity than other known markers such as alpha smooth muscle actin or podoplanin (3/11). Overall, this study identified PDGFRβ as a novel marker of stromal activation in OSCC, and further characterized a list of promising candidate CAF markers that may be relevant to other carcinomas. Our novel approach provides for a fast and accurate method to identify CAF markers without the need for

  9. Fibroblast PER2 Circadian Rhythmicity Depends on Cell Density

    PubMed Central

    Noguchi, Takako; Wang, Lexie L.; Welsh, David K.

    2013-01-01

    Like neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in the brain, single fibroblasts can function as independent oscillators. In the SCN, synaptic and paracrine signaling among cells creates a robust, synchronized circadian oscillation, whereas there is no evidence for such integration in fibroblast cultures. However, interactions among single-cell fibroblast oscillators cannot be completely excluded, because fibroblasts were not isolated in previous work. In this study, we tested the autonomy of fibroblasts as single-cell circadian oscillators in high and low density culture, by single-cell imaging of cells from PER2::LUC circadian reporter mice. We found greatly reduced PER2::LUC rhythmicity in low density cultures, which could result from lack of either constitutive or rhythmic paracrine signals from neighboring fibroblasts. To discriminate between these two possibilities, we mixed PER2::LUC wild type (WT) cells with non-luminescent, non-rhythmic Bmal1−/− cells, so that density of rhythmic cells was low but overall cell density remained high. In this condition, WT cells showed clear rhythmicity similar to high density cultures. We also mixed PER2::LUC WT cells with non-luminescent, long period Cry2−/− cells. In this condition, WT cells showed a period no different from cells cultured with rhythmic WT cells or non-rhythmic Bmal1−/− cells. In previous work, we found that low K+ suppresses fibroblast rhythmicity, and we and others have found that either low K+ or low Ca2+ suppresses SCN rhythmicity. Therefore, we attempted to rescue rhythmicity of low density fibroblasts with high K+ (21 mM), high Ca2+ (3.6 mM), or conditioned medium. Conditioned medium from high density fibroblast cultures rescued rhythmicity of low density cultures, whereas high K+ or Ca2+ medium did not consistently rescue rhythmicity. These data suggest that fibroblasts require paracrine signals from adjacent cells for normal expression of rhythmicity

  10. Efficient Generation of Chemically Induced Mesenchymal Stem Cells from Human Dermal Fibroblasts

    PubMed Central

    Lai, Pei-Lun; Lin, Hsuan; Chen, Shang-Fu; Yang, Shang-Chih; Hung, Kuo-Hsuan; Chang, Ching-Fang; Chang, Hsiang-Yi; Lu, Frank Leigh; Lee, Yi-Hsuan; Liu, Yu-Chuan; Huang, Hsiao-Chun; Lu, Jean

    2017-01-01

    Human mesenchymal stromal/stem cells (MSCs) are multipotent and currently undergoing hundreds of clinical trials for disease treatments. To date, no studies have generated induced MSCs from skin fibroblasts with chemicals or growth factors. Here, we established the first chemical method to convert primary human dermal fibroblasts into multipotent, induced MSC-like cells (iMSCs). The conversion method uses a defined cocktail of small molecules and growth factors, and it can achieve efficient conversion with an average rate of 38% in 6 days. The iMSCs have much higher clonogenicity than fibroblasts, and they can be maintained and expanded in regular MSC medium for at least 8 passages and further differentiated into osteoblasts, adipocytes, and chondrocytes. Moreover, the iMSCs can suppress LPS-mediated acute lung injury as effectively as bone marrow-derived mesenchymal stem cells. This finding may greatly benefit stem cell biology, cell therapy, and regenerative medicine. PMID:28303927

  11. Isolating stromal stem cells from periodontal granulation tissues.

    PubMed

    Hung, Tzu-Yuan; Lin, Hsiang-Chun; Chan, Ying-Jen; Yuan, Kuo

    2012-08-01

    Stem cell therapy is a promising area in regenerative medicine. Periodontal granulation tissues are often discarded during conventional surgery. If stromal stem cells can be isolated from these tissues, they can be used for subsequent surgery on the same patient. Fifteen human periodontal granulation tissue samples were obtained from intrabony defects during surgery. Immunohistochemistry (IHC) was carried out on five of the samples to identify STRO-1, a marker of mesenchymal stem cells. Five samples underwent flow cytometry analysis for the same marker. The remaining five samples were characterized by "colony formation unit-fibroblast" (CFU-f) assay and selected for proliferation assay, flow cytometry of stem cell markers, immunocytochemistry (ICC), multipotent differentiation assays, and repairing critical-size defects in mice. The ratio of STRO-1(+) cells detected by IHC was 5.91 ± 1.50%. The analysis of flow cytometry for STRO-1 was 6.70 ± 0.81%. Approximately two thirds of the CFU-f colonies had a strong reaction to STRO-1 in ICC staining. The cells were multipotent both in vitro and in vivo. Mice given bone grafts and stem cells showed significantly better bone healing than those without stem cells. Multipotent stromal stem cells can be isolated from human periodontal granulation tissues. These cells improve new bone formation when transplanted in mouse calvarial defects. Isolating stem cells from relatively accessible sites without extra procedures is clinically advantageous. This study demonstrated that human periodontal granulation tissues contain isolatable multipotent stem cells. The cells may be a good source for autotransplantation in subsequent treatment.

  12. Zfp423 promotes adipogenic differentiation of bovine stromal vascular cells.

    PubMed

    Huang, Yan; Das, Arun Kr; Yang, Qi-Yuan; Zhu, Mei-Jun; Du, Min

    2012-01-01

    Intramuscular fat or marbling is critical for the palatability of beef. In mice, very recent studies show that adipocytes and fibroblasts share a common pool of progenitor cells, with Zinc finger protein 423 (Zfp423) as a key initiator of adipogenic differentiation. To evaluate the role of Zfp423 in intramuscular adipogenesis and marbling in beef cattle, we sampled beef muscle for separation of stromal vascular cells. These cells were immortalized with pCI neo-hEST2 and individual clones were selected by G418. A total of 288 clones (3×96 well plates) were isolated and induced to adipogenesis. The presence of adipocytes was assessed by Oil-Red-O staining. Three clones with high and low adipogenic potential respectively were selected for further analyses. In addition, fibro/adipogenic progenitor cells were selected using a surface marker, platelet derived growth factor receptor (PDGFR) α. The expression of Zfp423 was much higher (307.4±61.9%, P<0.05) in high adipogenic cells, while transforming growth factor (TGF)-β was higher (156.1±48.7%, P<0.05) in low adipogenic cells. Following adipogenic differentiation, the expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) were much higher (239.4±84.1% and 310.7±138.4%, respectively, P<0.05) in high adipogenic cells. Over-expression of Zfp423 in stromal vascular cells and cloned low adipogenic cells dramatically increased their adipogenic differentiation, accompanied with the inhibition of TGF-β expression. Zfp423 knockdown by shRNA in high adipogenic cells largely prevented their adipogenic differentiation. The differential regulation of Zfp423 and TGF-β between low and high adipogenic cells is associated with the DNA methylation in their promoters. In conclusion, data show that Zfp423 is a critical regulator of adipogenesis in stromal vascular cells of bovine muscle, and Zfp423 may provide a molecular target for enhancing intramuscular adipogenesis

  13. The Role of the Transcriptional Regulation of Stromal Cells in Chronic Inflammation

    PubMed Central

    Valin, Alvaro; Pablos, José L.

    2015-01-01

    Chronic inflammation is a common process connecting pathologies that vary in their etiology and pathogenesis such as cancer, autoimmune diseases, and infections. The response of the immune system to tissue damage involves a carefully choreographed series of cellular interactions between immune and non-immune cells. In recent years, it has become clear that stromal resident cells have an essential role perpetuating the inflammatory environment and dictating in many cases the outcome of inflammatory based pathologies. Signal transduction pathways remain the main focus of study to understand how stimuli contribute to perpetuating the inflammatory response, mainly due to their potential role as therapeutic targets. However, molecular events orchestrated in the nucleus by transcription factors add additional levels of complexity and may be equally important for understanding the phenotypic differences of activated stromal components during the chronic inflammatory process. In this review, we focus on the contribution of transcription factors to the selective regulation of inducible proinflammatory genes, with special attention given to the regulation of the stromal fibroblastic cell function and response. PMID:26501341

  14. Targeting Stromal-Cancer Cell Crosstalk Networks in Ovarian Cancer Treatment

    PubMed Central

    Yeung, Tsz-Lun; Leung, Cecilia S.; Li, Fuhai; Wong, Stephen T. C.; Mok, Samuel C.

    2016-01-01

    Ovarian cancer is a histologically, clinically, and molecularly diverse disease with a five-year survival rate of less than 30%. It has been estimated that approximately 21,980 new cases of epithelial ovarian cancer will be diagnosed and 14,270 deaths will occur in the United States in 2015, making it the most lethal gynecologic malignancy. Ovarian tumor tissue is composed of cancer cells and a collection of different stromal cells. There is increasing evidence that demonstrates that stromal involvement is important in ovarian cancer pathogenesis. Therefore, stroma-specific signaling pathways, stroma-derived factors, and genetic changes in the tumor stroma present unique opportunities for improving the diagnosis and treatment of ovarian cancer. Cancer-associated fibroblasts (CAFs) are one of the major components of the tumor stroma that have demonstrated supportive roles in tumor progression. In this review, we highlight various types of signaling crosstalk between ovarian cancer cells and stromal cells, particularly with CAFs. In addition to evaluating the importance of signaling crosstalk in ovarian cancer progression, we discuss approaches that can be used to target tumor-promoting signaling crosstalk and how these approaches can be translated into potential ovarian cancer treatment. PMID:26751490

  15. Stromal expression of Fer suppresses tumor progression in renal cell carcinoma and is a predictor of survival

    PubMed Central

    Mitsunari, Kensuke; Miyata, Yasuyoshi; Watanabe, Shin-Ichi; Asai, Akihiro; Yasuda, Takuji; Kanda, Shigeru; Sakai, Hideki

    2017-01-01

    Fps/Fes related (Fer) is a non-receptor tyrosine kinase that is expressed in fibroblasts, immune cells and endothelial cells. Fer serves an important pathological role in cell survival, angiogenesis and the immune system. However, the pathological role of Fer expression in the stromal cells surrounding renal cell carcinoma (RCC) has not been previously investigated. In the present study, immunohistochemical analysis of Fer was performed using the formalin-fixed tissue samples of 152 patients with RCC. The proliferative and apoptotic indices were used to represent the percentage of proliferation marker protein Ki-67- and cleaved caspase-3-positive cells, respectively. The microvessel density was defined as the number of cluster of differentiation (CD) 31-positively stained vessels/mm2. In addition, CD57+ and CD68+ cells were counted using semi-quantification of natural killer (NK) cells and macrophages. Fer expression in stromal cells was negatively associated with Fuhrman grade, pathological tumor stage and metastasis (P<0.001). Fer expression in stromal cells was negatively associated with CD68+ macrophage density, whereas it was positively associated with CD57+ NK cell density. Kaplan-Meier estimators indicated that decreased stromal Fer expression was a predictive marker of decreased cause-specific survival rate (P<0.001). Furthermore, low expression of Fer was identified as being an independent marker of decreased cause-specific survival using multivariate analysis (hazard ratio, 7.4; 95% confidence interval, 1.7–33.0; P<0.001). The results of the present study suggested that low Fer expression in stromal cells is associated with increased malignant aggressiveness and decreased survival in patients with RCC. CD57+ NK cell and CD68+ macrophage regulation in cancer-stromal tissue is considered to affect RCC pathology. PMID:28356966

  16. Asporin is a stromally expressed marker associated with prostate cancer progression

    PubMed Central

    Rochette, Annie; Boufaied, Nadia; Scarlata, Eleonora; Hamel, Lucie; Brimo, Fadi; Whitaker, Hayley C; Ramos-Montoya, Antonio; Neal, David E; Dragomir, Alice; Aprikian, Armen; Chevalier, Simone; Thomson, Axel A

    2017-01-01

    Background: Prostate cancer shows considerable heterogeneity in disease progression and we propose that markers expressed in tumour stroma may be reliable predictors of aggressive tumour subtypes. Methods: We have used Kaplan–Meier, univariate and multivariate analysis to correlate the expression of Asporin (ASPN) mRNA and protein with prostate cancer progression in independent cohorts. We used immunohistochemistry and H scoring to document stromal localisation of ASPN in a tissue microarray and mouse prostate cancer model, and correlated expression with reactive stroma, defined using Masson Trichrome staining. We used cell cultures of primary prostate cancer fibroblasts treated with serum-free conditioned media from prostate cancer cell lines to examine regulation of ASPN mRNA in tumour stromal cells. Results: We observed increased expression of ASPN mRNA in a data set derived from benign vs tumour microdissected tissue, and a correlation with biochemical recurrence using Kaplan–Meier and Cox proportional hazard analysis. ASPN protein localised to tumour stroma and elevated expression of ASPN was correlated with decreased time to biochemical recurrence, in a cohort of 326 patients with a median follow up of 9.6 years. Univariate and multivariate analysis demonstrated that ASPN was correlated with progression, as were Gleason score, and clinical stage. Additionally, ASPN expression correlated with the presence of reactive stroma, suggesting that it may be a stromal marker expressed in response to the presence of tumour cells and particularly with aggressive tumour subtypes. We observed expression of ASPN in the stroma of tumours induced by p53 inhibition in a mouse model of prostate cancer, and correlation with neuroendocrine marker expression. Finally, we demonstrated that ASPN transcript expression in normal and cancer fibroblasts was regulated by conditioned media derived from the PC3, but not LNCaP, prostate cancer cell lines. Conclusions: Our results

  17. Distinctive Responsiveness to Stromal Signaling Accompanies Histologic Grade Programming of Cancer Cells

    PubMed Central

    Sayeed, Aejaz; Champion, Stacey; Goodson, William H.; Jeffrey, Stefanie S.; Xiao, Wenzhong; Mindrinos, Michael; Davis, Ronald W.; Dairkee, Shanaz H.

    2011-01-01

    Whether stromal components facilitate growth, invasion, and dissemination of cancer cells or suppress neoplastic lesions from further malignant progression is a continuing conundrum in tumor biology. Conceptualizing a dynamic picture of tumorigenesis is complicated by inter-individual heterogeneity. In the post genomic era, unraveling such complexity remains a challenge for the cancer biologist. Towards establishing a functional association between cellular crosstalk and differential cancer aggressiveness, we identified a signature of malignant breast epithelial response to stromal signaling. Proximity to fibroblasts resulted in gene transcript alterations of >2-fold for 107 probes, collectively designated as Fibroblast Triggered Gene Expression in Tumor (FTExT). The hazard ratio predicted by the FTExT classifier for distant relapse in patients with intermediate and high grade breast tumors was significant compared to routine clinical variables (dataset 1, n = 258, HR – 2.11, 95% CI 1.17–3.80, p-value 0.01; dataset 2, n = 171, HR - 3.07, 95% CI 1.21–7.83, p-value 0.01). Biofunctions represented by FTExT included inflammatory signaling, free radical scavenging, cell death, and cell proliferation. Unlike genes of the ‘proliferation cluster’, which are overexpressed in aggressive primary tumors, FTExT genes were uniquely repressed in such cases. As proof of concept for our correlative findings, which link stromal-epithelial crosstalk and tumor behavior, we show a distinctive differential in stromal impact on prognosis-defining functional endpoints of cell cycle progression, and resistance to therapy-induced growth arrest and apoptosis in low vs. high grade cancer cells. Our experimental data thus reveal aspects of ‘paracrine cooperativity’ that are exclusively contingent upon the histopathologically defined grade of interacting tumor epithelium, and demonstrate that epithelial responsiveness to the tumor microenvironment is a deterministic factor

  18. COX2 (PTGS2) gene methylation in epithelial, subepithelial lymphocyte and stromal tissue compartments in a spectrum of esophageal squamous neoplasia

    PubMed Central

    Dawsey, Sonja P.; Roth, Mark J.; Adams, Lisa; Hu, Nan; Wang, Quan-Hong; Taylor, Philip R.; Woodson, Karen

    2008-01-01

    Background Previous studies have shown important effects of stromal elements in carcinogenesis. To explore the tumor-stromal relationship in esophageal neoplasia, we examined methylation of COX-2 (PTGS2), a gene etiologically associated with the development of gastrointestinal cancers, in adjacent foci of epithelium, subepithelial lymphocytes and non-lymphocytic stromal cells found in sections of normal squamous epithelium, squamous dysplasia and invasive esophageal squamous cell carcinoma. Methods Adjacent foci of epithelium, subepithelial lymphocytic aggregates and non-lymphocytic stromal tissues were laser microdissected from six fully embedded, ethanol fixed, esophagectomy samples from Shanxi, China, a high-risk region for esophageal cancer. Promoter CpG site-specific hypermethylation status of COX-2 was determined using real-time methylation specific PCR (qMS-PCR) based on Taqman Chemistry. The methylation status of a subset of samples was confirmed by pyrosequencing. Results Forty-nine microdissected foci were analyzed. COX-2 gene methylation was significantly more common in subepithelial lymphocytes (12/16 (75% of all foci)) than in epithelial foci (3/16 (19%)) or foci of non-lymphocytic stromal tissues (3/17 (18%)) (Fisher’s Exact p=0.05). Two of three epithelial samples and all three stromal samples that showed COX-2 methylation were adjacent to foci of methylated subepithelial lymphocytes. Pyrosequencing confirmed the methylation status in a subset of samples. Conclusions In these esopohageal cancer patients, COX-2 gene methylation was more common in subepithelial lymphocytes than in adjacent epithelial or stromal cells in both grades of dysplasia and in foci of invasive cancer. These findings raise the possibility that methylation of subepithelial lymphocytes may be important for tumorigenesis. Future studies of gene methylation should consider separate evaluation of epithelial and non-epithelial cell populations. Condensed abstract COX2 (PTGS2) gene

  19. Expression of Siglec-11 by human and chimpanzee ovarian stromal cells, with uniquely human ligands: implications for human ovarian physiology and pathology.

    PubMed

    Wang, Xiaoxia; Chow, Renee; Deng, Liwen; Anderson, Dan; Weidner, Noel; Godwin, Andrew K; Bewtra, Chanda; Zlotnik, Albert; Bui, Jack; Varki, Ajit; Varki, Nissi

    2011-08-01

    Siglecs (Sialic acid-binding Immunoglobulin Superfamily Lectins) are cell surface signaling receptors of the I-type lectin group that recognize sialic acid-bearing glycans. CD33-related-Siglecs are a subset with expression primarily in cells of hematopoietic origin and functional relevance to immune reactions. Earlier we reported a human-specific gene conversion event that markedly changed the coding region for the extracellular domain of Siglec-11, associated with human-specific expression in microglia (Hayakawa T, Angata T, Lewis AL, Mikkelsen TS, Varki NM, Varki A. 2005. A human-specific gene in microglia. Science. 309:1693). Analyzing human gene microarrays to define new patterns of expression, we observed high levels of SIGLEC11 transcript in the ovary and adrenal cortex. Thus, we examined human and chimpanzee tissues using a well-characterized anti-Siglec-11 mouse monoclonal antibody. Although adrenal expression was variable and confined to infiltrating macrophages in capillaries, ovarian expression of Siglec-11 in both humans and chimpanzees was on fibroblasts, the first example of Siglec expression on mesenchyme-derived stromal cells. Cytokines from such ovarian stromal fibroblasts play important roles in follicle development and ovulation. Stable transfection of SIGLEC11 into a primary human ovarian stromal fibroblast cell line altered the secretion of growth-regulated oncogene α, interleukin (IL)-10, IL-7, transforming growth factor β1 and tumor necrosis factor-α, cytokines involved in ovarian physiology. Probing for Siglec-11 ligands revealed distinct and strong mast cell expression in human ovaries, contrasting to diffuse stromal ligands in chimpanzee ovaries. Interestingly, there was a trend of increased Siglec-11 expression in post-menopausal ovaries compared with pre-menopausal ones. Siglec-11 expression was also found on human ovarian stromal tumors and in polycystic ovarian syndrome, a human-specific disease. These results indicate potential

  20. Expression of Siglec-11 by human and chimpanzee ovarian stromal cells, with uniquely human ligands: implications for human ovarian physiology and pathology

    PubMed Central

    Wang, Xiaoxia; Chow, Renee; Deng, Liwen; Anderson, Dan; Weidner, Noel; Godwin, Andrew K; Bewtra, Chanda; Zlotnik, Albert; Bui, Jack; Varki, Ajit; Varki, Nissi

    2011-01-01

    Siglecs (Sialic acid-binding Immunoglobulin Superfamily Lectins) are cell surface signaling receptors of the I-type lectin group that recognize sialic acid-bearing glycans. CD33-related-Siglecs are a subset with expression primarily in cells of hematopoietic origin and functional relevance to immune reactions. Earlier we reported a human-specific gene conversion event that markedly changed the coding region for the extracellular domain of Siglec-11, associated with human-specific expression in microglia (Hayakawa T, Angata T, Lewis AL, Mikkelsen TS, Varki NM, Varki A. 2005. A human-specific gene in microglia. Science. 309:1693). Analyzing human gene microarrays to define new patterns of expression, we observed high levels of SIGLEC11 transcript in the ovary and adrenal cortex. Thus, we examined human and chimpanzee tissues using a well-characterized anti-Siglec-11 mouse monoclonal antibody. Although adrenal expression was variable and confined to infiltrating macrophages in capillaries, ovarian expression of Siglec-11 in both humans and chimpanzees was on fibroblasts, the first example of Siglec expression on mesenchyme-derived stromal cells. Cytokines from such ovarian stromal fibroblasts play important roles in follicle development and ovulation. Stable transfection of SIGLEC11 into a primary human ovarian stromal fibroblast cell line altered the secretion of growth-regulated oncogene α, interleukin (IL)-10, IL-7, transforming growth factor β1 and tumor necrosis factor-α, cytokines involved in ovarian physiology. Probing for Siglec-11 ligands revealed distinct and strong mast cell expression in human ovaries, contrasting to diffuse stromal ligands in chimpanzee ovaries. Interestingly, there was a trend of increased Siglec-11 expression in post-menopausal ovaries compared with pre-menopausal ones. Siglec-11 expression was also found on human ovarian stromal tumors and in polycystic ovarian syndrome, a human-specific disease. These results indicate potential

  1. Gastrointestinal Stromal Tumours: An Update

    PubMed Central

    Somerhausen, Nicolas De Saint Aubain

    1998-01-01

    Purpose. To study the evolution of concepts concerning gastrointestinal stromal tumours (GISTs) over 30 years. Discussion. GISTs have been, for more than 30 years, the subject of considerable controversy regarding their line of differentiation as well as the prediction of their behaviour. Furthermore, once they spread within the peritoneal cavity, they are extremely hard to control. The recent findings of c-Kit mutations and the immunohistochemical detection of the product of this gene, KIT or CD117, in the mainly non-myogenic subset of this family of tumours, has led to a reappraisal of this group of lesions, which, with some exceptions, is now thought to be derived from the interstitial cells of Cajal, and this has facilitated a clearer definition of their pathological spectrum. In this article, we review chronologically the evolution of the concept of GIST with the gradual application of electron microscopy, immunohistochemistry, DNA ploidy analysis. We discuss the impact of these techniques on the pathological assessment and clinical management of GISTs. PMID:18521245

  2. Ghrelin and gastrointestinal stromal tumors

    PubMed Central

    Zhu, Chang-Zhen; Liu, Dong; Kang, Wei-Ming; Yu, Jian-Chun; Ma, Zhi-Qiang; Ye, Xin; Li, Kang

    2017-01-01

    Ghrelin, as a kind of multifunctional protein polypeptide, is mainly produced in the fundus of the stomach and can promote occurrence and development of many tumors, including gastrointestinal tumors, which has been proved by the relevant researches. Most gastrointestinal stromal tumors (GISTs, about 80%), as the most common mesenchymal tumor, also develop in the fundus. Scientific research has confirmed that ghrelin, its receptors and mRNA respectively can be found in GISTs, which demonstrated the existence of a ghrelin autocrine/paracrine loop in GIST tissues. However, no reports to date have specified the mechanism whether ghrelin can promote the occurrence and development of GISTs. Studies of pulmonary artery endothelial cells in a low-oxygen environment and cardiac muscle cells in an ischemic environment have shown that ghrelin can activate the phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway. Moreover, some studies of GISTs have confirmed that activation of the PI3K/AKT/mTOR pathway can indeed promote the growth and progression of GISTs. Whether ghrelin is involved in the development or progression of GISTs through certain pathways remains unknown. Can we find a new target for the treatment of GISTs? This review explores and summaries the relationship among ghrelin, the PI3K/AKT/mTOR pathway and the development of GISTs. PMID:28348480

  3. Ghrelin and gastrointestinal stromal tumors.

    PubMed

    Zhu, Chang-Zhen; Liu, Dong; Kang, Wei-Ming; Yu, Jian-Chun; Ma, Zhi-Qiang; Ye, Xin; Li, Kang

    2017-03-14

    Ghrelin, as a kind of multifunctional protein polypeptide, is mainly produced in the fundus of the stomach and can promote occurrence and development of many tumors, including gastrointestinal tumors, which has been proved by the relevant researches. Most gastrointestinal stromal tumors (GISTs, about 80%), as the most common mesenchymal tumor, also develop in the fundus. Scientific research has confirmed that ghrelin, its receptors and mRNA respectively can be found in GISTs, which demonstrated the existence of a ghrelin autocrine/paracrine loop in GIST tissues. However, no reports to date have specified the mechanism whether ghrelin can promote the occurrence and development of GISTs. Studies of pulmonary artery endothelial cells in a low-oxygen environment and cardiac muscle cells in an ischemic environment have shown that ghrelin can activate the phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway. Moreover, some studies of GISTs have confirmed that activation of the PI3K/AKT/mTOR pathway can indeed promote the growth and progression of GISTs. Whether ghrelin is involved in the development or progression of GISTs through certain pathways remains unknown. Can we find a new target for the treatment of GISTs? This review explores and summaries the relationship among ghrelin, the PI3K/AKT/mTOR pathway and the development of GISTs.

  4. Comparative proteomic analysis of normal and tumor stromal cells by tissue on chip based mass spectrometry (toc-MS)

    PubMed Central

    2010-01-01

    In carcinoma tissues, genetic and metabolic changes not only occur at the tumor cell level, but also in the surrounding stroma. This carcinoma-reactive stromal tissue is heterogeneous and consists e.g. of non-epithelial cells such as fibroblasts or fibrocytes, inflammatory cells and vasculature-related cells, which promote carcinoma growth and progression of carcinomas. Nevertheless, there is just little knowledge about the proteomic changes from normal connective tissue to tumor stroma. In the present study, we acquired and analysed specific protein patterns of small stromal sections surrounding head and neck cell complexes in comparison to normal subepithelial connective tissue. To gain defined stromal areas we used laser-based tissue microdissection. Because these stromal areas are limited in size we established the highly sensitive 'tissue on chip based mass spectrometry' (toc-MS). Therefore, the dissected areas were directly transferred to chromatographic arrays and the proteomic profiles were subsequently analysed with mass spectrometry. At least 100 cells were needed for an adequate spectrum. The locating of differentially expressed proteins enables a precise separation of normal and tumor stroma. The newly described toc-MS technology allows an initial insight into proteomic differences between small numbers of exactly defined cells from normal and tumor stroma. PMID:20205871

  5. Progression-related loss of stromal Caveolin 1 levels fosters the growth of human PC3 xenografts and mediates radiation resistance

    PubMed Central

    Panic, Andrej; Ketteler, Julia; Reis, Henning; Sak, Ali; Herskind, Carsten; Maier, Patrick; Rübben, Herbert; Jendrossek, Verena; Klein, Diana

    2017-01-01

    Despite good treatment results in localized prostate tumors, advanced disease stages usually have a pronounced resistance to chemotherapy and radiotherapy. The membrane protein caveolin-1 (Cav1) functions here as an important oncogene. Therefore we examined the impact of stromal Cav1 expression for tumor growth and sensitivity to ionizing radiation (IR). Silencing of Cav1 expression in PC3 cells resulted in increased tumor growth and a reduced growth delay after IR when compared to tumors generated by Cav1-expressing PC3 cells. The increased radiation resistance was associated with increasing amounts of reactive tumor stroma and a Cav1 re-expression in the malignant epithelial cells. Mimicking the human situation these results were confirmed using co-implantation of Cav1-silenced PC3 cells with Cav1-silenced or Cav1-expressing fibroblasts. Immunohistochemically analysis of irradiated tumors as well as human prostate tissue specimen confirmed that alterations in stromal-epithelial Cav1 expressions were accompanied by a more reactive Cav1-reduced tumor stroma after radiation and within advanced prostate cancer tissues which potentially mediates the resistance to radiation treatment. Conclusively, the radiation response of human prostate tumors is critically regulated by Cav1 expression in stromal fibroblasts. Loss of stromal Cav1 expression in advanced tumor stages may thus contribute to resistance of these tumors to radiotherapy. PMID:28112237

  6. A complex 3D human tissue culture system based on mammary stromal cells and silk scaffolds for modeling breast morphogenesis and function.

    PubMed

    Wang, Xiuli; Sun, Lin; Maffini, Maricel V; Soto, Ana; Sonnenschein, Carlos; Kaplan, David L

    2010-05-01

    Epithelial-stromal interactions play a crucial role in normal embryonic development and carcinogenesis of the human breast while the underlying mechanisms of these events remain poorly understood. To address this issue, we constructed a physiologically relevant, three-dimensional (3D) culture surrogate of complex human breast tissue that included a tri-culture system made up of human mammary epithelial cells (MCF10A), human fibroblasts and adipocytes, i.e., the two dominant breast stromal cell types, in a Matrigel/collagen mixture on porous silk protein scaffolds. The presence of stromal cells inhibited MCF10A cell proliferation and induced both alveolar and ductal morphogenesis and enhanced casein expression. In contrast to the immature polarity exhibited by co-cultures with either fibroblasts or adipocytes, the alveolar structures formed by the tri-cultures exhibited proper polarity similar to that observed in breast tissue in vivo. Only alveolar structures with reverted polarity were observed in MCF10A monocultures. Consistent with their phenotypic appearance, more functional differentiation of epithelial cells was also observed in the tri-cultures, where casein alpha- and -beta mRNA expression was significantly increased. This in vitro tri-culture breast tissue system sustained on silk scaffold effectively represents a more physiologically relevant 3D microenvironment for mammary epithelial cells and stromal cells than either co-cultures or monocultures. This experimental model provides an important first step for bioengineering an informative human breast tissue system, with which to study normal breast morphogenesis and neoplastic transformation.

  7. Specific Age-Associated DNA Methylation Changes in Human Dermal Fibroblasts

    PubMed Central

    Lin, Qiong; Bork, Simone; Goergens, Maria; Joussen, Sylvia; Pallua, Norbert; Ho, Anthony D.; Zenke, Martin; Wagner, Wolfgang

    2011-01-01

    Epigenetic modifications of cytosine residues in the DNA play a critical role for cellular differentiation and potentially also for aging. In mesenchymal stromal cells (MSC) from human bone marrow we have previously demonstrated age-associated methylation changes at specific CpG-sites of developmental genes. In continuation of this work, we have now isolated human dermal fibroblasts from young (<23 years) and elderly donors (>60 years) for comparison of their DNA methylation profiles using the Infinium HumanMethylation27 assay. In contrast to MSC, fibroblasts could not be induced towards adipogenic, osteogenic and chondrogenic lineage and this is reflected by highly significant differences between the two cell types: 766 CpG sites were hyper-methylated and 752 CpG sites were hypo-methylated in fibroblasts in comparison to MSC. Strikingly, global DNA methylation profiles of fibroblasts from the same dermal region clustered closely together indicating that fibroblasts maintain positional memory even after in vitro culture. 75 CpG sites were more than 15% differentially methylated in fibroblasts upon aging. Very high hyper-methylation was observed in the aged group within the INK4A/ARF/INK4b locus and this was validated by pyrosequencing. Age-associated DNA methylation changes were related in fibroblasts and MSC but they were often regulated in opposite directions between the two cell types. In contrast, long-term culture associated changes were very consistent in fibroblasts and MSC. Epigenetic modifications at specific CpG sites support the notion that aging represents a coordinated developmental mechanism that seems to be regulated in a cell type specific manner. PMID:21347436

  8. 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...

  9. 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...

  10. 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...

  11. 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...

  12. What Happens After Treatment for Gastrointestinal Stromal Tumor?

    MedlinePlus

    ... Tumor Is No Longer Working Gastrointestinal Stromal Tumor (GIST) After Treatment What Happens After Treatment for Gastrointestinal ... For some people with a gastrointestinal stromal tumor (GIST), treatment may remove or destroy the cancer. Completing ...

  13. Analysis of the effects of stromal cells on the migration of lymphocytes into and through inflamed tissue using 3-D culture models.

    PubMed

    Jeffery, Hannah C; Buckley, Christopher D; Moss, Paul; Rainger, G Ed; Nash, Gerard B; McGettrick, Helen M

    2013-12-31

    Stromal cells may regulate the recruitment and behaviour of leukocytes during an inflammatory response, potentially through interaction with the endothelial cells (EC) and the leukocytes themselves. Here we describe new in vitro methodologies to characterise the effects of stromal cells on the migration of lymphocytes through endothelium and its underlying matrix. Three-dimensional tissue-like constructs were created in which EC were cultured above a stromal layer incorporating fibroblasts either as a monolayer on a porous filter or dispersed within a matrix of collagen type 1. A major advantage of these constructs is that they enable each step in leukocyte migration to be analysed in sequence (migration through EC and then stroma), as would occur in vivo. Migrated cells can also be retrieved from the constructs to identify which subsets traffic more effectively and how their functional responses evolve during migration. We found that culture of EC with dermal fibroblasts promoted lymphocyte transendothelial migration but not onward transit through matrix. A critical factor influencing the effect of fibroblasts on recruitment proved to be their proximity to the EC, with direct contact tending to disrupt migration. Comparison of the different approaches indicates that choice of an appropriate 3-D model enables the steps in lymphocyte entry into tissue to be studied in sequence, the regulatory mechanism to be dissected, and the effects of changes in stroma to be investigated.

  14. ADAMTS-1 metalloproteinase promotes tumor development through the induction of a stromal reaction in vivo.

    PubMed

    Rocks, Natacha; Paulissen, Geneviève; Quesada-Calvo, Florence; Munaut, Carine; Gonzalez, Maria-Luz Alvarez; Gueders, Maud; Hacha, Jonathan; Gilles, Christine; Foidart, Jean-Michel; Noel, Agnès; Cataldo, Didier D

    2008-11-15

    ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin motifs), the first described member of the ADAMTS family, is differentially expressed in various tumors. However, its exact role in tumor development and progression is still unclear. The aim of this study was to investigate the effects of ADAMTS-1 transfection in a bronchial epithelial tumor cell line (BZR) and its potential to modulate tumor development. ADAMTS-1 overexpression did not affect in vitro cell properties such as (a) proliferation in two-dimensional culture, (b) proliferation in three-dimensional culture, (c) anchorage-independent growth in soft agar, (d) cell migration and invasion in modified Boyden chamber assay, (e) angiogenesis in the aortic ring assay, and (f) cell apoptosis. In contrast, ADAMTS-1 stable transfection in BZR cells accelerated the in vivo tumor growth after s.c. injection into severe combined immunodeficient mice. It also promoted a stromal reaction characterized by myofibroblast infiltration and excessive matrix deposition. These features are, however, not observed in tumors derived from cells overexpressing a catalytically inactive mutant of ADAMTS-1. Conditioned media from ADAMTS-1-overexpressing cells display a potent chemotactic activity toward fibroblasts. ADAMTS-1 overexpression in tumors was associated with increased production of matrix metalloproteinase-13, fibronectin, transforming growth factor beta (TGF-beta), and interleukin-1beta (IL-1beta). Neutralizing antibodies against TGF-beta and IL-1beta blocked the chemotactic effect of medium conditioned by ADAMTS-1-expressing cells on fibroblasts, showing the contribution of these factors in ADAMTS-1-induced stromal reaction. In conclusion, we propose a new paradigm for catalytically active ADAMTS-1 contribution to tumor development, which consists of the recruitment of fibroblasts involved in tumor growth and tumor-associated stroma remodeling.

  15. Matrix metalloproteinase-8 regulates transforming growth factor-β1 levels in mouse tongue wounds and fibroblasts in vitro.

    PubMed

    Aström, Pirjo; Pirilä, Emma; Lithovius, Riitta; Heikkola, Heidi; Korpi, Jarkko T; Hernández, Marcela; Sorsa, Timo; Salo, Tuula

    2014-10-15

    Matrix metalloproteinase-8 (MMP-8)-deficient mice (Mmp8-/-) exhibit delayed dermal wound healing, but also partly contradicting results have been reported. Using the Mmp8-/- mice we investigated the role of MMP-8 in acute wound healing of the mobile tongue, and analyzed the function of tongue fibroblasts in vitro. Interestingly, in the early phase the tongue wounds of Mmp8-/- mice healed faster than those of wild type (wt) mice resulting in significant difference in wound widths (P=0.001, 6-24h). The Mmp8-/- wounds showed no change in myeloperoxidase positive myeloid cell count, but the level of transforming growth factor (TGF)-β1 was significantly increased (P=0.007) compared to the wt tongues. Fibroblasts cultured from wt tongues expressed MMP-8 and TGF-β1. However, higher TGF-β1 levels were detected in Mmp8-/- fibroblasts, and MMP-8 treatment decreased phosphorylated Smad-2 levels and α-smooth muscle actin expression in these fibroblasts suggesting reduced TGF-β1 signaling. Consistently, a degradation of recombinant TGF-β1 by MMP-8 decreased its ability to activate the signaling cascade in fibroblasts. Moreover, collagen gels with Mmp8-/- fibroblasts reduced more in size. We conclude that MMP-8 regulates tongue wound contraction rate and TGF-β1 levels. In vitro analyses suggest that MMP-8 may also play a role in regulating TGF-β1 signaling of stromal fibroblasts.

  16. Hepatic immune regulation by stromal cells.

    PubMed

    Schildberg, Frank A; Sharpe, Arlene H; Turley, Shannon J

    2015-02-01

    A metabolic organ, the liver also has a central role in tolerance induction. Stromal cells lining the hepatic sinusoids, such as liver sinusoidal endothelial cells (LSECs) and hepatic stellate cells (HSCs), are the first liver cells to encounter gut-derived and systemic antigens, thereby shaping local and systemic tolerance. Recent studies have demonstrated that stromal cells can modulate immune responses by antigen-dependent and independent mechanisms. Stromal cells interfere with the function of other antigen-presenting cells (APCs) and induce non-responsive T cells as well as regulatory T cells and myeloid-derived suppressor cells (MDSCs). The immunosuppressive microenvironment thus created provides a means to protect the liver from tissue damage. Such tolerized surroundings, however, can be exploited by certain pathogens, promoting persistent liver infections.

  17. SU-E-J-105: Stromal-Epithelial Responses to Fractionated Radiotherapy

    SciTech Connect

    Qayyum, M

    2014-06-01

    Purpose: The stromal-epithelial-cell interactions that are responsible for directing normal breast-tissue development and maintenance play a central role in the progression of breast cancer. In the present study, we developed three-dimensional (3-D) cell co-cultures used to study cancerous mammary cell responses to fractionated radiotherapy. In particular, we focused on the role of the reactive stroma in determining the therapeutic ratio for postsurgical treatment. Methods: Cancerous human mammary epithelial cells were cultured in a 3-D collagen matrix with human fibroblasts stimulated by various concentrations of transforming growth factor beta 1 (TGF-β1). These culture samples were designed to model the post-lumpectomy mammary stroma in the presence of residual cancer cells. We tracked over time the changes in medium stiffness, fibroblast-cell activation (conversion to cancer activated fibroblasts (CAF)), and proliferation of both cell types under a variety of fractionated radiotherapy protocols. Samples were exposed to 6 MV X-rays from a linear accelerator in daily fraction sizes of 90, 180 and 360 cGy over five days in a manner consistent with irradiation exposure during radiotherapy. Results: We found in fractionation studies with fibroblasts and CAF that higher doses per fraction may be more effective early on in deactivating cancer-harboring cellular environments. Higher-dose fraction schemes inhibit contractility in CAF and prevent differentiation of fibroblasts, thereby metabolically uncoupling tumor cells from their surrounding stroma. Yet, over a longer time period, the higher dose fractions may slow wound healing and increase ECM stiffening that could stimulate proliferation of surviving cancer cells. Conclusion: The findings suggest that dose escalation to the region with residual disease can deactivate the reactive stroma, thus minimizing the cancer promoting features of the cellular environment. Large-fraction irradiation may be used to sterilize

  18. 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.

  19. Melanoma-associated fibroblasts modulate NK cell phenotype and antitumor cytotoxicity

    PubMed Central

    Balsamo, Mirna; Scordamaglia, Francesca; Pietra, Gabriella; Manzini, Claudia; Cantoni, Claudia; Boitano, Monica; Queirolo, Paola; Vermi, William; Facchetti, Fabio; Moretta, Alessandro; Moretta, Lorenzo; Mingari, Maria Cristina; Vitale, Massimo

    2009-01-01

    Although the role of the tumor microenvironment in the process of cancer progression has been extensively investigated, the contribution of different stromal components to tumor growth and/or evasion from immune surveillance is still only partially defined. In this study we analyzed fibroblasts derived from metastatic melanomas and provide evidence for their strong immunosuppressive activity. In coculture experiments, melanoma-derived fibroblasts sharply interfered with NK cell functions including cytotoxicity and cytokine production. Thus, both the IL-2-induced up-regulation of the surface expression of NKp44, NKp30, and DNAM-1 triggering receptors and the acquisition of cytolytic granules were inhibited in NK cells. This resulted in an impairment of the NK cell-mediated killing of melanoma target cells. Transwell cocultures and the use of specific inhibitors suggested that cell-to-cell contact was required for inducing DNAM-1 modulation. In contrast, modulation of NKp44 and NKp30 was due to PGE2 released by fibroblasts during coculture. Normal skin fibroblasts could also partially affect NK cell phenotype and function. However, the inhibitory effect of tumor-derived fibroblasts was far stronger and directly correlated with their ability to produce PGE2 either constitutively or upon induction by NK cells. PMID:19934056

  20. Fibroblasts from phenotypically normal palmar fascia exhibit molecular profiles highly similar to fibroblasts from active disease in Dupuytren's Contracture

    PubMed Central

    2012-01-01

    Background Dupuytren's contracture (DC) is a fibroproliferative disorder characterized by the progressive development of a scar-like collagen-rich cord that affects the palmar fascia of the hand and leads to digital flexion contractures. DC is most commonly treated by surgical resection of the diseased tissue, but has a high reported recurrence rate ranging from 27% to 80%. We sought to determine if the transcriptomic profiles of fibroblasts derived from DC-affected palmar fascia, adjacent phenotypically normal palmar fascia, and non-DC palmar fascial tissues might provide mechanistic clues to understanding the puzzle of disease predisposition and recurrence in DC. Methods To achieve this, total RNA was obtained from fibroblasts derived from primary DC-affected palmar fascia, patient-matched unaffected palmar fascia, and palmar fascia from non-DC patients undergoing carpal tunnel release (6 patients in each group). These cells were grown on a type-1 collagen substrate (to better mimic their in vivo environments). Microarray analyses were subsequently performed using Illumina BeadChip arrays to compare the transcriptomic profiles of these three cell populations. Data were analyzed using Significance Analysis of Microarrays (SAM v3.02), hierarchical clustering, concordance mapping and Venn diagram. Results We found that the transcriptomic profiles of DC-disease fibroblasts and fibroblasts from unaffected fascia of DC patients exhibited a much greater overlap than fibroblasts derived from the palmar fascia of patients undergoing carpal tunnel release. Quantitative real time RT-PCR confirmed the differential expression of select genes validating the microarray data analyses. These data are consistent with the hypothesis that predisposition and recurrence in DC may stem, at least in part, from intrinsic similarities in the basal gene expression of diseased and phenotypically unaffected palmar fascia fibroblasts. These data also demonstrate that a collagen

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

    SciTech Connect

    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, although 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.

  2. 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

  3. Stromal-epithelial measurements of prostate cancer in native Japanese and Japanese-American men.

    PubMed

    Veltri, R W; Park, J; Miller, M C; Marks, L; Kojima, M; van Rootselaar, C; Khan, M A; Partin, A W

    2004-01-01

    We measured the histologic stromal and epithelial tissue components of the benign (normal) and malignant tissue compartments of Japanese-Americans (J-A) and native Japanese (NJ) men living in Japan. The patient cohort included 25 NJ men undergoing radical prostatectomy (RP) in Nagoya, Japan and 25 J-A (second or third generation US born). We conducted tissue image quantitation (in-house image software) of the stromal and epithelial compartments in malignant and adjacent normal tissue areas from a tissue microarray (TMA) selected from radical prostatectomy (RP) blocks. Stromal-epithelial (S-E) areas were determined using immunohistochemical stains for CAM-5.2 epithelial cytokeratin marker and the Masson trichrome stain to measure the stroma component. We observed differences in the volumes of normal and cancer epithelium and stroma within both the J-A and NJ study populations (P<0.01). Only the individual average cancer epithelium (CE) volume (JA=24.1 vs NJ=29.9) differed significantly between the NJ and J-A study populations (P=0.03). Consequently, the S-E ratio in NJ group was significantly different from that of J-A population (P=0.05). The decrease in S-E ratio noted in the malignant tissues of NJ prostate tissue may provide a biological marker for differentiation of the two groups and suggests a need for further investigations into the molecular basis for these histologic differences.

  4. Fibroblasts drive an immunosuppressive and growth-promoting microenvironment in breast cancer via secretion of Chitinase 3-like 1.

    PubMed

    Cohen, N; Shani, O; Raz, Y; Sharon, Y; Hoffman, D; Abramovitz, L; Erez, N

    2017-04-03

    Cancer-Associated Fibroblasts (CAFs) are the most prominent stromal cell type in breast tumors. CAFs promote tumor growth and metastasis by multiple mechanisms, including by mediating tumor-promoting inflammation. Immune modulation in the tumor microenvironment plays a central role in determining disease outcome. However, the functional interactions of CAFs with immune cells are largely unknown. Here we report a novel signaling axis between fibroblasts, cancer cells and immune cells in breast tumors that drives an immunosuppressive microenvironment, mediated by CAF-derived Chi3L1. We demonstrate that Chi3L1 is highly upregulated in CAFs isolated from mammary tumors and pulmonary metastases of transgenic mice, and in the stroma of human breast carcinomas. Genetic ablation of Chi3L1 in fibroblasts in vivo attenuated tumor growth, macrophage recruitment and reprogramming to an M2-like phenotype, enhanced tumor infiltration by CD8(+) and CD4(+) T cells and promoted a Th1 phenotype. These results indicate that CAF-derived Chi3L1 promotes tumor growth and shifts the balance of the immune milieu towards type 2 immunity. Taken together, our findings implicate fibroblast-derived Chi3L1 as a novel key player in the complex reciprocal interactions of stromal cells that facilitate tumor progression and metastasis, and suggest that targeting Chi3L1 may be clinically beneficial in breast cancer.Oncogene advance online publication, 3 April 2017; doi:10.1038/onc.2017.65.

  5. Origin of hemopoietic stromal progenitor cells in chimeras

    SciTech Connect

    Chertkov, J.L.; Drize, N.J.; Gurevitch, O.A.; Samoylova, R.S.

    1985-12-01

    Intravenously injected bone marrow cells do not participate in the regeneration of hemopoietic stromal progenitors in irradiated mice, nor in the curetted parts of the recipient's marrow. The hemopoietic stromal progenitors in allogeneic chimeras are of recipient origin. The adherent cell layer (ACL) of long-term cultures of allogeneic chimera bone marrow contains only recipient hemopoietic stromal progenitors. However, in ectopic hemopoietic foci produced by marrow implantation under the renal capsule and repopulated by the recipient hemopoietic cells after irradiation and reconstitution by syngeneic hemopoietic cells, the stromal progenitors were of implant donor origin, as were stromal progenitors of the ACL in long-term cultures of hemopoietic cells from ectopic foci. Our results confirm that the stromal and hemopoietic progenitors differ in origin and that hemopoietic stromal progenitors are not transplantable by the intravenous route in mice.

  6. Do Carcinoma-Associated Fibroblasts Adjacent to Breast Cancer Cells Originate from Circulating Mesenchymal Stem Cells?

    DTIC Science & Technology

    2007-08-01

    D., Gorringe , K., Haviv, I., Desmond, P.V., et al. (2003). Distinctive Patterns of Gene Expression in Premalignant Gastric Mucosa and Gastric Cancer...Hu, M., Shipitsin, M., Trivett, M., Thompson, E.R., Ramakrishna, M., Gorringe , K., et al. (2008). No evidence of genomic alterations in cancer

  7. Skull metastasis from rectal gastrointestinal stromal tumours.

    PubMed

    Gil-Arnaiz, Irene; Martínez-Trufero, Javier; Pazo-Cid, Roberto Antonio; Felipo, Francesc; Lecumberri, María José; Calderero, Verónica

    2009-09-01

    Gastrointestinal stromal tumours (GIST) are the most common mesenchymal neoplasm of the gastrointestinal tract. Rectum localisation is infrequent for these neoplasms, accounting for about 5% of all cases. Distant metastases of GIST are also rare. We present a patient with special features: the tumour is localised in rectum and it has an uncommon metastatic site, the skull, implying a complex differential diagnosis approach.

  8. 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

  9. 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.

  10. 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.

  11. Cathelicidin suppresses colon cancer development by inhibition of cancer associated fibroblasts

    PubMed Central

    Cheng, Michelle; Ho, Samantha; Yoo, Jun Hwan; Tran, Deanna Hoang-Yen; Bakirtzi, Kyriaki; Su, Bowei; Tran, Diana Hoang-Ngoc; Kubota, Yuzu; Ichikawa, Ryan; Koon, Hon Wai

    2015-01-01

    Background Cathelicidin (LL-37 in humans and mCRAMP in mice) represents a family of endogenous antimicrobial and anti-inflammatory peptides. Cancer-associated fibroblasts can promote the proliferation of colon cancer cells and growth of colon cancer tumors. Methods We examined the role of cathelicidin in the development of colon cancer, using subcutaneous human HT-29 colon-cancer-cell-derived tumor model in nude mice and azoxymethane- and dextran sulfate-mediated colon cancer model in C57BL/6 mice. We also determined the indirect antitumoral mechanism of cathelicidin via the inhibition of epithelial–mesenchymal transition (EMT) of colon cancer cells and fibroblast-supported colon cancer cell proliferation. Results Intravenous administration of cathelicidin expressing adeno-associated virus significantly reduced the size of tumors, tumor-derived collagen expression, and tumor-derived fibroblast expression in HT-29-derived subcutaneous tumors in nude mice. Enema administration of the mouse cathelicidin peptide significantly reduced the size and number of colonic tumors in azoxymethane- and dextran sulfate-treated mice without inducing apoptosis in tumors and the adjacent normal colonic tissues. Cathelicidin inhibited the collagen expression and vimentin-positive fibroblast expression in colonic tumors. Cathelicidin did not directly affect HT-29 cell viability, but did significantly reduce tumor growth factor-β1-induced EMT of colon cancer cells. Media conditioned by the human colonic CCD-18Co fibroblasts promoted human colon cancer HT-29 cell proliferation. Cathelicidin pretreatment inhibited colon cancer cell proliferation mediated by media conditioned by human colonic CCD-18Co fibroblasts. Cathelicidin disrupted tubulin distribution in colonic fibroblasts. Disruption of tubulin in fibroblasts reduced fibroblast-supported colon cancer cell proliferation. Conclusion Cathelicidin effectively inhibits colon cancer development by interfering with EMT and fibroblast

  12. Fibroblast heterogeneity: more than skin deep.

    PubMed

    Sorrell, J Michael; Caplan, Arnold I

    2004-02-15

    Dermal fibroblasts are a dynamic and diverse population of cells whose functions in skin in many respects remain unknown. Normal adult human skin contains at least three distinct subpopulations of fibroblasts, which occupy unique niches in the dermis. Fibroblasts from each of these niches exhibit distinctive differences when cultured separately. Specific differences in fibroblast physiology are evident in papillary dermal fibroblasts, which reside in the superficial dermis, and reticular fibroblasts, which reside in the deep dermis. Both of these subpopulations of fibroblasts differ from the fibroblasts that are associated with hair follicles. Fibroblasts engage in fibroblast-epidermal interactions during hair development and in interfollicular regions of skin. They also play an important role in cutaneous wound repair and an ever-increasing role in bioengineering of skin. Bioengineered skin currently performs important roles in providing (1) a basic understanding of skin biology, (2) a vehicle for testing topically applied products and (3) a resource for skin replacement.

  13. CTGF drives autophagy, glycolysis and senescence in cancer-associated fibroblasts via HIF1 activation, metabolically promoting tumor growth

    PubMed Central

    Capparelli, Claudia; Whitaker-Menezes, Diana; Guido, Carmela; Balliet, Renee; Pestell, Timothy G.; Howell, Anthony; Sneddon, Sharon; Pestell, Richard G.; Martinez-Outschoorn, Ubaldo; Lisanti, Michael P.; Sotgia, Federica

    2012-01-01

    Previous studies have demonstrated that loss of caveolin-1 (Cav-1) in stromal cells drives the activation of the TGF-β signaling, with increased transcription of TGF-β target genes, such as connective tissue growth factor (CTGF). In addition, loss of stromal Cav-1 results in the metabolic reprogramming of cancer-associated fibroblasts, with the induction of autophagy and glycolysis. However, it remains unknown if activation of the TGF-β / CTGF pathway regulates the metabolism of cancer-associated fibroblasts. Therefore, we investigated whether CTGF modulates metabolism in the tumor microenvironment. For this purpose, CTGF was overexpressed in normal human fibroblasts or MDA-MB-231 breast cancer cells. Overexpression of CTGF induces HIF-1α-dependent metabolic alterations, with the induction of autophagy/mitophagy, senescence, and glycolysis. Here, we show that CTGF exerts compartment-specific effects on tumorigenesis, depending on the cell-type. In a xenograft model, CTGF overexpressing fibroblasts promote the growth of co-injected MDA-MB-231 cells, without any increases in angiogenesis. Conversely, CTGF overexpression in MDA-MB-231 cells dramatically inhibits tumor growth in mice. Intriguingly, increased extracellular matrix deposition was seen in tumors with either fibroblast or MDA-MB-231 overexpression of CTGF. Thus, the effects of CTGF expression on tumor formation are independent of its extracellular matrix function, but rather depend on its ability to activate catabolic metabolism. As such, CTGF-mediated induction of autophagy in fibroblasts supports tumor growth via the generation of recycled nutrients, whereas CTGF-mediated autophagy in breast cancer cells suppresses tumor growth, via tumor cell self-digestion. Our studies shed new light on the compartment-specific role of CTGF in mammary tumorigenesis, and provide novel insights into the mechanism(s) generating a lethal tumor microenvironment in patients lacking stromal Cav-1. As loss of Cav-1 is a

  14. Prostacyclin analogs inhibit fibroblast migration.

    PubMed

    Kohyama, Tadashi; Liu, Xiangde; Kim, Hui Jung; Kobayashi, Tetsu; Ertl, Ronald F; Wen, Fu-Qiang; Takizawa, Hajime; Rennard, Stephen I

    2002-08-01

    The controlled accumulation of fibroblasts to sites of inflammation is crucial to effective tissue repair after injury. Either inadequate or excessive accumulation of fibroblasts could result in abnormal tissue function. Prostacyclin (PGI(2)) is a potent mediator in the coagulation and inflammatory processes. The aim of this study was to investigate the effect of PGI(2) on chemotaxis of human fetal lung fibroblasts (HFL-1). Using the blind well chamber technique, we found that the PGI(2) analog carbaprostacyclin (10(-6) M) inhibited HFL-1 chemotaxis to human plasma fibronectin (20 microg/ml) 58.0 +/- 13.2% (P < 0.05) and to platelet-derived growth factor (PDGF)-BB (10 ng/ml) 48.7 +/- 4.6% (P < 0.05). Checkerboard analysis demonstrated that carbaprostacyclin inhibits both directed and undirected migration. The inhibitory effect of the carbaprostacyclin was concentration dependent and blocked by the cAMP-dependent protein kinase (PKA) inhibitor KT-5720, suggesting that a cAMP-PKA pathway may be involved in the process. Two other PGI(2) analogs, ciprostene and dehydro-15-cyclohexyl carbaprostacyclin (both 10(-6) M), significantly inhibited fibroblast migration to fibronectin. In summary, PGI(2) appears to inhibit fibroblast chemotaxis to fibronectin and PDGF-BB. Such an effect may contribute to the regulation of fibroblasts in wound healing and could contribute to the pathogenesis of diseases characterized by abnormal tissue repair remodeling.

  15. Characterization of Mesenchymal Stem Cells from Human Vocal Fold Fibroblasts

    PubMed Central

    Hanson, Summer; Kim, Jaehyup; Quinchia Johnson, Beatriz H.; Bradley, Bridget; Breunig, Melissa; Hematti, Peiman; Thibeault, Susan L.

    2009-01-01

    Objective/Hypothesis Mesenchymal stem cells (MSCs) originally isolated from bone marrow, are fibroblast-looking cells that are now assumed to be present in the stromal component of many tissues. MSCs are characterized by a certain set of criteria including their growth culture characteristics, a combination of cell surface markers, and the ability to differentiate along multiple mesenchymal tissue lineages. We hypothesized that human vocal fold fibroblasts (hVFF) isolated from the lamina propria meet the criteria established to define MSCs and are functionally similar to MSCs derived from BM and adipose tissue. Study Design In vitro study Methods HVFF were previously derived from human vocal fold tissues. MSCs were derived from adipose tissue (AT), and BM of healthy donors, based on their attachment to culture dishes and their morphology, and expanded in culture. Cells were analyzed for standard cell surface markers identified on BM-derived MSCs as well as the ability to differentiate into cells of mesenchymal lineage, i.e. fat, bone and cartilage. We investigated the immunophenotype of these cells before and after interferon-γ (INF- γ) stimulation. Results HVFF displayed cell surface markers and multipotent differentiation capacity characteristic of MSCs. Furthermore, these cells exhibited similar patterns of expression of HLA and co-stimulatory molecules, after stimulation with INF- γ compared to MSCs derived from BM and AT. Conclusions Based on our findings hVFF derived from lamina propria have the same cell surface markers, immunophenotypic characteristics, and differentiation potential as BM- and AT-derived MSCs. We propose VF fibroblasts are MSCs resident in the vocal fold lamina propria. PMID:20131365

  16. A role for fibroblasts in mediating the effects of tobacco-induced epithelial cell growth and invasion.

    PubMed

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

    2008-07-01

    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.

  17. 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

    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.

  18. 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

  19. A murine stromal cell line promotes the proliferation of the human factor-dependent leukemic cell line UT-7.

    PubMed

    Auffray, I; Dubart, A; Izac, B; Vainchenker, W; Coulombel, L

    1994-05-01

    In long-term human bone marrow cultures, stromal cells of human origin are usually used on the assumption that human primitive progenitor cells do not respond to cytokines produced by stromal cells from other species. There is accumulating evidence, however, that murine stromal cells also promote maintenance and differentiation of very primitive human stem cells, which suggests the existence of novel stromal activities that cross species barriers. In this study, we show that a murine bone marrow-derived stromal cell line, MS-5, allows the proliferation of the human leukemic cell line UT-7. The long-term growth of UT-7 is usually supported only by human interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or erythropoietin (Epo). None of these three cytokines was involved in the observed effect, since murine GM-CSF and IL-3 do not act on human cells and MS-5 cells do not produce Epo. Soluble stem cell factor (SCF) induced UT-7 cell proliferation. However, S1/S1 mutant fibroblasts also supported UT-7 cell growth and anti-c-kit antibodies only partially abolished UT-7 cell proliferative response to MS-5 cells. These observations excluded a major role of SCF in this system. MS-5-derived growth-promoting activity was diffusible, but attempts to grow UT-7 cells in high levels of known soluble murine stromal-derived cytokines active on human cells showed no or minimal response, suggesting that MS-5's proliferative effect was not mediated by known cytokines. Finally, involvement of an autocrine loop of activation induced by MS-5 was excluded: RT-PCR analysis did not detect increased transcripts for GM-CSF, IL-3, IL-6, SCF, or Epo in UT-7 cells cocultured for 2 to 6 days with MS-5. In addition, UT-7 cell proliferation on MS-5 was not inhibited by neutralizing antibodies against the human GM-CSF receptor or the human IL-6 receptor alpha chain. Whether UT-7 cell proliferation triggered by MS-5 reflects the existence of novel stromal cytokines or

  20. PINOCYTOSIS IN FIBROBLASTS

    PubMed Central

    Steinman, Ralph M.; Silver, Jonathan M.; Cohn, Zanvil A.

    1974-01-01

    Horseradish peroxidase (HRP) was used as a marker to determine the rate of ongoing pinocytosis in several fibroblast cell lines. The enzyme was interiorized in the fluid phase without evidence of adsorption to the cell surface. Cytochemical reaction product was not found on the cell surface and was visualized only within intracellular vesicles and granules. Uptake was directly proportional to the administered concentration of HRP and to the duration of exposure. The rate of HRP uptake was 0.0032–0.0035% of the administered load per 106 cells per hour for all cells studied with one exception: L cells, after reaching confluence, progressively increased their pinocytic activity two- to fourfold. After uptake of HRP, L cells inactivated HRP with a half-life of 6–8 h. Certain metabolic requirements of pinocytosis were then studied in detail in L cells. Raising the environmental temperature increased pinocytosis over a range of 2–38°C. The Q10 was 2.7 and the activation energy, 17.6 kcal/mol. Studies on the levels of cellular ATP in the presence of various metabolic inhibitors (fluoride, 2-desoxyglycose, azide, and cyanide) showed that L cells synthesized ATP by both glycolytic and respiratory pathways. A combination of a glycolytic and a respiratory inhibitor was needed to depress cellular ATP levels as well as pinocytic activity to 10–20% of control values, whereas drugs administered individually had only partial effects. In spite of the availability of an accurate quantitative assay for fluid and solute uptake, the function of pinocytosis in tissue culture cells remains unknown. PMID:4140194

  1. 43 CFR 420.3 - Adjacent lands.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 43 Public Lands: Interior 1 2014-10-01 2014-10-01 false Adjacent lands. 420.3 Section 420.3 Public Lands: Interior Regulations Relating to Public Lands BUREAU OF RECLAMATION, DEPARTMENT OF THE INTERIOR OFF-ROAD VEHICLE USE § 420.3 Adjacent lands. When administratively feasible, the regulation of...

  2. 43 CFR 420.3 - Adjacent lands.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 43 Public Lands: Interior 1 2012-10-01 2011-10-01 true Adjacent lands. 420.3 Section 420.3 Public Lands: Interior Regulations Relating to Public Lands BUREAU OF RECLAMATION, DEPARTMENT OF THE INTERIOR OFF-ROAD VEHICLE USE § 420.3 Adjacent lands. When administratively feasible, the regulation of...

  3. 43 CFR 420.3 - Adjacent lands.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 43 Public Lands: Interior 1 2013-10-01 2013-10-01 false Adjacent lands. 420.3 Section 420.3 Public Lands: Interior Regulations Relating to Public Lands BUREAU OF RECLAMATION, DEPARTMENT OF THE INTERIOR OFF-ROAD VEHICLE USE § 420.3 Adjacent lands. When administratively feasible, the regulation of...

  4. 43 CFR 420.3 - Adjacent lands.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 43 Public Lands: Interior 1 2010-10-01 2010-10-01 false Adjacent lands. 420.3 Section 420.3 Public Lands: Interior Regulations Relating to Public Lands BUREAU OF RECLAMATION, DEPARTMENT OF THE INTERIOR OFF-ROAD VEHICLE USE § 420.3 Adjacent lands. When administratively feasible, the regulation of...

  5. 43 CFR 420.3 - Adjacent lands.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 43 Public Lands: Interior 1 2011-10-01 2011-10-01 false Adjacent lands. 420.3 Section 420.3 Public Lands: Interior Regulations Relating to Public Lands BUREAU OF RECLAMATION, DEPARTMENT OF THE INTERIOR OFF-ROAD VEHICLE USE § 420.3 Adjacent lands. When administratively feasible, the regulation of...

  6. Unilateral gynecomastia and pseudoangiomatous stromal hyperplasia in neurofibromatosis: case report and review of the literature.

    PubMed

    Kimura, Satoshi; Tanimoto, Akihide; Shimajiri, Shohei; Sasaguri, Takakazu; Yamada, Sohsuke; Wang, Ke-Yong; Guo, Xin; Sasaguri, Yasuyuki

    2012-05-15

    In this article, we describe unilateral gynecomastia and pseudoangiomatous stromal hyperplasia (PASH) in a case of type-1 neurofibromatosis (NF-1). It is important to distinguish PASH from fibroadenoma clinically, and from true blood capillaries and angiosarcoma histologically. In the present case, giant multinucleated cells lined the pseudovascular spaces, which was markedly different from that of conventional breast PASH. The origin of PASH has been reported to be either the fibroblast or the myofibroblast phenotype and may be affected by endocrine signaling because many cases have been reported in premenopausal women, and cases are often estrogen (ER) and progesterone receptor (PR) positive. However, previous reports have identified PASH in NF-1 in juvenile males only, and the cases were negative for α-SMA, ER and PR. The cause and prognosis of PASH in NF-1 may be distinguished from that of conventional PASH, and mast cells, histiocytes and CD54 may play roles.

  7. 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.

  8. 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.

  9. BRAF exon 15 mutations in pediatric renal stromal tumors: prevalence in metanephric stromal tumors.

    PubMed

    Marsden, Lily; Jennings, Lawrence J; Gadd, Samantha; Yu, Min; Perlman, Elizabeth J; Cajaiba, Mariana M

    2017-02-01

    Metanephric stromal tumors (MSTs) are rare renal stromal tumors that predominantly affect children. They belong to the metanephric family of tumors, along with metanephric adenofibroma and metanephric adenoma. The previous documentation of BRAF exon 15 mutations in 88% of metanephric adenomas and in isolated cases of metanephric adenofibroma prompted us to investigate the prevalence of these mutations in MSTs and in other pediatric renal stromal tumors. In this study, 17 MSTs, 22 congenital mesoblastic nephromas, and 6 ossifying renal tumors of infancy were selected for BRAF exon 15 testing. Tumor genomic DNA was extracted from formalin-fixed paraffin-embedded tissue, followed by polymerase chain reaction amplification and Sanger dideoxy sequencing with primers flanking the BRAF exon 15 gene. BRAF exon 15 mutations were found in 11 (65%) of the 17 cases of MST, all corresponding to a thymidine-to-adenine substitution at codon 600 (BRAF V600E). All other renal stromal tumors tested were negative for BRAF exon 15 mutations. In conclusion, BRAF V600E mutations are encountered in most MSTs, supporting a link with other metanephric tumors and suggesting a clonal event possibly affecting primordial renal cells. In addition, BRAF V600E mutations have been associated with oncogene-induced senescence in other benign tumors, providing clues to the pathogenesis of metanephric neoplasms in keeping with their overall benign behavior. Our results also suggest a potential diagnostic use for BRAF exon 15 mutations in differentiating MSTs from other pediatric renal stromal tumors, particularly in limited samples.

  10. Leptin as a mediator of tumor-stromal interactions promotes breast cancer stem cell activity.

    PubMed

    Giordano, Cinzia; Chemi, Francesca; Panza, Salvatore; Barone, Ines; Bonofiglio, Daniela; Lanzino, Marilena; Cordella, Angela; Campana, Antonella; Hashim, Adnan; Rizza, Pietro; Leggio, Antonella; Győrffy, Balázs; Simões, Bruno M; Clarke, Robert B; Weisz, Alessandro; Catalano, Stefania; Andò, Sebastiano

    2016-01-12

    Breast cancer stem cells (BCSCs) play crucial roles in tumor initiation, metastasis and therapeutic resistance. A strict dependency between BCSCs and stromal cell components of tumor microenvironment exists. Thus, novel therapeutic strategies aimed to target the crosstalk between activated microenvironment and BCSCs have the potential to improve clinical outcome. Here, we investigated how leptin, as a mediator of tumor-stromal interactions, may affect BCSC activity using patient-derived samples (n = 16) and breast cancer cell lines, and determined the potential benefit of targeting leptin signaling in these model systems. Conditioned media (CM) from cancer-associated fibroblasts and breast adipocytes significantly increased mammosphere formation in breast cancer cells and depletion of leptin from CM completely abrogated this effect. Mammosphere cultures exhibited increased leptin receptor (OBR) expression and leptin exposure enhanced mammosphere formation. Microarray analyses revealed a similar expression profile of genes involved in stem cell biology among mammospheres treated with CM and leptin. Interestingly, leptin increased mammosphere formation in metastatic breast cancers and expression of OBR as well as HSP90, a target of leptin signaling, were directly correlated with mammosphere formation in metastatic samples (r = 0.68/p = 0.05; r = 0.71/p = 0.036, respectively). Kaplan-Meier survival curves indicated that OBR and HSP90 expression were associated with reduced overall survival in breast cancer patients (HR = 1.9/p = 0.022; HR = 2.2/p = 0.00017, respectively). Furthermore, blocking leptin signaling by using a full leptin receptor antagonist significantly reduced mammosphere formation in breast cancer cell lines and patient-derived samples. Our results suggest that leptin/leptin receptor signaling may represent a potential therapeutic target that can block the stromal-tumor interactions driving BCSC-mediated disease progression.

  11. Functional differences in mesenchymal stromal cells from human dental pulp and periodontal ligament.

    PubMed

    Vasandan, Anoop Babu; Shankar, Shilpa Rani; Prasad, Priya; Sowmya Jahnavi, Vulugundam; Bhonde, Ramesh Ramachandra; Jyothi Prasanna, Susarla

    2014-02-01

    Clinically reported reparative benefits of mesenchymal stromal cells (MSCs) are majorly attributed to strong immune-modulatory abilities not exactly shared by fibroblasts. However, MSCs remain heterogeneous populations, with unique tissue-specific subsets, and lack of clear-cut assays defining therapeutic stromal subsets adds further ambiguity to the field. In this context, in-depth evaluation of cellular characteristics of MSCs from proximal oro-facial tissues: dental pulp (DPSCs) and periodontal ligament (PDLSCs) from identical donors provides an opportunity to evaluate exclusive niche-specific influences on multipotency and immune-modulation. Exhaustive cell surface profiling of DPSCs and PDLSCs indicated key differences in expression of mesenchymal (CD105) and pluripotent/multipotent stem cell-associated cell surface antigens: SSEA4, CD117, CD123 and CD29. DPSCs and PDLSCs exhibited strong chondrogenic potential, but only DPSCs exhibited adipogenic and osteogenic propensities. PDLSCs expressed immuno-stimulatory/immune-adhesive ligands like HLA-DR and CD50, upon priming with IFNγ, unlike DPSCs, indicating differential response patterns to pro-inflammatory cytokines. Both DPSCs and PDLSCs were hypo-immunogenic and did not elicit robust allogeneic responses despite exposure to IFNγ or TNFα. Interestingly, only DPSCs attenuated mitogen-induced lympho-proliferative responses and priming with either IFNγ or TNFα enhanced immuno-modulation capacity. In contrast, primed or unprimed PDLSCs lacked the ability to suppress polyclonal T cell blast responses. This study indicates that stromal cells from even topographically related tissues do not necessarily share identical MSC properties and emphasizes the need for a thorough functional testing of MSCs from diverse sources with respect to multipotency, immune parameters and response to pro-inflammatory cytokines before translational usage.

  12. Fibronectin functional domains coupled to hyaluronan stimulate adult human dermal fibroblast responses critical for wound healing.

    PubMed

    Ghosh, Kaustabh; Ren, Xiang-Dong; Shu, Xiao Zheng; Prestwich, Glenn D; Clark, Richard A F

    2006-03-01

    Fibronectin (FN) facilitates dermal fibroblast migration during normal wound healing. Proteolytic degradation of FN in chronic wounds hampers healing. Previously, three FN functional domains (FNfd) have been shown to be sufficient for optimal adult human dermal fibroblast migration. Here we report the development of an acellular hydrogel matrix comprised of the FNfds coupled to a hyaluronan (HA) backbone to stimulate wound repair. Employing Michael-type addition, the cysteine- tagged FNfds were first coupled to a homobifunctional PEG derivative. Thereafter, these PEG derivative FNfd solutions, containing bifunctional PEG-derivative crosslinker were coupled to thiol-modified HA (HA-DTPH) to obtain a crosslinked hydrogel matrix. When evaluated in vitro, these acellular hydrogels were completely cytocompatible. While spreading and proliferation of adult human dermal fibroblasts plateaued at higher FNfd bulk densities, their rapid and robust migration followed a typical bell-shaped response. When implanted in porcine cutaneous wounds, these acellular matrices, besides being completely biocompatible, induced rapid and en masse recruitment of stromal fibroblasts that was not observed with RGD-tethered or unmodified hydrogels. Such constructs might be of great benefit in clinical settings where rapid formation of new tissue is needed.

  13. Comparative proteomics of paired vocal fold and oral mucosa fibroblasts

    PubMed Central

    Karbiener, Michael; Darnhofer, Barbara; Frisch, Marie-Therese; Rinner, Beate; Birner-Gruenberger, Ruth; Gugatschka, Markus

    2017-01-01

    Injuries of the vocal folds frequently heal with scar formation, which can have lifelong detrimental impact on voice quality. Current treatments to prevent or resolve scars of the vocal fold mucosa are highly unsatisfactory. In contrast, the adjacent oral mucosa is mostly resistant to scarring. These differences in healing tendency might relate to distinct properties of the fibroblasts populating oral and vocal fold mucosae. We thus established the in vitro cultivation of paired, near-primary vocal fold fibroblasts (VFF) and oral mucosa fibroblasts (OMF) to perform a basic cellular characterization and comparative cellular proteomics. VFF were significantly larger than OMF, proliferated more slowly, and exhibited a sustained TGF-β1-induced elevation of pro-fibrotic interleukin 6. Cluster analysis of the proteomic data revealed distinct protein repertoires specific for VFF and OMF. Further, VFF displayed a broader protein spectrum, particularly a more sophisticated array of factors constituting and modifying the extracellular matrix. Conversely, subsets of OMF-enriched proteins were linked to cellular proliferation, nuclear events, and protection against oxidative stress. Altogether, this study supports the notion that fibroblasts sensitively adapt to the functional peculiarities of their respective anatomical location and presents several molecular targets for further investigation in the context of vocal fold wound healing. Biological significance Mammalian vocal folds are a unique but delicate tissue. A considerable fraction of people is affected by voice problems, yet many of the underlying vocal fold pathologies are sparsely understood at the molecular level. One such pathology is vocal fold scarring - the tendency of vocal fold injuries to heal with scar formation -, which represents a clinical problem with highly suboptimal treatment modalities. This study employed proteomics to obtain comprehensive insight into the protein repertoire of vocal fold

  14. Mesenchymal stromal cells for treatment of arthritis.

    PubMed

    Swart, J F; Wulffraat, N M

    2014-08-01

    Patients with refractory inflammatory arthritis can still respond favourable to autologous haematopoietic stem cell transplantation. However, this treatment has a high morbidity and even 5% mortality. Mesenchymal stromal cells (MSC), a subset of the non-haematopoietic stromal cells obtained from bone marrow, were found to have a strong immunosuppressive effect. MSC treatment is explored in many diseases like diabetes, SLE, MS and RA. This review covers all relevant literature regarding MSC treatment of inflammatory arthritis (RA and JIA). This review contains data of in vitro studies, animal studies and clinical studies. The following subjects will be discussed in detail: properties of MSC, presence of MSC in the joint, intra-articular versus intravenous route, autologous versus allogeneic, ideal source of MSC, distribution, transdifferentiation, engraftment, rejection, efficacy and toxicology. After reading this review the reader will be totally updated in this quickly evolving field of MSC therapy.

  15. Stromal SLIT2 impacts on pancreatic cancer-associated neural remodeling

    PubMed Central

    Secq, V; Leca, J; Bressy, C; Guillaumond, F; Skrobuk, P; Nigri, J; Lac, S; Lavaut, M-N; Bui, T-t; Thakur, A K; Callizot, N; Steinschneider, R; Berthezene, P; Dusetti, N; Ouaissi, M; Moutardier, V; Calvo, E; Bousquet, C; Garcia, S; Bidaut, G; Vasseur, S; Iovanna, J L; Tomasini, R

    2015-01-01

    Pancreatic ductal adenocarcinoma (PDA) is a critical health issue in the field of cancer, with few therapeutic options. Evidence supports an implication of the intratumoral microenvironment (stroma) on PDA progression. However, its contribution to the role of neuroplastic changes within the pathophysiology and clinical course of PDA, through tumor recurrence and neuropathic pain, remains unknown, neglecting a putative, therapeutic window. Here, we report that the intratumoral microenvironment is a mediator of PDA-associated neural remodeling (PANR), and we highlight factors such as ‘SLIT2' (an axon guidance molecule), which is expressed by cancer-associated fibroblasts (CAFs), that impact on neuroplastic changes in human PDA. We showed that ‘CAF-secreted SLIT2' increases neurite outgrowth from dorsal root ganglia neurons as well as from Schwann cell migration/proliferation by modulating N-cadherin/β-catenin signaling. Importantly, SLIT2/ROBO signaling inhibition disrupts this stromal/neural connection. Finally, we revealed that SLIT2 expression and CAFs are correlated with neural remodeling within human and mouse PDA. All together, our data demonstrate the implication of CAFs, through the secretion of axon guidance molecule, in PANR. Furthermore, it provides rationale to investigate the disruption of the stromal/neural compartment connection with SLIT2/ROBO inhibitors for the treatment of pancreatic cancer recurrence and pain. PMID:25590802

  16. Mouse granulated metrial gland cells require contact with stromal cells to maintain viability

    PubMed Central

    STEWART, I. J.

    2000-01-01

    Granulated metrial gland (GMG) cells differentiate in the uterine wall in pregnancy in mice but the mechanisms which control their differentiation and maintenance are unknown. In vivo, GMG cells share an intimate association with fibroblast-like stromal cells. The importance of this association has been assessed by examining the effects of withdrawal of stromal cell contact on GMG cell maintenance in vitro. When single cell suspensions of cells were prepared from mouse metrial glands there was a steady decline in numbers with days of culture but usually some remained at 7 d of culture. The ability of metrial gland cells to kill Wehi 164 target cells in 51Cr-release cytotoxicity assays was retained by cells cultured for at least 3 d. When explants of metrial gland were maintained in culture to allow GMG cell migration onto the culture flask, the attached GMG cells were lost by 1 d later. Overall, these results suggest that a juxtacrine regulatory mechanism maintains GMG cells. The rapid loss of unsupported GMG cells in culture has major implications in the design of assays to examine GMG cell function. PMID:11117633

  17. Mesenchymal Stromal Cells Can Regulate the Immune Response in the Tumor Microenvironment

    PubMed Central

    Poggi, Alessandro; Giuliani, Massimo

    2016-01-01

    The tumor microenvironment is a good target for therapy in solid tumors and hematological malignancies. Indeed, solid tumor cells’ growth and expansion can influence neighboring cells’ behavior, leading to a modulation of mesenchymal stromal cell (MSC) activities and remodeling of extracellular matrix components. This leads to an altered microenvironment, where reparative mechanisms, in the presence of sub-acute inflammation, are not able to reconstitute healthy tissue. Carcinoma cells can undergo epithelial mesenchymal transition (EMT), a key step to generate metastasis; these mesenchymal-like cells display the functional behavior of MSC. Furthermore, MSC can support the survival and growth of leukemic cells within bone marrow participating in the leukemic cell niche. Notably, MSC can inhibit the anti-tumor immune response through either carcinoma-associated fibroblasts or bone marrow stromal cells. Experimental data have indicated their relevance in regulating cytolytic effector lymphocytes of the innate and adaptive arms of the immune system. Herein, we will discuss some of the evidence in hematological malignancies and solid tumors. In particular, we will focus our attention on the means by which it is conceivable to inhibit MSC-mediated immune suppression and trigger anti-tumor innate immunity. PMID:27834810

  18. Mesenchymal Stromal Cells Can Regulate the Immune Response in the Tumor Microenvironment.

    PubMed

    Poggi, Alessandro; Giuliani, Massimo

    2016-11-08

    The tumor microenvironment is a good target for therapy in solid tumors and hematological malignancies. Indeed, solid tumor cells' growth and expansion can influence neighboring cells' behavior, leading to a modulation of mesenchymal stromal cell (MSC) activities and remodeling of extracellular matrix components. This leads to an altered microenvironment, where reparative mechanisms, in the presence of sub-acute inflammation, are not able to reconstitute healthy tissue. Carcinoma cells can undergo epithelial mesenchymal transition (EMT), a key step to generate metastasis; these mesenchymal-like cells display the functional behavior of MSC. Furthermore, MSC can support the survival and growth of leukemic cells within bone marrow participating in the leukemic cell niche. Notably, MSC can inhibit the anti-tumor immune response through either carcinoma-associated fibroblasts or bone marrow stromal cells. Experimental data have indicated their relevance in regulating cytolytic effector lymphocytes of the innate and adaptive arms of the immune system. Herein, we will discuss some of the evidence in hematological malignancies and solid tumors. In particular, we will focus our attention on the means by which it is conceivable to inhibit MSC-mediated immune suppression and trigger anti-tumor innate immunity.

  19. Extracellular Superoxide Dismutase Expression in Papillary Thyroid Cancer Mesenchymal Stem/Stromal Cells Modulates Cancer Cell Growth and Migration

    PubMed Central

    Parascandolo, Alessia; Rappa, Francesca; Cappello, Francesco; Kim, Jaehyup; Cantu, David A.; Chen, Herbert; Mazzoccoli, Gianluigi; Hematti, Peiman; Castellone, Maria Domenica; Salvatore, Marco; Laukkanen, Mikko O.

    2017-01-01

    Tumor stroma-secreted growth factors, cytokines, and reactive oxygen species (ROS) influence tumor development from early stages to the metastasis phase. Previous studies have demonstrated downregulation of ROS-producing extracellular superoxide dismutase (SOD3) in thyroid cancer cell lines although according to recent data, the expression of SOD3 at physiological levels stimulates normal and cancer cell proliferation. Therefore, to analyze the expression of SOD3 in tumor stroma, we characterized stromal cells from the thyroid. We report mutually exclusive desmoplasia and inflammation in papillary and follicular thyroid cancers and the presence of multipotent mesenchymal stem/stromal cells (MSCs) in non-carcinogenic thyroids and papillary thyroid cancer (PTC). The phenotypic and differentiation characteristics of Thyroid MSCs and PTC MSCs were comparable with bone marrow MSCs. A molecular level analysis showed increased FIBROBLAST ACTIVATING PROTEIN, COLLAGEN 1 TYPE A1, TENASCIN, and SOD3 expression in PTC MSCs compared to Thyroid MSCs, suggesting the presence of MSCs with a fibrotic fingerprint in papillary thyroid cancer tumors and the autocrine-paracrine conversion of SOD3 expression, which was enhanced by cancer cells. Stromal SOD3 had a stimulatory effect on cancer cell growth and an inhibitory effect on cancer cell migration, thus indicating that SOD3 might be a novel player in thyroid tumor stroma. PMID:28216675

  20. Podoplanin-rich stromal networks induce dendritic cell motility via activation of the C-type lectin receptor CLEC-2.

    PubMed

    Acton, Sophie E; Astarita, Jillian L; Malhotra, Deepali; Lukacs-Kornek, Veronika; Franz, Bettina; Hess, Paul R; Jakus, Zoltan; Kuligowski, Michael; Fletcher, Anne L; Elpek, Kutlu G; Bellemare-Pelletier, Angelique; Sceats, Lindsay; Reynoso, Erika D; Gonzalez, Santiago F; Graham, Daniel B; Chang, Jonathan; Peters, Anneli; Woodruff, Matthew; Kim, Young-A; Swat, Wojciech; Morita, Takashi; Kuchroo, Vijay; Carroll, Michael C; Kahn, Mark L; Wucherpfennig, Kai W; Turley, Shannon J

    2012-08-24

    To initiate adaptive immunity, dendritic cells (DCs) move from parenchymal tissues to lymphoid organs by migrating along stromal scaffolds that display the glycoprotein podoplanin (PDPN). PDPN is expressed by lymphatic endothelial and fibroblastic reticular cells and promotes blood-lymph separation during development by activating the C-type lectin receptor, CLEC-2, on platelets. Here, we describe a role for CLEC-2 in the morphodynamic behavior and motility of DCs. CLEC-2 deficiency in DCs impaired their entry into lymphatics and trafficking to and within lymph nodes, thereby reducing T cell priming. CLEC-2 engagement of PDPN was necessary for DCs to spread and migrate along stromal surfaces and sufficient to induce membrane protrusions. CLEC-2 activation triggered cell spreading via downregulation of RhoA activity and myosin light-chain phosphorylation and triggered F-actin-rich protrusions via Vav signaling and Rac1 activation. Thus, activation of CLEC-2 by PDPN rearranges the actin cytoskeleton in DCs to promote efficient motility along stromal surfaces.

  1. Cardiac Fibrosis: The Fibroblast Awakens

    PubMed Central

    Travers, Joshua G.; Kamal, Fadia A.; Robbins, Jeffrey; Yutzey, Katherine E.; Blaxall, Burns C.

    2016-01-01

    Myocardial fibrosis is a significant global health problem associated with nearly all forms of heart disease. Cardiac fibroblasts comprise an essential cell type in the heart that is responsible for the homeostasis of the extracellular matrix; however upon injury, these cells transform to a myofibroblast phenotype and contribute to cardiac fibrosis. This remodeling involves pathological changes that include chamber dilation, cardiomyocyte hypertrophy and apoptosis, and ultimately leads to the progression to heart failure. Despite the critical importance of fibrosis in cardiovascular disease, our limited understanding of this cell population impedes the development of potential therapies that effectively target this cell type and its pathological contribution to disease progression. This review summarizes current knowledge regarding the origins and roles of fibroblasts, mediators and signaling pathways known to influence fibroblast function after myocardial injury, as well as novel therapeutic strategies under investigation to attenuate cardiac fibrosis. PMID:26987915

  2. 3D Functional Corneal Stromal Tissue Equivalent Based on Corneal Stromal Stem Cells and Multi-Layered Silk Film Architecture

    PubMed Central

    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. PMID:28099503

  3. Sclerosing stromal tumor of the ovary in a premenarchal female.

    PubMed

    Fefferman, Nancy R; Pinkney, Lynne P; Rivera, Rafael; Popiolek, Dorota; Hummel-Levine, Pascale; Cosme, Jaqueline

    2003-01-01

    Sclerosing stromal tumor (SST) is a rare benign ovarian neoplasm of stromal origin with less than 100 cases reported in the literature. Unlike the other stromal tumors, thecomas and fibromas, which tend to occur in the fifth and sixth decades, sclerosing stromal tumors predominantly affect females in the second and third decades. Computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound findings have been described, but have not been reported previously in the pediatric literature. We present a case of SST of the ovary in a 10-year-old premenarchal female, the youngest patient to our knowledge reported in the literature, and describe the ultrasound and CT findings with pathologic correlation.

  4. Stromal Effects on Mammary Gland Development and Breast Cancer

    NASA Astrophysics Data System (ADS)

    Wiseman, Bryony S.; Werb, Zena

    2002-05-01

    Breast cancer manifests itself in the mammary epithelium, yet there is a growing recognition that mammary stromal cells also play an important role in tumorigenesis. During its developmental cycle, the mammary gland displays many of the properties associated with breast cancer, and many of the stromal factors necessary for mammary development also promote or protect against breast cancer. Here we review our present knowledge of the specific factors and cell types that contribute to epithelial-stromal crosstalk during mammary development. To find cures for diseases like breast cancer that rely on epithelial-stromal crosstalk, we must understand how these different cell types communicate with each other.

  5. Cutting edge: JAM-C controls homeostatic chemokine secretion in lymph node fibroblastic reticular cells expressing thrombomodulin.

    PubMed

    Frontera, Vincent; Arcangeli, Marie-Laure; Zimmerli, Claudia; Bardin, Florence; Obrados, Elodie; Audebert, Stéphane; Bajenoff, Marc; Borg, Jean-Paul; Aurrand-Lions, Michel

    2011-07-15

    The development and maintenance of secondary lymphoid organs, such as lymph nodes, occur in a highly coordinated manner involving lymphoid chemokine production by stromal cells. Although developmental pathways inducing lymphoid chemokine production during organogenesis are known, signals maintaining cytokine production in adults are still elusive. In this study, we show that thrombomodulin and platelet-derived growth factor receptor α identify a population of fibroblastic reticular cells in which chemokine secretion is controlled by JAM-C. We demonstrate that Jam-C-deficient mice and mice treated with Ab against JAM-C present significant decreases in stromal cell-derived factor 1α (CXCL12), CCL21, and CCL19 intranodal content. This effect is correlated with reduced naive T cell egress from lymph nodes of anti-JAM-C-treated mice.

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

    PubMed Central

    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.

    2016-01-01

    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. PMID:26980748

  7. 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.

  8. Tumor Associated Stromal Cells Play a Critical Role on the Outcome of the Oncolytic Efficacy of Conditionally Replicative Adenoviruses

    PubMed Central

    Lopez, M. Verónica; Viale, Diego L.; Cafferata, Eduardo G. A.; Bravo, Alicia I.; Carbone, Cecilia; Gould, David; Chernajovsky, Yuti; Podhajcer, Osvaldo L.

    2009-01-01

    The clinical efficacy of conditionally replicative oncolytic adenoviruses (CRAd) is still limited by the inefficient infection of the tumor mass. Since tumor growth is essentially the result of a continuous cross-talk between malignant and tumor-associated stromal cells, targeting both cell compartments may profoundly influence viral efficacy. Therefore, we developed SPARC promoter-based CRAds since the SPARC gene is expressed both in malignant cells and in tumor-associated stromal cells. These CRAds, expressing or not the Herpes Simplex thymidine kinase gene (Ad-F512 and Ad(I)-F512-TK, respectively) exerted a lytic effect on a panel of human melanoma cells expressing SPARC; but they were completely attenuated in normal cells of different origins, including fresh melanocytes, regardless of whether cells expressed or not SPARC. Interestingly, both CRAds displayed cytotoxic activity on SPARC positive-transformed human microendothelial HMEC-1 cells and WI-38 fetal fibroblasts. Both CRAds were therapeutically effective on SPARC positive-human melanoma tumors growing in nude mice but exhibited restricted efficacy in the presence of co-administered HMEC-1 or WI-38 cells. Conversely, co-administration of HMEC-1 cells enhanced the oncolytic efficacy of Ad(I)-F512-TK on SPARC-negative MIA PaCa-2 pancreatic cancer cells in vivo. Moreover, conditioned media produced by stromal cells pre-infected with the CRAds enhanced the in vitro viral oncolytic activity on pancreatic cancer cells, but not on melanoma cells. The whole data indicate that stromal cells might play an important role on the outcome of the oncolytic efficacy of conditionally replicative adenoviruses. PMID:19337591

  9. Induction of T Cell Development In Vitro by Delta-Like (Dll)-Expressing Stromal Cells.

    PubMed

    Mohtashami, Mahmood; Zarin, Payam; Zúñiga-Pflücker, Juan Carlos

    2016-01-01

    Recreating the thymic microenvironment in vitro poses a great challenge to immunologists. Until recently, the only approach was to utilize the thymic tissue in its three-dimensional form and to transfer the hematopoietic progenitors into this tissue to generate de novo T cells. With the advent of OP9-DL cells (bone marrow-derived cells that are transduced to express Notch ligand, Delta-like), hematopoietic stem cells (HSC) could be induced to differentiate into T cells in culture for the first time outside of the thymic tissue on a monolayer. We, as well as others, asked whether the ability to support T cell development in vitro in a monolayer is unique to BM-derived OP9 cells, and showed that provision of Delta-like expression to thymic epithelial cells and fibroblasts also allowed for T cell development. This provides the opportunity to design an autologous coculture system where the supportive stromal and the hematopoietic components are both derived from the same individual, which has obvious clinical implications. In this chapter, we describe methods for establishing a primary murine dermal fibroblast cell population that is transduced to express Delta-like 4, and describe the conditions for its coculture with HSCs to support T cell lineage initiation and expansion, while comparing it to the now classic OP9-DL coculture.

  10. Role of Nanog in the maintenance of marrow stromal stem cells during post natal bone regeneration

    SciTech Connect

    Bais, Manish V.; Shabin, Zabrina M.; Young, Megan; Einhorn, Thomas A.; Kotton, Darrell N.; Gerstnefeld, Louis C.

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Nanog is related to marrow stromal stem cell maintenance. Black-Right-Pointing-Pointer Increasing Nanog expression is seen during post natal surgical bone repair. Black-Right-Pointing-Pointer Nanog knockdown decreases post surgical bone regeneration. -- Abstract: Post natal bone repair elicits a regenerative mechanism that restores the injured tissue to its pre-injury cellular composition and structure and is believed to recapitulate the embryological processes of bone formation. Prior studies showed that Nanog, a central epigenetic regulator associated with the maintenance of embryonic stem cells (ESC) was transiently expressed during fracture healing, Bais et al. . In this study, we show that murine bone marrow stromal cells (MSCs) before they are induced to undergo osteogenic differentiation express {approx}50 Multiplication-Sign the background levels of Nanog seen in murine embryonic fibroblasts (MEFs) and the W20-17 murine marrow stromal cell line stably expresses Nanog at {approx}80 Multiplication-Sign the MEF levels. Nanog expression in this cell line was inhibited by BMP7 treatment and Nanog lentivrial shRNA knockdown induced the expression of the terminal osteogenic gene osteocalcin. Lentivrial shRNA knockdown or lentiviral overexpression of Nanog in bone MSCs had inverse effects on proliferation, with knockdown decreasing and overexpression increasing MSC cell proliferation. Surgical marrow ablation of mouse tibia by medullary reaming led to a {approx}3-fold increase in Nanog that preceded osteogenic differentiation during intramembranous bone formation. Lentiviral shRNA knockdown of Nanog after surgical ablation led to an initial overexpression of osteogenic gene expression with no initial effect on bone formation but during subsequent remodeling of the newly formed bone a {approx}50% decrease was seen in the expression of terminal osteogenic gene expression and a {approx}50% loss in trabecular bone mass. This

  11. In vitro analysis of multipotent mesenchymal stromal cells as potential cellular therapeutics in neurometabolic diseases in pediatric patients.

    PubMed

    Müller, Ingo; Kustermann-Kuhn, Birgit; Holzwarth, Christina; Isensee, Gesa; Vaegler, Martin; Harzer, Klaus; Krägeloh-Mann, Ingeborg; Handgretinger, Rupert; Bruchelt, Gernot

    2006-10-01

    Multipotent mesenchymal stromal cells (MSCs) play an important role in stromal support for hematopoietic stem cells, immune modulation, and tissue regeneration. We investigated their potential as cellular therapeutic tools in neurometabolic diseases as a growing number of affected children undergo to bone marrow transplantation. MSCs were isolated from bone marrow aspirates and expanded ex vivo under various culture conditions. MSCs under optimal good medical practice (GMP)-conform culture conditions showed the typical morphology, immunophenotype, and plasticity. Biochemically, the activities of beta-hexosaminidase A, total beta-hexosaminidase, arylsulfatase A (ASA), and beta-galactosidase measured in MSCs were comparable to those in fibroblasts of healthy donors. These four enzymes were interesting for their expression in MSCs, as each of them is defective, respectively, in well-known neurometabolic diseases. We found that MSCs released significant amounts of ASA into the media. In coculture experiments, fibroblasts from patients with metachromatic leukodystrophy, who are deficient for ASA, took up a substantial amount of ASA that was released into the media from MSCs. Mannose-6-phosphate (M6P) inhibited this uptake, which was in accordance with the M6P receptor-mediated uptake of lysosomal enzymes. Taken together, we show that MSCs produce appreciable amounts of lysosomal enzyme activities, making these cells first-choice candidates for providing metabolic correction when given to enzyme-deficient patients. With the example of ASA, it was also shown that an enzyme secreted from MSCs is taken up by enzyme-deficient patient fibroblasts. Given the plasticity of MSCs, these cells represent an interesting add-on option for cellular therapy in children undergoing bone marrow transplantation for lysosomal storage diseases and other neurometabolic diseases.

  12. Epithelial and Stromal Spectral Imaging for Rapid Surgical Margin Analysis

    DTIC Science & Technology

    2012-03-01

    for key diagnostic classes. The optical system samples mesoscopic tissue volumes; therefore, microscopic segmentation of glandular , stromal and...heterogeneous, but imaging-pathology correlates revealed that this variation had a spatial pattern that reflected the organization of glandular structures...characterized by marked expansion of glandular units by neo-plastic cells, compressing (but not invading) the surrounding stromal environment

  13. Stromal cell-derived factor 1 (SDF-1) and its receptor CXCR4 in the formation of postburn hypertrophic scar (HTS).

    PubMed

    Ding, Jie; Hori, Keijiro; Zhang, Rainny; Marcoux, Yvonne; Honardoust, Dariush; Shankowsky, Heather A; Tredget, Edward E

    2011-01-01

    Recent data support the involvement of stromal cell-derived factor 1 (SDF-1) in the homing of bone marrow-derived stem cells to wound sites during skeletal, myocardial, vascular, lung, and skin wound repair as well as some fibrotic disorders via its receptor CXCR4. In this study, the role of SDF-1/CXCR4 signaling in the formation of hypertrophic scar (HTS) following burn injury and after treatment with systemic interferon α2b (IFNα2b) is investigated. Studies show SDF-1/CXCR4 signaling was up-regulated in burn patients, including SDF-1 level in HTS tissue and serum as well as CD14+ CXCR4+ cells in the peripheral blood mononuclear cells. In vitro, dermal fibroblasts constitutively expressed SDF-1 and deep dermal fibroblasts expressed more SDF-1 than superficial fibroblasts. Lipopolysaccharide increased SDF-1 gene expression in fibroblasts. Also, recombinant SDF-1 and lipopolysaccharide stimulated fibroblast-conditioned medium up-regulated peripheral blood mononuclear cell mobility. In the burn patients with HTS who received subcutaneous IFNα2b treatment, increased SDF-1/CXCR4 signaling was found prior to treatment which was down-regulated after IFNα2b administration, coincident with enhanced remodeling of their HTS. Our results suggest that SDF-1/CXCR4 signaling is involved in the development of HTS by promoting migration of activated CD14+ CXCR4+ cells from the bloodstream to wound sites, where they may differentiate into fibrocyte and myofibroblasts and contribute to the development of HTS.

  14. Hic-5’s Regulatory Role in TGFB Signaling in Prostate Stroma

    DTIC Science & Technology

    2012-06-01

    TGFβ) signaling, transdifferentiation into a myofibroblast phenotype by stromal fibroblasts, and an increased production of reactive oxygen species (ROS...β1 induced transdifferentiation of stromal cells leads to increased androgenicity of DHEA in the cancer cells, as measured by increased PSA...inherent regulatory mechanism offered by the adjacent stromal cells regardless of their state of transdifferentiation . The role of estrogens in the

  15. On the time-course of adjacent and non-adjacent transposed-letter priming

    PubMed Central

    Ktori, Maria; Kingma, Brechtsje; Hannagan, Thomas; Holcomb, Phillip J.; Grainger, Jonathan

    2014-01-01

    We compared effects of adjacent (e.g., atricle-ARTICLE) and non-adjacent (e.g., actirle-ARTICLE) transposed-letter (TL) primes in an ERP study using the sandwich priming technique. TL priming was measured relative to the standard double-substitution condition. We found significantly stronger priming effects for adjacent transpositions than non-adjacent transpositions (with 2 intervening letters) in behavioral responses (lexical decision latencies), and the adjacent priming effects emerged earlier in the ERP signal, at around 200 ms post-target onset. Non-adjacent priming effects emerged about 50 ms later and were short-lived, being significant only in the 250-300 ms time-window. Adjacent transpositions on the other hand continued to produce priming in the N400 time-window (300-500 ms post-target onset). This qualitatively different pattern of priming effects for adjacent and non-adjacent transpositions is discussed in the light of different accounts of letter transposition effects, and the utility of drawing a distinction between positional flexibility and positional noise. PMID:25364497

  16. Tumor-associated Endo180 requires stromal-derived LOX to promote metastatic prostate cancer cell migration on human ECM surfaces.

    PubMed

    Caley, Matthew P; King, Helen; Shah, Neel; Wang, Kai; Rodriguez-Teja, Mercedes; Gronau, Julian H; Waxman, Jonathan; Sturge, Justin

    2016-02-01

    The diverse composition and structure of extracellular matrix (ECM) interfaces encountered by tumor cells at secondary tissue sites can influence metastatic progression. Extensive in vitro and in vivo data has confirmed that metastasizing tumor cells can adopt different migratory modes in response to their microenvironment. Here we present a model that uses human stromal cell-derived matrices to demonstrate that plasticity in tumor cell movement is controlled by the tumor-associated collagen receptor Endo180 (CD280, CLEC13E, KIAA0709, MRC2, TEM9, uPARAP) and the crosslinking of collagen fibers by stromal-derived lysyl oxidase (LOX). Human osteoblast-derived and fibroblast-derived ECM supported a rounded 'amoeboid-like' mode of cell migration and enhanced Endo180 expression in three prostate cancer cell lines (PC3, VCaP, DU145). Genetic silencing of Endo180 reverted PC3 cells from their rounded mode of migration towards a bipolar 'mesenchymal-like' mode of migration and blocked their translocation on human fibroblast-derived and osteoblast-derived matrices. The concomitant decrease in PC3 cell migration and increase in Endo180 expression induced by stromal LOX inhibition indicates that the Endo180-dependent rounded mode of prostate cancer cell migration requires ECM crosslinking. In conclusion, this study introduces a realistic in vitro model for the study of metastatic prostate cancer cell plasticity and pinpoints the cooperation between tumor-associated Endo180 and the stiff microenvironment imposed by stromal-derived LOX as a potential target for limiting metastatic progression in prostate cancer.

  17. Stromal cells can contribute oncogenic signals

    NASA Technical Reports Server (NTRS)

    Tlsty, T. D.

    2001-01-01

    The majority of studies of neoplastic transformation have focused attention on events that occur within transformed cells. These cell autonomous events result in the disruption of molecular pathways that regulate basic activities of the cells such as proliferation, death, movement and genomic integrity. Other studies have addressed the microenvironment of tumor cells and documented its importance in supporting tumor progression. Recent work has begun to expand on these initial studies of tumor microenvironment and now provide novel insights into the possible initiation and progression of malignant cells. This review will address the transforming effect of stromal cells on epithelial components. Copyright 2001 Academic Press.

  18. Schwann cells induce neuronal differentiation of bone marrow stromal cells.

    PubMed

    Zurita, Mercedes; Vaquero, Jesús; Oya, Santiago; Miguel, Miriam

    2005-04-04

    Bone marrow stromal cells are multipotent stem cells that have the potential to differentiate into bone, cartilage, fat and muscle. Recently, bone marrow stromal cells have been shown to have the capacity to differentiate into neurons under specific experimental conditions, using chemical factors. We now describe how bone marrow stromal cells can be induced to differentiate into neuron-like cells when they are co-cultured with Schwann cells. When compared with chemical differentiation, expression of neuronal differentiation markers begins later, but one week after beginning co-culture, most bone marrow stromal cells showed a typical neuronal morphology. Our present findings support the transdifferentiation of bone marrow stromal cells, and the potential utility of these cells for the treatment of degenerative and acquired disorders of the nervous system.

  19. Stromal networking: cellular connections in the germinal centre.

    PubMed

    Denton, Alice E; Linterman, Michelle A

    2017-03-17

    Secondary lymphoid organs are organized into distinct zones, governed by different types of mesenchymal stromal cells. These stromal cell subsets are critical for the generation of protective humoral immunity because they direct the migration of, and interaction between, multiple immune cell types to form the germinal centre. The germinal centre response generates long-lived antibody-secreting plasma cells and memory B cells which can provide long-term protection against re-infection. Stromal cell subsets mediate this response through control of immune cell trafficking, activation, localization and antigen access within the secondary lymphoid organ. Further, distinct populations of stromal cells underpin the delicate spatial organization of immune cells within the germinal centre. Because of this, the interactions between immune cells and stromal cells in secondary lymphoid organs are fundamental to the germinal centre response. Herein we review how this unique relationship leads to effective germinal centre responses.

  20. Giant fibroepithelial stromal polyp of the vulva: largest case reported

    PubMed Central

    2013-01-01

    Background Fibroepithelial stromal polyps are site-specific mesenchymal lesions that are commonly found in the vulvovaginal region in premenopausal females. These polyps usually are less than 5 cm in diameter and are most commonly identified during routine gynecological examination. Although the stromal polyp is benign, its differential diagnosis includes some malignant vulva lesions making it critical to ensure that an accurate pathologic diagnosis is made. Case We present a case of a 21 year old female with a giant fibroepithelial stromal polyp of the vulva. Upon review of the literature this is the largest reported fibroepithelial stromal polyp to date. Conclusion Fibroepithelial stromal polyps can grow as large as 390 grams and can be 18.5-cm in diameter. Microscopic evaluation of the polyp is critical in the exclusion of malignancy with this diagnosis. PMID:23842282

  1. 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.

  2. Expression of Fibroblast Activating Protein and Correlation with Histological Grade, Mitotic Index and Ki67 Expression in Canine Mast Cell Tumours.

    PubMed

    Giuliano, A; Dos Santos Horta, R; Constantino-Casas, F; Hoather, T; Dobson, J

    2017-01-01

    Fibroblast activating protein (FAP) is a membrane serine protease expressed by activated fibroblasts, particularly tumour associated fibroblasts (TAFs). FAP expression has not been reported in canine mast cell tumours (MCTs). The objective of this study was to evaluate the expression of FAP in TAFs and its correlation with histological grade, mitotic index and Ki67 expression in canine MCTs. FAP expression was evaluated by immunohistochemistry (IHC) in 30 canine MCTs. Twenty-eight (90%) of the MCTs expressed FAP in the stroma, 16 cases showed low to intermediate FAP score and 14 cases had a high FAP score. FAP was correlated positively with both Patnaik (P = 0.007) and Kiupel (P = 0.008) grading systems, mitotic index (P = 0.0008) and Ki67 expression (P = 0.009). High stromal FAP expression could be a potential negative prognostic factor in canine MCTs.

  3. In vitro quantitation of lethal and physiologic effects of total body irradiation on stromal and hematopoietic stem cells in continuous bone marrow cultures from Rf mice

    SciTech Connect

    Greenberger, J.S.; Eckner, R.J.; Otten, J.A.; Tennant, R.W.

    1982-07-01

    The effects of in vivo total body irradiation (TBI) and interval from TBI to explant of marrow on: stromal cell proliferation in vitro; stromal cell support of hematopoiesis in continuous bone marrow culture; and generation of WEHI-3 growth factor (GF)-dependent lines of hematopoietic progenitor cells were evaluated. Explant of marrow at 2, 4, 5, or 6 months after single fraction TBI (300-800 rad) was associated with decreased longevity of hemopoiesis and a decrease in the proliferative capacity of fibroblastic adherent-stromal colony forming cells (CFUf) as measured by colony size at 14 days and number of colonies per 10/sup 6/ cells plated. In contrast, explant of marrow 8 to 24 months after TBI produced cultures with longevity that was indistinguishable from age-matched control cultures (19-24 weeks). Marrow from irradiated first and second generation recipients of serially transferred marrow demonstrated a similar 7-month in vivo recovery period; however, the plateau maximum duration of hemopoiesis did not return to control levels. Purified stromal cell cultures were prepared by corticosteroid-deprivation of explanted marrow for 28 days and were then engrafted in vitro with marrow from C57BL/6J or RfM/UN mice that had been irradiated 1 month previously. Hemopoiesis in these cultures was restored, and they produced GM-CFUc and granulocytes for 15-24 weeks. Thus, healthy stroma supported growth of recently irradiated hemopoietic cells in vitro. Indirect effects of x-irradiation on hemopoietic stem cells through damage and repair in the stromal cell compartment can be effectively studied with the present bone marrow culture system. (JMT)

  4. The characterization of fibrocyte-like cells: a novel fibroblastic cell of the placenta.

    PubMed

    Riddell, M R; Winkler-Lowen, B; Chakrabarti, S; Dunk, C; Davidge, S T; Guilbert, L J

    2012-03-01

    The placenta is a highly vascularized organ thus angiogenesis is a key process in placental development. The contribution that different cells in the villous stroma play in placental angiogenesis is largely unknown. In this study we identified a novel stromal cell type in sections of term placenta which is morphologically fibroblastic and expressing the fibroblast marker TE-7 but also positive for the monocytic markers CD115 and CD14 and designated these cells as fibrocyte-like cells. Populations of fibrocyte-like cells from the placenta were isolated by two methods: culture of adherence-selected placental cells and, for higher purity, by CD45 fluorescence activated cell sorting (FACS). Fibrocyte-like cell conditioned medium increased endothelial tubule-like structure formation 2-fold versus control medium. Both pro-angiogenic growth factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) and the anti-angiogenic factor soluble-Flt were found in the conditioned medium. Neutralizing antibodies against VEGF and b-FGF reduced endothelial cell tubule-like structures to control levels. These data suggests that fibrocyte-like cells, a previously unidentified cell of the villous stroma, may play an important role in the regulation of placental angiogenesis.

  5. AHNAK enables mammary carcinoma cells to produce extracellular vesicles that increase neighboring fibroblast cell motility

    PubMed Central

    Dzik, Luciana M.; Iglesia, Rebeca P.; Cruz, Mário C.; Zelanis, André; de Siqueira, Adriane S.; Serrano, Solange M.T.; Goldberg, Gary S.; Jaeger, Ruy G.; Freitas, Vanessa M.

    2016-01-01

    Extracellular vesicles play important roles in tumor development. Many components of these structures, including microvesicles and exosomes, have been defined. However, mechanisms by which extracellular vesicles affect tumor progression are not fully understood. Here, we investigated vesicular communication between mammary carcinoma cells and neighboring nontransformed mammary fibroblasts. Nonbiased proteomic analysis found that over 1% of the entire proteome is represented in these vesicles, with the neuroblast differentiation associated protein AHNAK and annexin A2 being the most abundant. In particular, AHNAK was found to be the most prominent component of these vesicles based on peptide number, and appeared necessary for their formation. In addition, we report here that carcinoma cells produce vesicles that promote the migration of recipient fibroblasts. These data suggest that AHNAK enables mammary carcinoma cells to produce and release extracellular vesicles that cause disruption of the stroma by surrounding fibroblasts. This paradigm reveals fundamental mechanisms by which vesicular communication between carcinoma cells and stromal cells can promote cancer progression in the tumor microenvironment. PMID:27374178

  6. MYC mediates large oncosome-induced fibroblast reprogramming in prostate cancer.

    PubMed

    Minciacchi, Valentina R; Spinelli, Cristiana; Reis-Sobreiro, Mariana; Cavallini, Lorenzo; You, Sungyong; Zandian, Mandana; Li, Xiaohong; Chiarugi, Paola; Adam, Rosalyn M; Posadas, Edwin M; Viglietto, Giuseppe; Freeman, Michael R; Cocucci, Emanuele; Bhowmick, Neil A; Di Vizio, Dolores

    2017-02-15

    Communication between cancer cells and the tumor microenvironment results in the modulation of complex signaling networks that facilitate tumor progression. Here we describe a new mechanism of intercellular communication originating from large oncosomes (LO), which are cancer cell-derived, atypically large (1-10 μm) extracellular vesicles (EV). We demonstrate that, in the context of prostate cancer, LO harbor sustained AKT1 kinase activity, nominating them as active signaling platforms. Active AKT1 was detected in circulating EV from the plasma of metastatic prostate cancer patients and was LO specific. LO internalization induced reprogramming of human normal prostate fibroblasts as reflected by high levels of α-SMA, IL-6, and MMP9. In turn, LO-reprogrammed normal prostate fibroblasts stimulated endothelial tube formation in vitro and promoted tumor growth in mice. Activation of stromal MYC was critical for this reprogramming and for the sustained cellular responses elicited by LO both in vitro and in vivo in an AKT1-dependent manner. Inhibition of LO internalization prevented activation of MYC and impaired the tumor supporting properties of fibroblasts. Overall, our data show that prostate cancer-derived LO powerfully promote establishment of a tumor supportive environment by inducing a novel reprogramming of the stroma. This mechanism offers potential alternative options for patient treatment.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27827906

  8. Co-Culture of Tumor Spheroids and Fibroblasts in a Collagen Matrix-Incorporated Microfluidic Chip Mimics Reciprocal Activation in Solid Tumor Microenvironment

    PubMed Central

    Jeong, Su-Yeong; Lee, Ji-Hyun; Shin, Yoojin; Chung, Seok; Kuh, Hyo-Jeong

    2016-01-01

    Multicellular 3D culture and interaction with stromal components are considered essential elements in establishing a ‘more clinically relevant’ tumor model. Matrix-embedded 3D cultures using a microfluidic chip platform can recapitulate the microscale interaction within tumor microenvironments. As a major component of tumor microenvironment, cancer-associated fibroblasts (CAFs) play a role in cancer progression and drug resistance. Here, we present a microfluidic chip-based tumor tissue culture model that integrates 3D tumor spheroids (TSs) with CAF in proximity within a hydrogel scaffold. HT-29 human colorectal carcinoma cells grew into 3D TSs and the growth was stimulated when co-cultured with fibroblasts as shown by 1.5-folds increase of % changes in diameter over 5 days. TS cultured for 6 days showed a reduced expression of Ki-67 along with increased expression of fibronectin when co-cultured with fibroblasts compared to mono-cultured TSs. Fibroblasts were activated under co-culture conditions, as demonstrated by increases in α-SMA expression and migratory activity. When exposed to paclitaxel, a survival advantage was observed in TSs co-cultured with activated fibroblasts. Overall, we demonstrated the reciprocal interaction between TSs and fibroblasts in our 7-channel microfluidic chip. The co-culture of 3D TS-CAF in a collagen matrix-incorporated microfluidic chip may be useful to study the tumor microenvironment and for evaluation of drug screening and evaluation. PMID:27391808

  9. Staphylococcus aureus Blepharitis Associated with Multiple Corneal Stromal Microabscess, Stromal Edema, and Uveitis.

    PubMed

    Boto-de-los-Bueis, Ana; del Hierro Zarzuelo, Almudena; García Perea, Adela; de Pablos, Manuela; Pastora, Natalia; Noval, Susana

    2015-04-01

    We report a case of an immunocompetent woman with atypical marginal keratitis. She presented with recurrent episodes of multiples microabscess distributed in a triangular pattern associated with stromal oedema and anterior chamber uveitis, affecting both eyes, but not simultaneously. The episodes responded to steroid drops, corneal inflammation was coincidental with a worsening of her blepharitis in the affected eye and S. aureus was isolated from the lids.

  10. Immunological hallmarks of stromal cells in the tumour microenvironment.

    PubMed

    Turley, Shannon J; Cremasco, Viviana; Astarita, Jillian L

    2015-11-01

    A dynamic and mutualistic interaction between tumour cells and the surrounding stroma promotes the initiation, progression, metastasis and chemoresistance of solid tumours. Far less understood is the relationship between the stroma and tumour-infiltrating leukocytes; however, emerging evidence suggests that the stromal compartment can shape antitumour immunity and responsiveness to immunotherapy. Thus, there is growing interest in elucidating the immunomodulatory roles of the stroma that evolve within the tumour microenvironment. In this Review, we discuss the evidence that stromal determinants interact with leukocytes and influence antitumour immunity, with emphasis on the immunological attributes of stromal cells that may foster their protumorigenic function.

  11. 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.

  12. Bilateral Sclerosing Stromal Ovarian Tumor in an Adolescent

    PubMed Central

    Naidu, Anjani; Chung, Betty; Simon, Mitchell; Marshall, Ian

    2015-01-01

    Sclerosing stromal tumor of the ovary is a rare, benign, sex cord stromal tumor occurring predominantly in younger women in the 2nd and 3rd decades of life. It typically presents unilaterally with only 2 previously reported cases of bilateral presentation. Common clinical presentations include pelvic or abdominal pain, a mass, or menstrual changes. Although occasionally presenting with hormonal manifestations, virilization as a result of androgen production by the tumor is rare. Here we present an extremely rare case of a sclerosing stromal ovarian tumor in a 14-year-old patient with bilateral presentation and with clinical and biochemical evidence of hyperandrogenemia. PMID:26064755

  13. Cell motility and local viscoelasticity of fibroblasts.

    PubMed

    Park, S; Koch, D; Cardenas, R; Käs, J; Shih, C K

    2005-12-01

    Viscoelastic changes of the lamellipodial actin cytoskeleton are a fundamental element of cell motility. Thus, the correlation between the local viscoelastic properties of the lamellipodium (including the transitional region to the cell body) and the speed of lamellipodial extension is studied for normal and malignantly transformed fibroblasts. Using our atomic force microscopy-based microrheology technique, we found different mechanical properties between the lamellipodia of malignantly transformed fibroblasts (H-ras transformed and SV-T2 fibroblasts) and normal fibroblasts (BALB 3T3 fibroblasts). The average elastic constants, K, in the leading edge of SV-T2 fibroblasts (0.48 +/- 0.51 kPa) and of H-ras transformed fibroblasts (0.42 +/- 0.35 kPa) are significantly lower than that of BALB 3T3 fibroblasts (1.01 +/- 0.40 kPa). The analysis of time-lapse phase contrast images shows that the decrease in the elastic constant, K, for malignantly transformed fibroblasts is correlated with the enhanced motility of the lamellipodium. The measured mean speeds are 6.1 +/- 4.5 microm/h for BALB 3T3 fibroblasts, 13.1 +/- 5.2 microm/h for SV-T2 fibroblasts, and 26.2 +/- 11.5 microm/h for H-ras fibroblasts. Furthermore, the elastic constant, K, increases toward the cell body in many instances which coincide with an increase in actin filament density toward the cell body. The correlation between the enhanced motility and the decrease in viscoelastic moduli supports the Elastic Brownian Ratchet model for driving lamellipodia extension.

  14. Gastrointestinal stromal tumors: the histology report.

    PubMed

    Dei Tos, Angelo P; Laurino, Licia; Bearzi, Italo; Messerini, Luca; Farinati, Fabio

    2011-03-01

    Gastrointestinal stromal tumors (GISTs) represent a mesenchymal neoplasm occurring primarily in the gastrointestinal tract, and showing differentiation toward the interstitial cell of Cajal. Its incidence is approximately 15 case/100,000/year. Stomach and small bowel are the most frequently affected anatomic sites. GIST represents a morphological, immunophenotypical and molecular distinct entity, the recognition of which has profound therapeutic implications. In fact, they have shown an exquisite sensitivity to treatment with the tyrosine kinase inhibitor imatinib. Diagnosis relies upon morphology along with immunodetection of KIT and/or DOG1. When dealing with KIT negative cases, molecular analysis of KIT/PDGFRA genes may help in confirming diagnosis. Molecular evaluation of both genes are in any case recommended as mutational status provides key predictive information. Pathologists also play a key role in providing an estimation of the risk of biological aggressiveness, which is currently based on anatomic location of the tumor, size, and mitotic activity.

  15. Pseudoangiomatous stromal hyperplasia causing massive breast enlargement.

    PubMed

    Bourke, Anita Geraldine; Tiang, Stephen; Harvey, Nathan; McClure, Robert

    2015-10-16

    Pseudoangiomatous stromal hyperplasia (PASH) of the breast is a benign mesenchymal proliferative process, initially described by Vuitch et al. We report an unusual case of a 46-year-old woman who presented with a 6-week history of bilateral massive, asymmetrical, painful enlargement of her breasts, without a history of trauma. On clinical examination, both breasts were markedly enlarged and oedematous, but there were no discrete palpable masses. Preoperative image-guided core biopsies and surgery showed PASH. PASH is increasingly recognised as an incidental finding on image-guided core biopsy performed for screen detected lesions. There are a few reported cases of PASH presenting as rapid breast enlargement. In our case, the patient presented with painful, asymmetrical, massive breast enlargement. Awareness needs to be raised of this entity as a differential diagnosis in massive, painful breast enlargement.

  16. Update on Gastrointestinal Stromal Tumors for Radiologists

    PubMed Central

    Baheti, Akshay D.; Tirumani, Harika; O'Neill, Ailbhe; Jagannathan, Jyothi P.

    2017-01-01

    The management of gastrointestinal stromal tumors (GISTs) has evolved significantly in the last two decades due to better understanding of their biologic behavior as well as development of molecular targeted therapies. GISTs with exon 11 mutation respond to imatinib whereas GISTs with exon 9 or succinate dehydrogenase subunit mutations do not. Risk stratification models have enabled stratifying GISTs according to risk of recurrence and choosing patients who may benefit from adjuvant therapy. Assessing response to targeted therapies in GIST using conventional response criteria has several potential pitfalls leading to search for alternate response criteria based on changes in tumor attenuation, volume, metabolic and functional parameters. Surveillance of patients with GIST in the adjuvant setting is important for timely detection of recurrences. PMID:28096720

  17. Laparoscopic resection of duodenal gastrointestinal stromal tumour

    PubMed Central

    Zioni, Tammy; Dizengof, Vitaliy; Kirshtein, Boris

    2017-01-01

    Only a few studies have revealed using laparoscopic technique with limited resection of gastrointestinal stromal tumour (GIST) of the duodenum. A 68-year-old man was admitted to the hospital due to upper gastrointestinal (GI) bleeding. Evaluation revealed an ulcerated, bleeding GI tumour in the second part of the duodenum. After control of bleeding during gastroduodenoscopy, he underwent a laparoscopic wedge resection of the area. During 1.5 years of follow-up, the patient is disease free, eats drinks well, and has regained weight. Surgical resection of duodenal GIST with free margins is the main treatment of this tumour. Various surgical treatment options have been reported. Laparoscopic resection of duodenal GIST is an advanced and challenging procedure requiring experience and good surgical technique. The laparoscopic limited resection of duodenal GIST is feasible and safe, reducing postoperative morbidity without compromising oncologic results. PMID:28281485

  18. The Expression of Fibroblast Activation Protein in Clear Cell Renal Cell Carcinomas Is Associated with Synchronous Lymph Node Metastases

    PubMed Central

    Errarte, Peio; Guarch, Rosa; Pulido, Rafael; Blanco, Lorena; Nunes-Xavier, Caroline E.; Beitia, Maider; Gil, Javier; Angulo, Javier C.; López, José I.; Larrinaga, Gorka

    2016-01-01

    Clear cell renal cell carcinoma (CCRCC) is a heterogeneous and complex disease that frequently develops distant metastases. Fibroblast activation protein (FAP) is a serine peptidase the expression of which in cancer-associated fibroblasts has been associated with higher risk of metastases and poor survival. The objective of this study was to evaluate the role of FAP in metastatic CCRCC (mCCRCC). A series of 59 mCCRCC retrospectively collected was included in the study. Metastases developed either synchronous (n = 14) or metachronous to renal disease (n = 45). Tumor specimens were obtained from both primary lesion (n = 59) and metastases (n = 54) and FAP expression was immunohistochemically analyzed. FAP expression in fibroblasts from primary tumors correlated with FAP expression in the corresponding metastatic lesions. Also, primary and metastatic FAP expression was correlated with large tumor diameter (>7cm), high grade (G3/4), high stage (pT3/4), tumor necrosis and sarcomatoid transformation. The expression of FAP in primary tumors and in their metastases was associated both with synchronous metastases and also with metastases to the lymph nodes. FAP expression in the primary tumor was correlated with worse 10-year overall survival. Immunohistochemical detection of FAP in the stromal tumor fibroblasts could be a biomarker of early lymph node metastatic status and therefore could account for the poor prognosis of FAP positive CCRCC. PMID:28033421

  19. Adjacent Segment Pathology after Anterior Cervical Fusion

    PubMed Central

    Chung, Jae Yoon; Park, Jong-Beom; Seo, Hyoung-Yeon

    2016-01-01

    Anterior cervical fusion has become a standard of care for numerous pathologic conditions of the cervical spine. However, subsequent development of clinically significant disc disease at levels adjacent to fused discs is a serious long-term complication of this procedure. As more patients live longer after surgery, it is foreseeable that adjacent segment pathology (ASP) will develop in increasing numbers of patients. Also, ASP has been studied more intensively with the recent popularity of motion preservation technologies like total disc arthroplasty. The true nature and scope of ASP remains poorly understood. The etiology of ASP is most likely multifactorial. Various factors including altered biomechanical stresses, surgical disruption of soft tissue and the natural history of cervical disc disease contribute to the development of ASP. General factors associated with disc degeneration including gender, age, smoking and sports may play a role in the development of ASP. Postoperative sagittal alignment and type of surgery are also considered potential causes of ASP. Therefore, a spine surgeon must be particularly careful to avoid unnecessary disruption of the musculoligamentous structures, reduced risk of direct injury to the disc during dissection and maintain a safe margin between the plate edge and adjacent vertebrae during anterior cervical fusion. PMID:27340541

  20. Tissue Digestion for Stromal Cell and Leukocyte Isolation.

    PubMed

    Nayar, Saba; Campos, Joana; Steinthal, Nathalie; Barone, Francesca

    2017-01-01

    Tissue mechanical disruption is often not sufficient to disrupt cell-to-cell interactions; this is particularly relevant for stromal cells that are embedded within the extracellular matrix. For this reason, different enzyme combinations have been described to enable the isolation of single-cell populations, particularly stromal cells. This chapter aims to describe different methods used for enzymatic digestion of stromal cell and leukocyte populations from secondary and tertiary lymphoid organs. Collagenase D and P and collagenase D and dispase protocols provide a good yield of stromal cells, while a collagenase dispase-only protocol should be used if the main aim of the technique is to retrieve leukocyte populations. However, for isolation of both stroma and leukocyte populations the collagenase D and P protocol would provide the best results. Protocols for these techniques and illustrative results from flow cytometry analysis can be found in this chapter.

  1. Effect of hydrocortisone on multipotent human mesenchymal stromal cells.

    PubMed

    Shipunova, N N; Petinati, N A; Drize, N I

    2013-05-01

    We studied the effect of natural glucocorticosteroid hydrocortisone on total cell production, cloning efficiency, and expression of genes important for the function of mesenchymal stromal cells. Addition of hydrocortisone to the culture medium reduces the total cell yield by 2 times and significantly increased cloning efficiency by 2-3 times; this effect was more pronounced in multipotent mesenchymal stromal cells obtained from female donors. Hydrocortisone had no effect on the expression of immunomodulatory factors produced by multipotent mesenchymal stromal cells. Hydrocortisone inhibits the expression of bone differentiation markers, increases the expression of the early adipocyte differentiation marker at the beginning of culturing, and dramatically stimulates the expression of the late adipocyte differentiation marker throughout the culturing period. The findings suggest that hydrocortisone activates multipotent mesenchymal stromal cells.

  2. Stromal infrastructure of the lymph node and coordination of immunity.

    PubMed

    Chang, Jonathan E; Turley, Shannon J

    2015-01-01

    The initiation of adaptive immune responses depends upon the careful maneuvering of lymphocytes and antigen into and within strategically placed lymph nodes (LNs). Non-hematopoietic stromal cells form the cellular infrastructure that directs this process. Once regarded as merely structural features of lymphoid tissues, these cells are now appreciated as essential regulators of immune cell trafficking, fluid flow, and LN homeostasis. Recent advances in the identification and in vivo targeting of specific stromal populations have resulted in striking new insights to the function of stromal cells and reveal a level of complexity previously unrealized. We discuss here recent discoveries that highlight the pivotal role that stromal cells play in orchestrating immune cell homeostasis and adaptive immunity.

  3. Stromal-epithelial dynamics in response to fractionated radiotherapy

    NASA Astrophysics Data System (ADS)

    Qayyum, Muqeem Abdul

    effective at treating the reactive stroma. We can kill the cancer cells at the standard rate (180 cGy/fraction), but we have found the larger fractions specifically inhibit wound healing mechanisms by inactivating stromal fibroblasts. The long term goal would be to reduce recurrence rates for early stage breast cancer by treating postsurgical regions most likely to harbor residual tumor cells. Ionizing radiation stress and its effect on ECM mediated cellular functions continues to be an evolving area of research. This study is an initial step in my career plans to study stromal modulation of epithelial tumors. It is also my career goal to integrate basic science experiments and engineering tools into clinical practice.

  4. Senescent stromal cells induce cancer cell migration via inhibition of RhoA/ROCK/myosin-based cell contractility

    PubMed Central

    Aifuwa, Ivie; Giri, Anjil; Longe, Nick; Lee, Sang Hyuk; An, Steven S.; Wirtz, Denis

    2015-01-01

    Cells induced into senescence exhibit a marked increase in the secretion of pro-inflammatory cytokines termed senescence-associated secretory phenotype (SASP). Here we report that SASP from senescent stromal fibroblasts promote spontaneous morphological changes accompanied by an aggressive migratory behavior in originally non-motile human breast cancer cells. This phenotypic switch is coordinated, in space and time, by a dramatic reorganization of the actin and microtubule filament networks, a discrete polarization of EB1 comets, and an unconventional front-to-back inversion of nucleus-MTOC polarity. SASP-induced morphological/migratory changes are critically dependent on microtubule integrity and dynamics, and are coordinated by the inhibition of RhoA and cell contractility. RhoA/ROCK inhibition reduces focal adhesions and traction forces, while promoting a novel gliding mode of migration. PMID:26483365

  5. 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.

  6. Stromal p16 expression is significantly increased in endometrial carcinoma.

    PubMed

    Yoon, Gun; Koh, Chang Won; Yoon, Nara; Kim, Ji-Ye; Kim, Hyun-Soo

    2017-01-17

    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.

  7. Dexamethasone induction of osteoblast mRNAs in rat marrow stromal cell cultures.

    PubMed

    Leboy, P S; Beresford, J N; Devlin, C; Owen, M E

    1991-03-01

    osteoblast phenotype in cultures of rat marrow stromal fibroblasts.

  8. 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

  9. Ethanol and Cancer Induce Similar Changes on Protein Expression Pattern of Human Fibroblast Cell

    PubMed Central

    Zamanian–Azodi, Mona; Rezaei-Tavirani, Mostafa; Rahmati-Rad, Sara; Rezaei Tavirani, Majid

    2016-01-01

    Ethanol has a vast consumption around the world. Many researches confirmed some adverse effect of this component on human health. In addition, recent studies showed significant alteration in both cellular population, and protein profile of human foreskin fibroblast cell line (HFFF2) in the specific dosage of ethanol. Here, the role and interaction of some proteins (characterized by significant alteration in expression due to ethanol effect) analyzed by proteomics and evaluated by considering cancerous case. 2D-electrophoresis findings of comparison of normal fibroblast cells and treated fibroblast with 270 mM dosage of ethanol analyzed by using SameSpots software, R software, and Cytoscape for protein-protein interaction (PPI) investigation. Six proteins with significantly altered expression associated with fundamental properties in a cell identified in ethanol-treated sample. These include AnnexinA5, Heterogeneous nuclear ribonucleoprotein A1, Rho-GDP dissociation inhibitor, Cathepsin L, Cu/Zn-SOD, Rho-GDP dissociation inhibitor, and Serpin peptidase inhibitor. Surprisingly, all these proteins were down-regulated and this pattern is similar to nasopharyngeal carcinoma-associated stromal fibroblast sample. Additionally, protein-protein interaction (PPI) indicates that HNRNPA1, SERPINE1 are hub proteins. Once their expression alters, it can impose vast changes on other molecular function. Based on this approach, ethanol may target same pathways that are related to cancer onset. In addition, some epidemiologic studies proved that ethanol consumption is related to increment of cancer risk. Therefore, more investigation is required in this regard to elicit the feasible relationship. PMID:28228815

  10. Mesenchymal Stromal Cells are Readily Recoverable from Lung Tissue, but not the Alveolar Space, in Healthy Humans.

    PubMed

    Sinclair, K A; Yerkovich, S T; Chen, T; McQualter, J L; Hopkins, P M-A; Wells, C A; Chambers, D C

    2016-10-01

    Stromal support is critical for lung homeostasis and the maintenance of an effective epithelial barrier. Despite this, previous studies have found a positive association between the number of mesenchymal stromal cells (MSCs) isolated from the alveolar compartment and human lung diseases associated with epithelial dysfunction. We hypothesised that bronchoalveolar lavage derived MSCs (BAL-MSCs) are dysfunctional and distinct from resident lung tissue MSCs (LT-MSCs). In this study, we comprehensively interrogated the phenotype and transcriptome of human BAL-MSCs and LT-MSCs. We found that MSCs were rarely recoverable from the alveolar space in healthy humans, but could be readily isolated from lung transplant recipients by bronchoalveolar lavage. BAL-MSCs exhibited a CD90(Hi) , CD73(Hi) , CD45(Neg) , CD105(Lo) immunophenotype and were bipotent, lacking adipogenic potential. In contrast, MSCs were readily recoverable from healthy human lung tissue and were CD90(Hi or Lo) , CD73(Hi) , CD45(Neg) , CD105(Int) and had full tri-lineage potential. Transcriptional profiling of the two populations confirmed their status as bona fide MSCs and revealed a high degree of similarity between each other and the archetypal bone-marrow MSC. 105 genes were differentially expressed; 76 of which were increased in BAL-MSCs including genes involved in fibroblast activation, extracellular matrix deposition and tissue remodelling. Finally, we found the fibroblast markers collagen 1A1 and α-smooth muscle actin were increased in BAL-MSCs. Our data suggests that in healthy humans, lung MSCs reside within the tissue, but in disease can differentiate to acquire a profibrotic phenotype and migrate from their in-tissue niche into the alveolar space. Stem Cells 2016;34:2548-2558.

  11. Murine Dermal Fibroblast Isolation by FACS.

    PubMed

    Walmsley, Graham G; Maan, Zeshaan N; Hu, Michael S; Atashroo, David A; Whittam, Alexander J; Duscher, Dominik; Tevlin, Ruth; Marecic, Owen; Lorenz, H Peter; Gurtner, Geoffrey C; Longaker, Michael T

    2016-01-07

    Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin(-)) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

  12. KIT and PDGFRA mutations and PDGFRA immunostaining in gastrointestinal stromal tumors.

    PubMed

    Barreca, Antonella; Fornari, Alessandro; Bonello, Lisa; Tondat, Fabrizio; Chiusa, Luigi; Lista, Patrizia; Pich, Achille

    2011-01-01

    In the present study, we investigated the association of PDGFRA and KIT mutations as well as PDGFRA immunohistochemical expression with clinicopathologic features and prognosis in a series of gastrointestinal stromal tumors (GISTs). Tumor DNA from 40 GISTs was sequenced for the presence of mutations in KIT exons 9, 11, 13 and 17, and in PDGFRA exons 12 and 18. Tissue sections were stained with polyclonal anti-PDGFRA antibody. KIT mutations occurred in 26 cases. There were 13 deletions, 6 substitutions, 3 deletion-substitutions, 3 duplications and 1 insertion. Tumors with KIT deletions/insertion were large with a high mitotic index (MI), and were associated with a high rate of symptoms at diagnosis, invasion into adjacent organs, distant metastasis, relapse and a short disease-free survival (DFS). PDGFRA mutations occurred in 6 gastric GISTs. There were 4 deletions and 2 substitutions. Tumors with PDGFRA mutations were small, with a low MI and Ki67 score, and were associated with a very low rate of symptoms at diagnosis, invasion into adjacent organs and distant metastasis. PDGFRA immunopositivity was found in 23 cases: a peculiar 'dotlike' staining was found in 5 out of 6 PDGFRA mutated cases. Patients with positive PDGFRA immunostaining had a longer DFS than those with negative staining. Our data confirm that the type of KIT mutation is associated with various clinicopathologic features of GISTs, and indicate that PDGFRA mutations are associated with rather indolent tumors. PDGFRA immunopositivity reflects PDGFRA mutational status and is associated with a favorable outcome.

  13. A 3D in vitro model of patient-derived prostate cancer xenograft for controlled interrogation of in vivo tumor-stromal interactions.

    PubMed

    Fong, Eliza L S; Wan, Xinhai; Yang, Jun; Morgado, Micaela; Mikos, Antonios G; Harrington, Daniel A; Navone, Nora M; Farach-Carson, Mary C

    2016-01-01

    Patient-derived xenograft (PDX) models better represent human cancer than traditional cell lines. However, the complex in vivo environment makes it challenging to employ PDX models to investigate tumor-stromal interactions, such as those that mediate prostate cancer (PCa) bone metastasis. Thus, we engineered a defined three-dimensional (3D) hydrogel system capable of supporting the co-culture of PCa PDX cells and osteoblastic cells to recapitulate the PCa-osteoblast unit within the bone metastatic microenvironment in vitro. Our 3D model not only maintained cell viability but also preserved the typical osteogenic phenotype of PCa PDX cells. Additionally, co-culture cellularity was maintained over that of either cell type cultured alone, suggesting that the PCa-osteoblast cross-talk supports PCa progression in bone, as is hypothesized to occur in patients with prostatic bone metastasis. Strikingly, osteoblastic cells co-cultured with PCa PDX tumoroids organized around the tumoroids, closely mimicking the architecture of PCa metastases in bone. Finally, tumor-stromal signaling mediated by the fibroblast growth factor axis tightly paralleled that in the in vivo counterpart. Together, these findings indicate that this 3D PCa PDX model recapitulates important pathological properties of PCa bone metastasis, and validate the use of this model for controlled and systematic interrogation of complex in vivo tumor-stromal interactions.

  14. Role of human pulp fibroblasts in angiogenesis.

    PubMed

    Tran-Hung, L; Mathieu, S; About, I

    2006-09-01

    After pulp amputation, complete pulp healing requires not only reparative dentin production but also fibroblast proliferation, nerve fiber growth, and neoangiogenesis. This study was designed to investigate the role of pulp fibroblasts in angiogenesis. Human pulp fibroblasts from third molars co-cultured with human umbilical vein endothelial cells induced the organization of endothelial cells and the formation of tubular structures corresponding to capillaries in vivo. The direct contact between both cells was not necessary to induce angiogenesis, and the observed effect was due to soluble factors. This was confirmed with neutralizing antibodies against FGF-2 and VEGF, which decreased the angiogenic effects of these soluble factors. Immunohistochemistry showed that both FGF-2 and VEGF were expressed in human dental pulp fibroblasts, and this expression increased after injury. These results suggest that the pulp fibroblasts secrete angiogenic factors, which are necessary for complete pulp healing, particularly at the pulp injury site.

  15. Aberrant DNA methylation in non-small cell lung cancer-associated fibroblasts

    PubMed Central

    Vizoso, Miguel; Puig, Marta; Carmona, F.Javier; Maqueda, María; Velásquez, Adriana; Gómez, Antonio; Labernadie, Anna; Lugo, Roberto; Gabasa, Marta; Rigat-Brugarolas, Luis G.; Trepat, Xavier; Ramírez, Josep; Moran, Sebastian; Vidal, Enrique; Reguart, Noemí; Perera, Alexandre; Esteller, Manel; Alcaraz, Jordi

    2015-01-01

    Epigenetic changes through altered DNA methylation have been implicated in critical aspects of tumor progression, and have been extensively studied in a variety of cancer types. In contrast, our current knowledge of the aberrant genomic DNA methylation in tumor-associated fibroblasts (TAFs) or other stromal cells that act as critical coconspirators of tumor progression is very scarce. To address this gap of knowledge, we conducted genome-wide DNA methylation profiling on lung TAFs and paired control fibroblasts (CFs) from non-small cell lung cancer patients using the HumanMethylation450 microarray. We found widespread DNA hypomethylation concomitant with focal gain of DNA methylation in TAFs compared to CFs. The aberrant DNA methylation landscape of TAFs had a global impact on gene expression and a selective impact on the TGF-β pathway. The latter included promoter hypermethylation-associated SMAD3 silencing, which was associated with hyperresponsiveness to exogenous TGF-β1 in terms of contractility and extracellular matrix deposition. In turn, activation of CFs with exogenous TGF-β1 partially mimicked the epigenetic alterations observed in TAFs, suggesting that TGF-β1 may be necessary but not sufficient to elicit such alterations. Moreover, integrated pathway-enrichment analyses of the DNA methylation alterations revealed that a fraction of TAFs may be bone marrow-derived fibrocytes. Finally, survival analyses using DNA methylation and gene expression datasets identified aberrant DNA methylation on the EDARADD promoter sequence as a prognostic factor in non-small cell lung cancer patients. Our findings shed light on the unique origin and molecular alterations underlying the aberrant phenotype of lung TAFs, and identify a stromal biomarker with potential clinical relevance. PMID:26449251

  16. Cancer-associated-fibroblasts and tumour cells: a diabolic liaison driving cancer progression.

    PubMed

    Cirri, Paolo; Chiarugi, Paola

    2012-06-01

    Several recent papers have now provided compelling experimental evidence that the progression of tumours towards a malignant phenotype does not depend exclusively on the cell-autonomous properties of cancer cells themselves but is also deeply influenced by tumour stroma reactivity, thereby undergoing a strict environmental control. Tumour microenvironmental elements include structural components such as the extracellular matrix or hypoxia as well as stromal cells, either resident cells or recruited from circulating precursors, as macrophages and other inflammatory cells, endothelial cells and cancer-associated fibroblasts (CAFs). All these elements synergistically play a specific role in cancer progression. This review summarizes our current knowledge on the role of CAFs in tumour progression, with a particular focus on the biunivocal interplay between CAFs and cancer cells leading to the activation of the epithelial-mesenchymal transition programme and the achievement of stem cell traits, as well as to the metabolic reprogramming of both stromal and cancer cells. Recent advances on the role of CAFs in the preparation of metastatic niche, as well as the controversial origin of CAFs, are discussed in light of the new emerging therapeutic implications of targeting CAFs.

  17. Adjacent-level arthroplasty following cervical fusion.

    PubMed

    Rajakumar, Deshpande V; Hari, Akshay; Krishna, Murali; Konar, Subhas; Sharma, Ankit

    2017-02-01

    OBJECTIVE Adjacent-level disc degeneration following cervical fusion has been well reported. This condition poses a major treatment dilemma when it becomes symptomatic. The potential application of cervical arthroplasty to preserve motion in the affected segment is not well documented, with few studies in the literature. The authors present their initial experience of analyzing clinical and radiological results in such patients who were treated with arthroplasty for new or persistent arm and/or neck symptoms related to neural compression due to adjacent-segment disease after anterior cervical discectomy and fusion (ACDF). METHODS During a 5-year period, 11 patients who had undergone ACDF anterior cervical discectomy and fusion (ACDF) and subsequently developed recurrent neck or arm pain related to adjacent-level cervical disc disease were treated with cervical arthroplasty at the authors' institution. A total of 15 devices were implanted (range of treated levels per patient: 1-3). Clinical evaluation was performed both before and after surgery, using a visual analog scale (VAS) for pain and the Neck Disability Index (NDI). Radiological outcomes were analyzed using pre- and postoperative flexion/extension lateral radiographs measuring Cobb angle (overall C2-7 sagittal alignment), functional spinal unit (FSU) angle, and range of motion (ROM). RESULTS There were no major perioperative complications or device-related failures. Statistically significant results, obtained in all cases, were reflected by an improvement in VAS scores for neck/arm pain and NDI scores for neck pain. Radiologically, statistically significant increases in the overall lordosis (as measured by Cobb angle) and ROM at the treated disc level were observed. Three patients were lost to follow-up within the first year after arthroplasty. In the remaining 8 cases, the duration of follow-up ranged from 1 to 3 years. None of these 8 patients required surgery for the same vertebral level during the follow

  18. Small role with big impact: miRNAs as communicators in the cross-talk between cancer-associated fibroblasts and cancer cells

    PubMed Central

    Wang, Zhanhuai; Tan, Yinuo; Yu, Wei; Zheng, Shu; Zhang, Suzhan; Sun, Lifeng; Ding, Kefeng

    2017-01-01

    Cancer microenvironment is composed of numerous components that can support cancer cell proliferation, promote cancer progression and contribute to cancer treatment resistance. The major components of caner microenvironment are fibroblasts, endothelial cells, immune cells as well as cytokines, chemokines, and extracellular matrix (ECM) all of which surround tumor cells as the core and cross talk with each other. Among them, cancer-associated fibroblasts (CAFs) play an important role in promoting cancer progression by secreting various pro-inflammatory factors. MicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate protein expression both in cancer cell and normal stromal cells. Changes of miRNAs expression in cancer-associated fibroblasts can be induced both by cancer cells and other stromal cells. This change can arise through direct interaction or by secreted paracrine factors or even by secreted miRNAs. The desregulated miRNAs in cancer-associated fibroblasts then enhance the CAFs phenotype and assist their cancer promotion ability. Explore the regulatory function of miRNAs in the complex communication between cancer cells and cancer microenvironment is important to understand the process of tumor progression and may help to develop new therapeutic strategies. This review provides an updated content of latest research advances about the relevance of miRNAs in the interaction between cancer cells and the CAFs. We will describe miRNAs which are differential expressed by NFs and CAFs, their function in regulating fibroblasts activation as well as miRNAs expressed in CAFs as prognostic factors in cancer stroma in recent studies. We will also discuss miRNA as an important player in CAFs mediated regulation of cancer progression and metastasis, cancer metabolism, cancer stem cell property and chemoresistance. PMID:28367098

  19. Fibroblast miR-210 overexpression is independently associated with clinical failure in Prostate Cancer – a multicenter (in situ hybridization) study

    PubMed Central

    Andersen, Sigve; Richardsen, Elin; Moi, Line; Donnem, Tom; Nordby, Yngve; Ness, Nora; Holman, Marte Eilertsen; Bremnes, Roy M.; Busund, Lill-Tove

    2016-01-01

    There is a need for better prognostication in prostate cancer (PC). “The micromanager of hypoxia”, microRNA-210 (miR-210) is directly linked to hypoxia, is overexpressed in PC and has been implied in tumor cell-fibroblast crosstalk. We investigated the prognostic impact of miR-210 in tumor cells and fibroblasts in PC. Tumor and stromal samples from a multicenter PC cohort of 535 prostatectomy patients were inserted into tissue microarrays. To investigate the expression of miR-210, we used in situ hybridization and two pathologists semiquantitatively scored its expression. Overexpression of miR-210 in tumor cells was not associated to biochemical failure-free survival (BFFS, p = 0.85) or clinical failure-free survival (CFFS, p = 0.09). However, overexpression of miR-210 in fibroblasts was significantly associated to a poor CFFS (p = 0.001), but not BFFS (p = 0.232). This feature was validated in both cohorts. Overexpression of miR-210 was independently associated with a reduced CFFS (HR = 2.76, CI 95% 1.25–6.09, p = 0.012). Overexpression of miR-210 in fibroblasts is independently associated with a poor CFFS. This highlights the importance of fibroblasts and cellular compartment crosstalk in PC. miR-210 is a candidate prognostic marker and potential therapeutic target in PC. PMID:27824162

  20. The Aged Microenvironment Influences Prostate Carcinogenesis

    DTIC Science & Technology

    2008-12-01

    staining for smooth- muscle cells (anti-smooth- muscle -actin) and fibroblast (anti-vimentin), we determined that 95% of the adjacent stromal cells...stained positive for smooth- muscle actin and only 5% stained positive for vimentin (Figure 2). Thus, the majority of the adjacent cellular stroma is...represented by smooth muscle cells and few fibroblasts. The latter finding is of importance inasmuch as the stromal population targeted for capture in

  1. LAPAROSCOPIC RESECTION OF GASTROINTESTINAL STROMAL TUMORS (GIST)

    PubMed Central

    LOUREIRO, Marcelo de Paula; de ALMEIDA, Rômulo Augusto Andrade; CLAUS, Christiano Marlo Paggi; BONIN, Eduardo Aimoré; CURY-FILHO,, Antônio Moris; DIMBARRE, Daniellson; da COSTA, Marco Aurélio Raeder; VITAL, Marcílio Lisboa

    2016-01-01

    Background Gastrointestinal mesenchymal or stromal tumors (GIST) are lesions originated on digestive tract walls, which are treated by surgical resection. Several laparoscopic techniques, from gastrectomies to segmental resections, have been used successfully. Aim Describe a single center experience on laparoscopic GIST resection. Method Charts of 15 operated patients were retrospectively reviewed. Thirteen had gastric lesions, of which ten were sub epithelial, ranging from 2-8 cm; and three were pure exofitic growing lesions. The remaining two patients had small bowel lesions. Surgical laparoscopic treatment consisted of two distal gastrectomies, 11 wedge gastric resections and two segmental enterectomies. Mechanical suture was used in the majority of patients except on six, which underwent resection and closure using manual absorbable sutures. There were no conversions to open technique. Results Mean operative time was 1h 29 min±92 (40-420 min). Average lenght of hospital stay was three days (2-6 days). There were no leaks, postoperative bleeding or need for reintervention. Mean postoperative follow-up was 38±17 months (6-60 months). Three patients underwent adjuvant Imatinib treatment, one for recurrence five months postoperatively and two for tumors with moderate risk for recurrence . Conclusion Laparoscopic GIST resection, not only for small lesions but also for tumors above 5 cm, is safe and acceptable technique. PMID:27120729

  2. Mesenchymal stromal cells in myeloid malignancies

    PubMed Central

    Geyh, Stefanie; Germing, Ulrich; Haas, Rainer

    2016-01-01

    Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are clonal myeloid disorders characterized by hematopoietic insufficiency. As MDS and AML are considered to originate from genetic and molecular defects of hematopoietic stem and progenitor cells (HSPC), the main focus of research in this field has focused on the characterization of these cells. Recently, the contribution of BM microenvironment to the pathogenesis of myeloid malignancies, in particular MDS and AML has gained more interest. This is based on a better understanding of its physiological role in the regulation of hematopoiesis. Additionally, it was demonstrated as a ‘proof of principle’ that genetic disruption of cells of the mesenchymal or osteoblastic lineage can induce MDS, MPS or AML in mice. In this review, we summarize the current knowledge about the contribution of the BM microenvironment, in particular mesenchymal stromal cells (MSC) to the pathogenesis of AML and MDS. Furthermore, potential models integrating the BM microenvironment into the pathophysiology of these myeloid disorders are discussed. Finally, strategies to therapeutically exploit this knowledge and to interfere with the crosstalk between clonal hematopoietic cells and altered stem cell niches are introduced. PMID:28090484

  3. Giant gastrointestinal stromal tumor of the stomach.

    PubMed

    Ionescu, Sever; Barbu, Emil; Ionescu, Călin; Costache, Adrian; Bălăşoiu, Maria

    2015-01-01

    Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal malignancies of the digestive tract. Gastric localization is the most frequent. The aim of this study is to evaluate the importance of immunohistochemical factors (CD117, CD34, α-SMA, vimentin, p53, Ki67) in diagnostic and size tumor and mitotic activity as prognostic factors for these tumors. We present the case of a 66-year-old male patient with a giant gastric GIST. Like in the vast majority, the symptomatology in this patient has long been faint, despite the large tumor size, and when it became manifest, it was nonspecific. Imagery wise, the computer tomography (CT) scan was the most efficient, showing the origin of the tumor from the greater curvature of the stomach, its dimensions, as well as the relations with the other abdominal viscera. Surgery in this patient was en-bloc, according to the principles of GIST. The histological aspect is characterized by a proliferation of spindle cells positive for CD117 and CD34. Despite complete microscopic resection, the size of the tumor (25×20×27 cm) and the mitotic activity (21÷5 mm2) remains important relapse factor.

  4. Targeted therapy of gastrointestinal stromal tumours

    PubMed Central

    Jakhetiya, Ashish; Garg, Pankaj Kumar; Prakash, Gaurav; Sharma, Jyoti; Pandey, Rambha; Pandey, Durgatosh

    2016-01-01

    Gastrointestinal stromal tumours (GISTs) are mesenchymal neoplasms originating in the gastrointestinal tract, usually in the stomach or the small intestine, and rarely elsewhere in the abdomen. The malignant potential of GISTs is variable ranging from small lesions with a benign behaviour to fatal sarcomas. The majority of the tumours stain positively for the CD-117 (KIT) and discovered on GIST-1 (DOG-1 or anoctamin 1) expression, and they are characterized by the presence of a driver kinase-activating mutation in either KIT or platelet-derived growth factor receptor α. Although surgery is the primary modality of treatment, almost half of the patients have disease recurrence following surgery, which highlights the need for an effective adjuvant therapy. Traditionally, GISTs are considered chemotherapy and radiotherapy resistant. With the advent of targeted therapy (tyrosine kinase inhibitors), there has been a paradigm shift in the management of GISTs in the last decade. We present a comprehensive review of targeted therapy in the management of GISTs. PMID:27231512

  5. Imatinib treatment for gastrointestinal stromal tumour (GIST).

    PubMed

    Lopes, Lisandro F; Bacchi, Carlos E

    2010-01-01

    Gastrointestinal stromal tumour (GIST) is the most common mesenchymal neoplasm of the gastrointestinal tract. GISTs are believed to originate from intersticial cells of Cajal (the pacemaker cells of the gastrointestinal tract) or related stem cells, and are characterized by KIT or platelet-derived growth factor receptor alpha (PDGFRA) activating mutations. The use of imatinib has revolutionized the management of GIST and altered its natural history, substantially improving survival time and delaying disease progression in many patients. The success of imatinib in controlling advanced GIST led to interest in the neoadjuvant and adjuvant use of the drug. The neoadjuvant (preoperative) use of imatinib is recommended to facilitate resection and avoid mutilating surgery by decreasing tumour size, and adjuvant therapy is indicated for patients at high risk of recurrence. The molecular characterization (genotyping) of GISTs has become an essential part of the routine management of the disease as KIT and PDGFRA mutation status predicts the likelihood of achieving response to imatinib. However, the vast majority of patients who initially responded to imatinib will develop tumour progression (secondary resistance). Secondary resistance is often related to secondary KIT or PDGFRA mutations that interfere with drug binding. Multiple novel tyrosine kinase inhibitors may be potentially useful for the treatment of imatinib-resistant GISTs as they interfere with KIT and PDGFRA receptors or with the downstream-signalling proteins.

  6. Imatinib treatment for gastrointestinal stromal tumour (GIST)

    PubMed Central

    Lopes, Lisandro F; Bacchi, Carlos E

    2010-01-01

    Abstract Gastrointestinal stromal tumour (GIST) is the most common mesenchymal neoplasm of the gastrointestinal tract. GISTs are believed to originate from intersticial cells of Cajal (the pacemaker cells of the gastrointestinal tract) or related stem cells, and are characterized by KIT or platelet-derived growth factor receptor alpha (PDGFRA) activating mutations. The use of imatinib has revolutionized the management of GIST and altered its natural history, substantially improving survival time and delaying disease progression in many patients. The success of imatinib in controlling advanced GIST led to interest in the neoadjuvant and adjuvant use of the drug. The neoadjuvant (preoperative) use of imatinib is recommended to facilitate resection and avoid mutilating surgery by decreasing tumour size, and adjuvant therapy is indicated for patients at high risk of recurrence. The molecular characterization (genotyping) of GISTs has become an essential part of the routine management of the disease as KIT and PDGFRA mutation status predicts the likelihood of achieving response to imatinib. However, the vast majority of patients who initially responded to imatinib will develop tumour progression (secondary resistance). Secondary resistance is often related to secondary KIT or PDGFRA mutations that interfere with drug binding. Multiple novel tyrosine kinase inhibitors may be potentially useful for the treatment of imatinib-resistant GISTs as they interfere with KIT and PDGFRA receptors or with the downstream-signalling proteins. PMID:19968734

  7. Gut Mesenchymal Stromal Cells in Immunity

    PubMed Central

    Messina, Valeria; Buccione, Carla; Marotta, Giulia; Ziccheddu, Giovanna; Signore, Michele; Mattia, Gianfranco; Puglisi, Rossella; Sacchetti, Benedetto; Biancone, Livia

    2017-01-01

    Mesenchymal stromal cells (MSCs), first found in bone marrow (BM), are the structural architects of all organs, participating in most biological functions. MSCs possess tissue-specific signatures that allow their discrimination according to their origin and location. Among their multiple functions, MSCs closely interact with immune cells, orchestrating their activity to maintain overall homeostasis. The phenotype of tissue MSCs residing in the bowel overlaps with myofibroblasts, lining the bottom walls of intestinal crypts (pericryptal) or interspersed within intestinal submucosa (intercryptal). In Crohn's disease, intestinal MSCs are tightly stacked in a chronic inflammatory milieu, which causes their enforced expression of Class II major histocompatibility complex (MHC). The absence of Class II MHC is a hallmark for immune-modulator and tolerogenic properties of normal MSCs and, vice versa, the expression of HLA-DR is peculiar to antigen presenting cells, that is, immune-activator cells. Interferon gamma (IFNγ) is responsible for induction of Class II MHC expression on intestinal MSCs. The reversal of myofibroblasts/MSCs from an immune-modulator to an activator phenotype in Crohn's disease results in the formation of a fibrotic tube subverting the intestinal structure. Epithelial metaplastic areas in this context can progress to dysplasia and cancer. PMID:28337224

  8. Intestinal gastrointestinal stromal tumor in a cat

    PubMed Central

    SUWA, Akihisa; SHIMODA, Tetsuya

    2017-01-01

    A 12-year-old, 3.6-kg, spayed female domestic shorthaired cat had a 2-month history of anorexia and weight loss. Abdominal ultrasonography and computed tomography revealed an exophytic mass originating from the jejunum with very poor central and poor peripheral contrast enhancement. On day 14, surgical resection of the jejunum and mass with 5-cm margins and an end-to-end anastomosis were performed. Histopathological examination revealed the mass was a transmural, invasive cancer showing exophytic growth and originating from the small intestinal muscle layer. Immunohistochemical analysis of tumor cells revealed diffuse positivity for KIT protein and negativity for desmin and S-100. The mass was diagnosed as a gastrointestinal stromal tumor (GIST). Ultrasonographic findings indicated the tumor probably metastasized to the liver and omentum, as seen in humans and dogs. The owner rejected further treatment at the last visit on day 192. To our knowledge, this is the first report of intestinal tumor and metastasis in feline GIST and its imaging features. PMID:28163271

  9. Transcriptional control of cardiac fibroblast plasticity.

    PubMed

    Lighthouse, Janet K; Small, Eric M

    2016-02-01

    Cardiac fibroblasts help maintain the normal architecture of the healthy heart and are responsible for scar formation and the healing response to pathological insults. Various genetic, biomechanical, or humoral factors stimulate fibroblasts to become contractile smooth muscle-like cells called myofibroblasts that secrete large amounts of extracellular matrix. Unfortunately, unchecked myofibroblast activation in heart disease leads to pathological fibrosis, which is a major risk factor for the development of cardiac arrhythmias and heart failure. A better understanding of the molecular mechanisms that control fibroblast plasticity and myofibroblast activation is essential to develop novel strategies to specifically target pathological cardiac fibrosis without disrupting the adaptive healing response. This review highlights the major transcriptional mediators of fibroblast origin and function in development and disease. The contribution of the fetal epicardial gene program will be discussed in the context of fibroblast origin in development and following injury, primarily focusing on Tcf21 and C/EBP. We will also highlight the major transcriptional regulatory axes that control fibroblast plasticity in the adult heart, including transforming growth factor β (TGFβ)/Smad signaling, the Rho/myocardin-related transcription factor (MRTF)/serum response factor (SRF) axis, and Calcineurin/transient receptor potential channel (TRP)/nuclear factor of activated T-Cell (NFAT) signaling. Finally, we will discuss recent strategies to divert the fibroblast transcriptional program in an effort to promote cardiomyocyte regeneration. This article is a part of a Special Issue entitled "Fibrosis and Myocardial Remodeling".

  10. Insufficient stromal support in MDS results from molecular and functional deficits of mesenchymal stromal cells.

    PubMed

    Geyh, S; Oz, S; Cadeddu, R-P; Fröbel, J; Brückner, B; Kündgen, A; Fenk, R; Bruns, I; Zilkens, C; Hermsen, D; Gattermann, N; Kobbe, G; Germing, U; Lyko, F; Haas, R; Schroeder, T

    2013-09-01

    Ineffective hematopoiesis is a major characteristic of myelodysplastic syndromes (MDS) causing relevant morbidity and mortality. Mesenchymal stromal cells (MSC) have been shown to physiologically support hematopoiesis, but their contribution to the pathogenesis of MDS remains elusive. We show that MSC from patients across all MDS subtypes (n=106) exhibit significantly reduced growth and proliferative capacities accompanied by premature replicative senescence. Osteogenic differentiation was significantly reduced in MDS-derived MSC, indicated by cytochemical stainings and reduced expressions of Osterix and Osteocalcin. This was associated with specific methylation patterns that clearly separated MDS-MSC from healthy controls and showed a strong enrichment for biological processes associated with cellular phenotypes and transcriptional regulation. Furthermore, in MDS-MSC, we detected altered expression of key molecules involved in the interaction with hematopoietic stem and progenitor cells (HSPC), in particular Osteopontin, Jagged1, Kit-ligand and Angiopoietin as well as several chemokines. Functionally, this translated into a significantly diminished ability of MDS-derived MSC to support CD34+ HSPC in long-term culture-initiating cell assays associated with a reduced cell cycle activity. Taken together, our comprehensive analysis shows that MSC from all MDS subtypes are structurally, epigenetically and functionally altered, which leads to impaired stromal support and seems to contribute to deficient hematopoiesis in MDS.

  11. Fibroblast migration in fibrin gel matrices.

    PubMed Central

    Brown, L. F.; Lanir, N.; McDonagh, J.; Tognazzi, K.; Dvorak, A. M.; Dvorak, H. F.

    1993-01-01

    In healing wounds and many solid tumors, locally increased microvascular permeability results in extravasation of fibrinogen and its extravascular coagulation to form a fibrin gel, with concomitant covalent cross-linking of fibrin by factor XIIIa. Subsequently, inflammatory cells, fibroblasts, and endothelial cells migrate into the gel and organize it into granulation tissue and later into mature collagenous connective tissue. To gain insight into some of the cell migration events associated with these processes, we developed a quantitative in vitro assay that permits the study of fibroblast migration in fibrin gels. Early passage human or rat fibroblasts were allowed to attach to tissue culture dishes and then were overlaid with a thin layer of fibrinogen that was clotted with thrombin. Fibroblasts began to migrate upwards into the fibrin within 24 hours and their numbers and the distance migrated were quantified over several days. The extent of fibroblast migration was affected importantly by the nature of the fibrin clot. Fibroblasts migrated optimally into gels prepared from fibrinogen at concentrations of -3 mg/ml; ie, near normal plasma fibrinogen levels. Migration was greatly enhanced by extensive cross-linking of the fibrin alpha-chains by factor XIIIa, as occurs when clotting takes place in vivo. When fibrinogen was clotted in Dulbecco's modified Eagle's medium, gamma-chains were cross-linked, but alpha-chain cross-linking was strikingly inhibited, and fibroblasts migrated poorly. Gels prepared from factor XIII-depleted fibrinogen exhibited neither alpha-nor gamma-chain cross-linking and did not support fibroblast migration. Further purification of fibrinogen by anion exchange high pressure liquid chromatography depleted fibrinogen of fibronectin, plasminogen, and other impurities; this purified fibrinogen clotted to form fibrin gels that supported reproducible fibroblast migration. Images Figure 1 Figure 2 Figure 4 Figure 6 PMID:8424460

  12. Molecular mechanism of fluoroquinolones modulation on corneal fibroblast motility.

    PubMed

    Chen, Tsan-Chi; Tsai, Tzu-Yun; Chang, Shu-Wen

    2016-04-01

    Topical fluoroquinolones are widely used to prevent ocular infections after ophthalmic surgery. However, they have been shown to affect the corneal cell motility, whose mechanism remains indefinite. The purpose of this study was to investigate how fluoroquinolones affect corneal stromal cell motility. Human corneal fibroblasts (HCFs) were incubated in ciprofloxacin (CIP), levofloxacin (LEV), or moxifloxacin (MOX) at 0, 10, 50, and 100 μg/ml for up to 3 days. Effect of CIP, LEV, or MOX on HCF migration was monitored using migration assay. HCF viability was determined by WST-1 assay. Expression of focal adhesion kinase (FAK), paxillin (PXN), and their phosphorylated forms were analyzed by immunoblotting. Binding affinity between FAK and PXN was determined by co-immunoprecipitation. Our results revealed that CIP and MOX, but not LEV, noticeably retarded HCF migration. HCF proliferation was significantly reduced by CIP (38.2%), LEV (29.5%), and MOX (21.3%), respectively (p = 0.002). CIP and MOX suppressed the phosphorylation of PXN at tyrosines (10.2 ± 4.3%, p < 0.001; 11.7 ± 2.4%, p < 0.001, respectively), including tyrosine 118 (33.3 ± 5.2%, p < 0.001; 34.0 ± 4.4%, p < 0.001, respectively). CIP and MOX diminished the binding affinity between FAK and PXN (8.2 ± 1.8%, p < 0.001; 9.0 ± 4.5%, p < 0.001, respectively). Nevertheless, tyrosine dephosphorylation and FAK dissociation of PXN were not found in LEV-treated HCFs. None of these fluoroquinolones affect phosphorylation of FAK-Y397. We conclude that CIP and MOX, but not LEV, might delay corneal fibroblast migration via interfering with recruitment of PXN to focal adhesions and dephosphorylation of PXN at the tyrosines.

  13. 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

  14. CREB modulates calcium signaling in cAMP-induced bone marrow stromal cells (BMSCs).

    PubMed

    Zhang, Linxia; Liu, Li; Thompson, Ryan; Chan, Christina

    2014-10-01

    Calcium signaling has a versatile role in many important cellular functions. Despite its importance, regulation of calcium signaling in bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) has not been explored extensively. Our previous study revealed that cyclic adenosine monophosphate (cAMP) enabled BMSCs to generate calcium signal upon stimulation by dopamine, KCl and glutamate. Concurrently, cAMP transiently activated the transcription factor cAMP response element binding protein (CREB) in BMSCs. Activity of CREB can be modulated by the calcium/calmodulin-dependent kinase signaling pathway, however, whether the calcium signaling observed in cAMP-induced BMSCs requires CREB has not been investigated. In an effort to uncover the role of CREB in the generation of calcium signaling in response to modulators such as dopamine and KCl, we knocked down CREB activity in BMSCs. Our study indicated that BMSCs, but not its close relative fibroblasts, are responsive to dopamine and KCl after cAMP treatment. Calcium signal elicited by dopamine depends, in part, on calcium influx whereas that elicited by KCl depends completely on calcium influx. Knock-down of CREB activity significantly reduced or abolished the cAMP-induced calcium response, and reintroducing a constitutively active CREB partially restored the calcium response.

  15. Mechanisms of tumor escape from immune system: role of mesenchymal stromal cells.

    PubMed

    Poggi, Alessandro; Musso, Alessandra; Dapino, Irene; Zocchi, Maria Raffaella

    2014-01-01

    Tumor microenvironment represents the site where the tumor tries to survive and escape from immune system-mediated recognition. Indeed, to proliferate tumor cells can divert the immune response inducing the generation of myeloid derived suppressor cells and regulatory T cells which can limit the efficiency of effector antitumor lymphocytes in eliminating neoplastic cells. Many components of the tumor microenvironment can serve as a double sword for the tumor and the host. Several types of fibroblast-like cells, which herein we define mesenchymal stromal cells (MSC), secrete extracellular matrix components and surrounding the tumor mass can limit the expansion of the tumor. On the other hand, MSC can interfere with the immune recognition of tumor cells producing immunoregulatory cytokines as transforming growth factor (TGF)ß, releasing soluble ligands of the activating receptors expressed on cytolytic effector cells as decoy molecules, affecting the correct interaction among lymphocytes and tumor cells. MSC can also serve as target for the same anti-tumor effector lymphocytes or simply impede the interaction between these lymphocytes and neoplastic cells. Thus, several evidences point out the role of MSC, both in epithelial solid tumors and hematological malignancies, in regulating tumor cell growth and immune response. Herein, we review these evidences and suggest that MSC can be a suitable target for a more efficient anti-tumor therapy.

  16. Isolation, characterization and cardiac differentiation of human thymus tissue derived mesenchymal stromal cells.

    PubMed

    Lin, Ze Bang; Qian, Bo; Yang, Yu Zhong; Zhou, Kai; Sun, Jian; Mo, Xu Ming; Wu, Kai Hong

    2015-07-01

    Mesenchymal stromal cells (MSCs) are promising candidate donor cells for replacement of cardiomyocyte loss during ischemia and in vitro generation of myocardial tissue. We have successfully isolated MSCs from the discarded neonatal thymus gland during cardiac surgery. The thymus MSCs were characterized by cell-surface antigen expression. These cells have high ability for proliferation and are able to differentiate into osteoblasts and adipocytes in vitro. For cardiac differentiation, the cells were divided into 3 groups: untreated control; 5-azacytidine group and sequential exposure to 5-azacytidine, bone morphogenetic protein 4, and basic fibroblast growth factor. Thymus MSCs showed a fibrolast-like morphology and some differentiated cells increased in size, formed a ball-like appearance over time and spontaneously contracting cells were observed in sequential exposure group. Immunostaining studies, cardiac specific genes/protein expression confirmed the cardiomyocyte phenotype of the differentiated cells. These results demonstrate that thymus MSCs can be a promising cellular source for cardiac cell therapy and tissue engineering.

  17. Quantitative microplate assay for studying mesenchymal stromal cell-induced neuropoiesis.

    PubMed

    Aizman, Irina; McGrogan, Michael; Case, Casey C

    2013-03-01

    Transplanting mesenchymal stromal cells (MSCs) or their derivatives in a neurodegenerative environment is believed to be beneficial because of the trophic support, migratory guidance, and neurogenic stimuli they provide. There is a growing need for in vitro models of mesenchymal-neural cell interactions to enable identification of mediators of the MSC activity and quantitative assessment of neuropoietic potency of MSC preparations. Here, we characterize a microplate-format coculture system in which primary embryonic rat cortex cells are directly cocultured with human MSCs on cell-derived extracellular matrix (ECM) in the absence of exogenous growth factors. In this system, expression levels of the rat neural stem/early progenitor marker nestin, as well as neuronal and astrocytic markers, directly depended on MSC dose, whereas an oligodendrogenic marker exhibited a biphasic MSC-dose response, as measured using species-specific quantitative reverse transcription-polymerase chain reaction in total cell lysates and confirmed using immunostaining. Both neural cell proliferation and differentiation contributed to the MSC-mediated neuropoiesis. ECM's heparan sulfate proteoglycans were essential for the growth of the nestin-positive cell population. Neutralization studies showed that MSC-derived fibroblast growth factor 2 was a major and diffusible inducer of rat nestin, whereas MSC-derived bone morphogenetic proteins (BMPs), particularly, BMP4, were astrogenesis mediators, predominantly acting in a coculture setting. This system enables analysis of multifactorial MSC-neural cell interactions and can be used for elucidating the neuropoietic potency of MSCs and their derivative preparations.

  18. TLR4 signalling in pulmonary stromal cells is critical for inflammation and immunity in the airways.

    PubMed

    Perros, Frederic; Lambrecht, Bart N; Hammad, Hamida

    2011-09-24

    Inflammation of the airways, which is often associated with life-threatening infection by Gram-negative bacteria or presence of endotoxin in the bioaerosol, is still a major cause of severe airway diseases. Moreover, inhaled endotoxin may play an important role in the development and progression of airway inflammation in asthma. Pathologic changes induced by endotoxin inhalation include bronchospasm, airflow obstruction, recruitment of inflammatory cells, injury of the alveolar epithelium, and disruption of pulmonary capillary integrity leading to protein rich fluid leak in the alveolar space. Mammalian Toll-like receptors (TLRs) are important signalling receptors in innate host defense. Among these receptors, TLR4 plays a critical role in the response to endotoxin. Lungs are a complex compartmentalized organ with separate barriers, namely the alveolar-capillary barrier, the microvascular endothelium, and the alveolar epithelium. An emerging theme in the field of lung immunology is that structural cells (SCs) of the airways such as epithelial cells (ECs), endothelial cells, fibroblasts and other stromal cells produce activating cytokines that determine the quantity and quality of the lung immune response. This review focuses on the role of TLR4 in the innate and adaptive immune functions of the pulmonary SCs.

  19. Developmental Reprogramming in Mesenchymal Stromal Cells of Human Subjects with Idiopathic Pulmonary Fibrosis

    PubMed Central

    Chanda, Diptiman; Kurundkar, Ashish; Rangarajan, Sunad; Locy, Morgan; Bernard, Karen; Sharma, Nirmal S.; Logsdon, Naomi J.; Liu, Hui; Crossman, David K.; Horowitz, Jeffrey C.; De Langhe, Stijn; Thannickal, Victor J.

    2016-01-01

    Cellular plasticity and de-differentiation are hallmarks of tissue/organ regenerative capacity in diverse species. Despite a more restricted capacity for regeneration, humans with age-related chronic diseases, such as cancer and fibrosis, show evidence of a recapitulation of developmental gene programs. We have previously identified a resident population of mesenchymal stromal cells (MSCs) in the terminal airways-alveoli by bronchoalveolar lavage (BAL) of human adult lungs. In this study, we characterized MSCs from BAL of patients with stable and progressive idiopathic pulmonary fibrosis (IPF), defined as <5% and ≥10% decline, respectively, in forced vital capacity over the preceding 6-month period. Gene expression profiles of MSCs from IPF subjects with progressive disease were enriched for genes regulating lung development. Most notably, genes regulating early tissue patterning and branching morphogenesis were differentially regulated. Network interactive modeling of a set of these genes indicated central roles for TGF-β and SHH signaling. Importantly, fibroblast growth factor-10 (FGF-10) was markedly suppressed in IPF subjects with progressive disease, and both TGF-β1 and SHH signaling were identified as critical mediators of this effect in MSCs. These findings support the concept of developmental gene re-activation in IPF, and FGF-10 deficiency as a potentially critical factor in disease progression. PMID:27869174

  20. Adult rat bone marrow stromal cells express genes associated with dopamine neurons

    SciTech Connect

    Kramer, Brian C.; Woodbury, Dale . E-mail: WOODBURYDL@AOL.COM; Black, Ira B.

    2006-05-19

    An intensive search is underway to identify candidates to replace the cells that degenerate in Parkinson's disease (PD). To date, no suitable substitute has been found. We have recently found that adult rat bone marrow stromal cells (MSCs) can be induced to assume a neuronal phenotype in vitro. These findings may have particular relevance to the treatment of PD. We now report that adult MSCs express multiple dopaminergic genes, suggesting that they are potential candidates for cell therapy. Using RT-PCR, we have examined families of genes that are associated with the development and/or survival of dopaminergic neurons. MSCs transcribe a variety of dopaminergic genes including patched and smoothened (components of the Shh receptor), Gli-1 (downstream mediator of Shh), and Otx-1, a gene associated with formation of the mesencephalon during development. Furthermore, Shh treatment elicits a 1.5-fold increase in DNA synthesis in cultured MSCs, suggesting the presence of a functional Shh receptor complex. We have also found that MSCs transcribe and translate Nurr-1, a nuclear receptor essential for the development of dopamine neurons. In addition, MSCs express a variety of growth factor receptors including the glycosyl-phosphatidylinositol-anchored ligand-binding subunit of the GDNF receptor, GFR{alpha}1, as well as fibroblast growth factor receptors one and four. The expression of genes that are associated with the development and survival of dopamine neurons suggests a potential role for these cells in the treatment of Parkinson's disease.

  1. 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.

  2. 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.

  3. Parenchymal and Stromal Cells Contribute to Pro-Inflammatory Myocardial Environment at Early Stages of Diabetes: Protective Role of Resveratrol

    PubMed Central

    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

    2016-01-01

    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. PMID:27854328

  4. Limited Functional Effects of Subacute Syngeneic Bone Marrow Stromal Cell Transplantation After Rat Spinal Cord Contusion Injury.

    PubMed

    Sandner, Beatrice; Ciatipis, Mareva; Motsch, Melanie; Soljanik, Irina; Weidner, Norbert; Blesch, Armin

    2016-01-01

    Cell transplantation might be one means to improve motor, sensory, or autonomic recovery after traumatic spinal cord injury (SCI). Among the different cell types evaluated to date, bone marrow stromal cells (BMSCs) have received considerable interest due to their potential neuroprotective properties. However, uncertainty exists whether the efficacy of BMSCs after intraspinal transplantation justifies an invasive procedure. In the present study, we analyzed the effect of syngeneic BMSC transplantation following a moderate to severe rat spinal cord injury. Adult Fischer 344 rats underwent a T9 contusion injury (200 kDy) followed by grafting of GFP-expressing BMSCs 3 days postinjury. Animals receiving a contusion injury without cellular grafts or an injury followed by grafts of syngeneic GFP-expressing fibroblasts served as control. Eight weeks posttransplantation, BMSC-grafted animals showed only a minor effect in one measure of sensorimotor recovery, no significant differences in tissue sparing, and no changes in the recovery of bladder function compared to both control groups in urodynamic measurements. Both cell types survived in the lesion site with fibroblasts displaying a larger graft volume. Thus, contrary to some reports using allogeneic or xenogeneic transplants, subacute intraparenchymal grafting of syngeneic BMSCs has only a minor effect on functional recovery.

  5. 30 CFR 56.9103 - Clearance on adjacent tracks.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Clearance on adjacent tracks. 56.9103 Section..., Hauling, and Dumping Traffic Safety § 56.9103 Clearance on adjacent tracks. Railcars shall not be left on side tracks unless clearance is provided for traffic on adjacent tracks....

  6. 30 CFR 57.9103 - Clearance on adjacent tracks.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Clearance on adjacent tracks. 57.9103 Section..., Hauling, and Dumping Traffic Safety § 57.9103 Clearance on adjacent tracks. Railcars shall not be left on side tracks unless clearance is provided for traffic on adjacent tracks....

  7. 49 CFR 236.404 - Signals at adjacent control points.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ..., INSPECTION, MAINTENANCE, AND REPAIR OF SIGNAL AND TRAIN CONTROL SYSTEMS, DEVICES, AND APPLIANCES Traffic Control Systems Standards § 236.404 Signals at adjacent control points. Signals at adjacent controlled... 49 Transportation 4 2011-10-01 2011-10-01 false Signals at adjacent control points....

  8. 49 CFR 236.404 - Signals at adjacent control points.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ..., INSPECTION, MAINTENANCE, AND REPAIR OF SIGNAL AND TRAIN CONTROL SYSTEMS, DEVICES, AND APPLIANCES Traffic Control Systems Standards § 236.404 Signals at adjacent control points. Signals at adjacent controlled... 49 Transportation 4 2010-10-01 2010-10-01 false Signals at adjacent control points....

  9. 33 CFR 80.1395 - Puget Sound and adjacent waters.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Puget Sound and adjacent waters... INTERNATIONAL NAVIGATION RULES COLREGS DEMARCATION LINES Thirteenth District § 80.1395 Puget Sound and adjacent waters. The 72 COLREGS shall apply on all waters of Puget Sound and adjacent waters, including Lake...

  10. 33 CFR 80.1395 - Puget Sound and adjacent waters.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 1 2014-07-01 2014-07-01 false Puget Sound and adjacent waters... INTERNATIONAL NAVIGATION RULES COLREGS DEMARCATION LINES Thirteenth District § 80.1395 Puget Sound and adjacent waters. The 72 COLREGS shall apply on all waters of Puget Sound and adjacent waters, including Lake...

  11. 33 CFR 80.1395 - Puget Sound and adjacent waters.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 1 2013-07-01 2013-07-01 false Puget Sound and adjacent waters... INTERNATIONAL NAVIGATION RULES COLREGS DEMARCATION LINES Thirteenth District § 80.1395 Puget Sound and adjacent waters. The 72 COLREGS shall apply on all waters of Puget Sound and adjacent waters, including Lake...

  12. 33 CFR 80.1395 - Puget Sound and adjacent waters.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 1 2011-07-01 2011-07-01 false Puget Sound and adjacent waters... INTERNATIONAL NAVIGATION RULES COLREGS DEMARCATION LINES Thirteenth District § 80.1395 Puget Sound and adjacent waters. The 72 COLREGS shall apply on all waters of Puget Sound and adjacent waters, including Lake...

  13. 33 CFR 80.1395 - Puget Sound and adjacent waters.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 1 2012-07-01 2012-07-01 false Puget Sound and adjacent waters... INTERNATIONAL NAVIGATION RULES COLREGS DEMARCATION LINES Thirteenth District § 80.1395 Puget Sound and adjacent waters. The 72 COLREGS shall apply on all waters of Puget Sound and adjacent waters, including Lake...

  14. Seismicity in Azerbaijan and Adjacent Caspian Sea

    SciTech Connect

    Panahi, Behrouz M.

    2006-03-23

    So far no general view on the geodynamic evolution of the Black Sea to the Caspian Sea region is elaborated. This is associated with the geological and structural complexities of the region revealed by geophysical, geochemical, petrologic, structural, and other studies. A clash of opinions on geodynamic conditions of the Caucasus region, sometimes mutually exclusive, can be explained by a simplified interpretation of the seismic data. In this paper I analyze available data on earthquake occurrences in Azerbaijan and the adjacent Caspian Sea region. The results of the analysis of macroseismic and instrumental data, seismic regime, and earthquake reoccurrence indicate that a level of seismicity in the region is moderate, and seismic event are concentrated in the shallow part of the lithosphere. Seismicity is mostly intra-plate, and spatial distribution of earthquake epicenters does not correlate with the plate boundaries.

  15. 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

  16. Antitumor activity of the MEK inhibitor trametinib on intestinal polyp formation in ApcΔ716 mice involves stromal COX-2

    PubMed Central

    Fujishita, Teruaki; Kajino-Sakamoto, Rie; Kojima, Yasushi; Taketo, Makoto Mark; Aoki, Masahiro

    2015-01-01

    Extracellular signal-regulated kinase is an MAPK that is most closely associated with cell proliferation, and the MEK/ERK signaling pathway is implicated in various human cancers. Although epidermal growth factor receptor, KRAS, and BRAF are considered major targets for colon cancer treatment, the precise roles of the MEK/ERK pathway, one of their major downstream effectors, during colon cancer development remain to be determined. Using ApcΔ716 mice, a mouse model of familial adenomatous polyposis and early-stage sporadic colon cancer formation, we show that MEK/ERK signaling is activated not only in adenoma epithelial cells, but also in tumor stromal cells including fibroblasts and vascular endothelial cells. Eight-week treatment of ApcΔ716 mice with trametinib, a small-molecule MEK inhibitor, significantly reduced the number of polyps in the large size class, accompanied by reduced angiogenesis and tumor cell proliferation. Trametinib treatment reduced the COX-2 level in ApcΔ716 tumors in vivo and in primary culture of intestinal fibroblasts in vitro. Antibody array analysis revealed that trametinib and the COX-2 inhibitor rofecoxib both reduced the level of CCL2, a chemokine known to be essential for the growth of Apc mutant polyps, in intestinal fibroblasts in vitro. Consistently, trametinib treatment reduced the Ccl2 mRNA level in ApcΔ716 tumors in vivo. These results suggest that MEK/ERK signaling plays key roles in intestinal adenoma formation in ApcΔ716 mice, at least in part, through COX-2 induction in tumor stromal cells. PMID:25855137

  17. 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

  18. Morphological Characterization of Organized Extracellular Matrix Deposition by Ascorbic Acid-Stimulated Human Corneal Fibroblasts

    PubMed Central

    Guo, Xiaoqing; Hutcheon, Audrey E. K.; Melotti, Suzanna A.; Zieske, James D.; Trinkaus-Randall, Vickery; Ruberti, Jeffrey W.

    2016-01-01

    Purpose To characterize the structure and morphology of extracellular matrix (ECM) synthesized by untransformed, cultured human corneal fibroblasts in long-term cultures. Methods Human corneal stromal keratocytes were expanded in transwell culture in the presence of fetal bovine serum and a stable derivative of Vitamin C. The cells were allowed to synthesize a fibrillar ECM for up to five weeks. Constructs were assessed via light (phase contrast and differential interference contrast) and transmission (standard and quick freeze/deep etch) microscopy. Results Electron micrographs revealed stratified constructs with multiple parallel layers of cells and an extracellular matrix comprising parallel arrays of small, polydisperse fibrils (27–51 nm) which often alternate in direction. Differential interference contrast images demonstrated oriented ECM fibril arrays parallel to the plane of the construct while quick-freeze deep etch micrographs showed the details of the matrix interaction with fibroblasts via arrays of membrane surface structures. Conclusions Human keratocytes, cultured in a stable Vitamin C derivative, are capable of assembling extracellular matrix which comprise parallel arrays of ECM fibrils. The resulting constructs, which are highly cellular, exhibit morphology similar to the developing mammalian stroma where organized matrix is derived. The appearance of arrays of structures on the cell membranes suggest a role in the local organization of synthesized ECM. This model could provide critical insight into the fundamental processes which govern the genesis of organized connective tissues such as the cornea and may provide a scaffolding suitable for tissue-engineering a biomimetic stroma. PMID:17724187

  19. Gastrointestinal stromal tumors (GISTs) and second malignancies

    PubMed Central

    Rodriquenz, Maria Grazia; Rossi, Sabrina; Ricci, Riccardo; Martini, Maurizio; Larocca, Mario; Dipasquale, Angelo; Quirino, Michela; Schinzari, Giovanni; Basso, Michele; D’Argento, Ettore; Strippoli, Antonia; Barone, Carlo; Cassano, Alessandra

    2016-01-01

    Abstract Several evidences showed that patients with gastrointestinal stromal tumors (GISTs) develop additional malignancies. However, thorough incidence of second tumors remains uncertain as the possibility of a common molecular pathogenesis. A retrospective series of 128 patients with histologically proven GIST treated at our institution was evaluated. Molecular analysis of KIT and PDGFR-α genes was performed in all patients. Following the involvement of KRAS mutation in many tumors’ pathogenesis, analysis of KRAS was performed in patients with also second neoplasms. Forty-six out of 128 GIST patients (35.9%) had a second neoplasm. Most second tumors (52%) raised from gastrointestinal tract and 19.6% from genitourinary tract. Benign neoplasms were also included (21.7%). Molecular analysis was available for 29/46 patients with a second tumor: wild-type GISTs (n. 5), exon 11 (n. 16), exon 13 (n. 1), exon 9 (n. 1) KIT mutations, exon 14 PDGFR-α mutation (n. 2) and exon 18 PDGFR-α mutation (n. 4). KIT exon 11 mutations were more frequent between patients who developed a second tumor (P = 0.0003). Mutational analysis of KRAS showed a wild-type sequence in all cases. In metachronous cases, the median time interval between GIST and second tumor was 21.5 months. The high frequency of second tumors suggests that an unknown common molecular mechanism might play a role, but it is not likely that KRAS is involved in this common pathogenesis. The short interval between GIST diagnosis and the onset of second neoplasms asks for a careful follow-up, particularly in the first 3 years after diagnosis. PMID:27661019

  20. 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.

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

    SciTech Connect

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

    2010-10-01

    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 Nglycosylated 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.

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

    SciTech Connect

    Sassoli, Chiara; Nosi, Daniele; Tani, Alessia; Chellini, Flaminia; Mazzanti, Benedetta; Quercioli, Franco; Zecchi-Orlandini, Sandra; Formigli, Lucia

    2014-05-01

    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 single 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.

  3. Tumeur stromale rectale: à propos d'une observation

    PubMed Central

    Rejab, Haitham; Kridis, Wala Ben; Ben Ameur, Hazem; Feki, Jihene; Frikha, Mounir; Beyrouti, Mohamed Issam

    2014-01-01

    Les tumeurs stromales gastro-intestinales sont des tumeurs mésenchymateuses peu fréquentes. Elles sont localisées préférentiellement eu niveau de l'estomac. La localisation rectale reste rare. A un nouveau cas de tumeur stromale du rectum ainsi qu'une bref revue de la littérature, on se propose d’étudier les particularités cliniques, radiologiques et thérapeutiques de cette entité rare. PMID:25120863

  4. Heterogeneity of multipotent mesenchymal stromal cells: from stromal cells to stem cells and vice versa.

    PubMed

    Dominici, Massimo; Paolucci, Paolo; Conte, Pierfranco; Horwitz, Edwin M

    2009-05-15

    Discovered more than 40 years ago, the biological features of multipotent mesenchymal stromal cells (MSC) were progressively compared first with hematopoietic stem cells (HSC) and, more recently, with embryonic stem cells (ESC). Although these comparisons have been crucial in helping to clarify their nature, there is now a robust amount of data indicating that MSC in vitro represent an independent and heterogeneous group of progenitors with distinct self-renewal properties and established differentiation potentials. However, research developments both in humans and animals have progressively revealed the limits that MSC may face in vivo. To recognize these issues and challenge MSC stemness may seem to be a step backward. Nevertheless, it might also represent the beginning of a phase in which the introduction of novel preclinical approaches could provide better characterization and standardization of the in vivo factors influencing cell engraftment and survival, allowing a more successful impact of mesenchymal progenitors in several clinical settings.

  5. 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

  6. Stromal cues regulate the pancreatic cancer epigenome and metabolome

    PubMed Central

    Sherman, Mara H.; Yu, Ruth T.; Tseng, Tiffany W.; Sousa, Cristovao M.; Liu, Sihao; Truitt, Morgan L.; He, Nanhai; Ding, Ning; Liddle, Christopher; Atkins, Annette R.; Leblanc, Mathias; Collisson, Eric A.; Asara, John M.; Kimmelman, Alec C.; Downes, Michael; Evans, Ronald M.

    2017-01-01

    A fibroinflammatory stromal reaction cooperates with oncogenic signaling to influence pancreatic ductal adenocarcinoma (PDAC) initiation, progression, and therapeutic outcome, yet the mechanistic underpinning of this crosstalk remains poorly understood. Here we show that stromal cues elicit an adaptive response in the cancer cell including the rapid mobilization of a transcriptional network implicated in accelerated growth, along with anabolic changes of an altered metabolome. The close overlap of stroma-induced changes in vitro with those previously shown to be regulated by oncogenic Kras in vivo suggests that oncogenic Kras signaling—a hallmark and key driver of PDAC—is contingent on stromal inputs. Mechanistically, stroma-activated cancer cells show widespread increases in histone acetylation at transcriptionally enhanced genes, implicating the PDAC epigenome as a presumptive point of convergence between these pathways and a potential therapeutic target. Notably, inhibition of the bromodomain and extraterminal (BET) family of epigenetic readers, and of Bromodomain-containing protein 2 (BRD2) in particular, blocks stroma-inducible transcriptional regulation in vitro and tumor progression in vivo. Our work suggests the existence of a molecular “AND-gate” such that tumor activation is the consequence of mutant Kras and stromal cues, providing insight into the role of the tumor microenvironment in the origin and treatment of Ras-driven tumors. PMID:28096419

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

    SciTech Connect

    Nakamura, Ryosuke; Kayamori, Kou; Oue, Erika; Sakamoto, Kei; Harada, Kiyoshi; Yamaguchi, Akira

    2015-03-20

    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 the 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

  8. Effect of coating Straumann Bone Ceramic with Emdogain on mesenchymal stromal cell hard tissue formation.

    PubMed

    Mrozik, Krzysztof Marek; Gronthos, Stan; Menicanin, Danijela; Marino, Victor; Bartold, P Mark

    2012-06-01

    Periodontal tissue engineering requires a suitable biocompatible scaffold, cells with regenerative capacity, and instructional molecules. In this study, we investigated the capacity of Straumann Bone Ceramic coated with Straumann Emdogain, a clinical preparation of enamel matrix protein (EMP), to aid in hard tissue formation by post-natal mesenchymal stromal cells (MSCs) including bone marrow stromal cells (BMSCs) and periodontal ligament fibroblasts (PDLFs). MSCs were isolated and ex vivo-expanded from human bone marrow and periodontal ligament and, in culture, allowed to attach to Bone Ceramic in the presence or absence of Emdogain. Gene expression of bone-related proteins was investigated by real time RT-PCR for 72 h, and ectopic bone formation was assessed histologically in subcutaneous implants of Bone Ceramic containing MSCs with or without Emdogain in NOD/SCID mice. Alkaline phosphatase activity was also assessed in vitro, in the presence or absence of Emdogain. Collagen-I mRNA was up-regulated in both MSC populations over the 72-h time course with Emdogain. Expression of BMP-2 and the osteogenic transcription factor Cbfa-1 showed early stimulation in both MSC types after 24 h. In contrast, expression of BMP-4 was consistently down-regulated in both MSC types with Emdogain. Up-regulation of osteopontin and periostin mRNA was restricted to BMSCs, while higher levels of bone sialoprotein-II were observed in PDLFs with Emdogain. Furthermore, alkaline phosphatase activity levels were reduced in both BMSCs and PDLFs in the presence of Emdogain. Very little evidence was found for ectopic bone formation following subcutaneous implantation of MSCs with Emdogain-coated or -uncoated Bone Ceramic in NOD/SCID mice. The early up-regulation of several important bone-related genes suggests that Emdogain may have a significant stimulatory effect in the commitment of mesenchymal cells to osteogenic differentiation in vitro. While Emdogain inhibited AP activity and appeared

  9. Cancer-associated fibroblasts in digestive tumors

    PubMed Central

    Huang, Lei; Xu, A-Man; Liu, Sha; Liu, Wei; Li, Tuan-Jie

    2014-01-01

    The significant influence of tumor stroma on malignant cells has been extensively investigated in this era of targeted therapy. The tumor microenvironment, as a dynamic system, is orchestrated by various cells including tumor vascular composing cells, inflammatory cells and fibroblasts. As a major and important component in tumor stroma, increasing evidence has shown that spindle-shaped cancer-associated fibroblasts (CAFs) are a significant modifier of cancer evolution, and promote tumorigenesis, tumor invasion and metastasis by stimulating angiogenesis, malignant cell survival, epithelial-mesenchymal transition (EMT) and proliferation via direct cell-to-cell contact or secretion of soluble factors in most digestive solid tumors. CAFs are thought to be activated, characterized by the expression of α-smooth muscle actin, fibroblast activated protein, fibroblast specific protein, vimentin, fibronectin, etc. They are hypothesized to originate from normal or aged fibroblasts, bone marrow-derived mesenchymal cells, or vascular endothelial cells. EMT may also be an important process generating CAFs, and most probably, CAFs may originate from multiple cells. A close link exists between EMT, tumor stem cells, and chemo-resistance of tumor cells, which is largely orchestrated by CAFs. CAFs significantly induce immunosuppression, and may be a prognostic marker in various malignancies. Targeted therapy toward CAFs has displayed promising anticancer efficacy, which further reinforces the necessity to explore the relationship between CAFs and their hosts. PMID:25548479

  10. Fibroblast TGF-Beta Signaling in Breast Development and Cancer

    DTIC Science & Technology

    2012-09-01

    determine the effect of loss of TGFβ signaling within stromal cells on mammary carcinoma development. Study Design: This will be accomplished using...elucidated that autonomous TGFb signaling within epithelial cells suppresses carcinoma formation early, but promotes cancer formation later. To...signaling within stromal cells on mammary carcinoma development. BODY The research findings of three years will be detailed according the

  11. Low-dose gamma-irradiation inhibits IL-6 secretion from human lung fibroblasts that promotes bronchial epithelial cell transformation by cigarette-smoke carcinogen.

    PubMed

    Chen, Wenshu; Xu, Xiuling; Bai, Lang; Padilla, Mabel T; Gott, Katherine M; Leng, Shuguang; Tellez, Carmen S; Wilder, Julie A; Belinsky, Steven A; Scott, Bobby R; Lin, Yong

    2012-07-01

    Despite decades of research in defining the health effects of low-dose (<100 mGy) ionizing photon radiation (LDR), the relationship between LDR and human cancer risk remains elusive. Because chemical carcinogens modify the tumor microenvironment, which is critical for cancer development, we investigated the role and mechanism of LDR in modulating the response of stromal cells to chemical carcinogen-induced lung cancer development. Secretion of proinflammatory cytokines such as interleukin-6 (IL-6), CXCL1 and CXCL5 from human lung fibroblasts was induced by cigarette-smoke carcinogen benzo[a]pyrene diol epoxide (BPDE), which was inhibited by a single dose of LDR. The activation of NF-κB, which is important for BPDE-induced IL-6 secretion, was also effectively suppressed by LDR. In addition, conditioned media from BPDE-treated fibroblasts activated STAT3 in the immortalized normal human bronchial epithelial cell line Beas-2B, which was blocked with an IL-6 neutralizing antibody. Conditioned medium from LDR-primed and BPDE-treated fibroblast showed diminished capacity in activating STAT3. Furthermore, IL-6 enhanced BPDE-induced Beas-2B cell transformation in vitro. These results suggest that LDR inhibits cigarette smoke-induced lung carcinogenesis by suppressing secretion of cytokines such as IL-6 from fibroblasts in lung tumor-prone microenvironment.

  12. Application of cell sheet technology to bone marrow stromal cell transplantation for rat brain infarct.

    PubMed

    Ito, Masaki; Shichinohe, Hideo; Houkin, Kiyohiro; Kuroda, Satoshi

    2017-02-01

    Bone marrow stromal cells (BMSC) transplantation enhances functional recovery after cerebral infarct, but the optimal delivery route is undetermined. This study was aimed to assess whether a novel cell-sheet technology non-invasively serves therapeutic benefits to ischemic stroke. First, the monolayered cell sheet was engineered by culturing rat BMSCs on a temperature-responsive dish. The cell sheet was analysed histologically and then transplanted onto the ipsilateral neocortex of rats subjected to permanent middle cerebral artery occlusion at 7 days after the insult. Their behaviours and histology were compared with those in the animals treated with direct injection of BMSCs or vehicle over 4 weeks post-transplantation. The cell sheet was 27.9 ± 8.0 μm thick and was composed of 9.8 ± 2.4 × 10(5) cells. Cell sheet transplantation significantly improved motor function when compared with the vehicle-injected animals. Histological analysis revealed that the BMSCs were densely distributed to the neocortex adjacent to the cerebral infarct and expressed neuronal phenotype in the cell sheet-transplanted animals. These findings were almost equal to those for the animals treated with direct BMSC injection. The attachment of the BMSC sheet to the brain surface did not induce reactive astrocytes in the adjacent neocortex, although direct injection of BMSCs profoundly induced reactive astrocytes around the injection site. These findings suggest that the BMSCs in cell sheets preserve their biological capacity of migration and neural differentiation. Cell-sheet technology may enhance functional recovery after ischaemic stroke, using a less invasive method. Copyright © 2014 John Wiley & Sons, Ltd.

  13. [Abberant methylation of p16, HIC1, N33 and GSTP1 genes in tumor epitelium and tumor-associated stromal cells of prostate cancer].

    PubMed

    Kekeeva, T V; Popova, O P; Shegaĭ, P V; Alekseev, B Ia; Adnreeva, Iu Iu; Zaletaev, D V; Nemtsova, M V

    2007-01-01

    The methylation status of four genes significant in prostate carcinogenesis p16, HIC1, N33 and GSTP1, were evaluated using quantitative methylationsensitive polymerase chain reaction. Tumor epithelia, tumor-associated stroma, normal epithelia, foci of PIN and benign prostate hyperplasia, and stroma adjacent to tumor tissues were isolated from whole-mount prostatectomy specimens of patients with localized prostate cancer by using laser capture microdissection. We found high levels of gene methylation in the tumor epithelium and tumor-associated stromal cells and some methylation in both hyperplastic epithelium and stromal cells in normal-appearing tissues located adjacent to tumors. Promoter methylation in the non-neoplastic cells of the prostate tumor microenvironment may play an important role in cancer development and progression. We examined the promoter methylation status of pl6, HIC1, N33 and GSTP1 in prostate biopsy fragments and prostate tissues after radical prostatectomy from patients with adenocarcinoma without laser capture microdissection. Methylation frequencies of all genes in tumor samples were considerably lower than frequencies in microdissected tumour samples (HIC1, 71 versus 89%; p16, 22 versus 78%; GSTP1, 32 versus 100%; N33, 20 versus 33%). The laser capture microdissection is required procedure in methylation studies taking into account multifocality and heterogenity of prostate cancer tissue.

  14. Development of enterohepatic fistula after embolization in ileal gastrointestinal stromal tumor: a case report.

    PubMed

    Lee, Yun Ho; Koo, Ja Seol; Jung, Chang Ho; Chung, Sang Yoon; Lee, Jae Joong; Kim, Seung Young; Hyun, Jong Jin; Jung, Sung Woo; Choung, Rok Seon; Lee, Sang Woo; Choi, Jai Hyun

    2013-11-21

    Gastrointestinal stromal tumor (GIST) is a rare mesenchymal tumor of the gastrointestinal tract that has been associated with the formation of fistulas to adjacent organs in few case reports. However, GIST with enterohepatic fistula has not been reported. Here we report the case of an enterohepatic fistula that occurred after embolization of a liver mass originating in the distal ileum. An 87-year-old woman was hospitalized for melena. On initial conventional endoscopy, a bleeding focus in the gastrointestinal tract was not found. Because of massive hematochezia, enteroscopy was performed through the anus. A protruding, ulcerative mass was found in the distal ileum that was suspected to be the source of the bleeding; a biopsy sample was taken. Electrocoagulation was not successful in controlling the bleeding; therefore, embolization was performed. After embolization, the patient developed a high fever and severe abdominal tenderness with rebound tenderness. Follow-up abdominopelvic computed tomography revealed an enterohepatic fistula between the liver and distal ileum. The fistula was treated surgically by segmental resection of the distal ileum and unlooping of the liver mass.

  15. A Rare Collision Tumour of Uterus- Squamous Cell Carcinoma and Endometrial Stromal Sarcoma

    PubMed Central

    Gupta, Bindiya; Pathre, Abhishek; Rajaram, Shalini; Goyal, Neerja

    2017-01-01

    Collision tumours are defined by co-existence of two tumours in the same or adjacent organs which are topographically and histologically distinct with minimal or no histological admixture. Collision tumours have been described in many organs notably thyroid, brain, adrenal gland, stomach and rarely uterus. Most of the collision tumours reported in uterus have two components; an adenocarcinoma and a sarcoma. We report a case of a 60-year-old lady who presented with complaints of post-menopausal bleeding. A cervical biopsy was performed which showed a non-keratinizing squamous cell carcinoma of cervix. Intra-operatively the uterus was bulky with a 6 cm x 5 cm polypoidal mass in the endometrial canal along with a 2 cm friable cervical growth. The fleshy uterine cavity mass was a spindle cell tumour with moderate pleomorphism and frequent mitosis. It was immunopositive for CD10 and negative for smooth muscle actin and cytokeratin 5/6. The other growth showed non-keratinizing squamous cell carcinoma which was positive for cytokeratin 5/6. Based on the distinct topographical location and limited areas of tumour admixture of the two tumours, a diagnosis of collision tumour of uterus comprising of endometrial stromal sarcoma (high grade) uterus and squamous cell carcinoma cervix was made. PMID:28384878

  16. Testosterone metabolism of fibroblasts grown from prostatic carcinoma, benign prostatic hyperplasia and skin fibroblasts

    SciTech Connect

    Schweikert, H.U.; Hein, H.J.; Romijn, J.C.; Schroeder, F.H.

    1982-02-01

    The metabolism of (1,2,6,7-3H)testosterone was assessed in fibroblast monolayers derived from tissue of 5 prostates with benign hyperplasia (BPH), 4 prostates with carcinoma (PC), and 3 biopsy samples of skin, 2 nongenital skin (NG) and 1 genital skin. The following metabolites could be identified: androstanedione androstenedione, dihydrotestosterone, androsterone, epiandrosterone, androstane-3 alpha, 17 beta-diol and androstane-3 beta, 17 beta-diol. Testosterone was metabolized much more rapidly in fibroblasts originating from prostatic tissue than in fibroblasts derived from NG. A significantly higher formation of 5 alpha-androstanes and 3 alpha-hydroxysteroids could be observed in fibroblasts from BPH as compared to PC. 17-ketosteroid formation exceeded 5 alpha-androstane formation in BPH, whereas 5 alpha-reduction was the predominant pathway in fibroblasts grown from PC and NG. Since testosterone metabolism in fibroblasts of prostatic origin therefore resembles in many aspects that in whole prostatic tissue, fibroblasts grown from prostatic tissues might be a valuable tool for further investigation of the pathogenesis of human BPH and PC.

  17. Targeting of metastasis-promoting tumor-associated fibroblasts and modulation of pancreatic tumor-associated stroma with a carboxymethylcellulose-docetaxel nanoparticle.

    PubMed

    Ernsting, Mark J; Hoang, Bryan; Lohse, Ines; Undzys, Elijus; Cao, Pinjiang; Do, Trevor; Gill, Bethany; Pintilie, Melania; Hedley, David; Li, Shyh-Dar

    2015-05-28

    Pancreatic ductal adenocarcinomas are characterized by the desmoplastic reaction, a dense fibrous stroma that has been shown to be supportive of tumor cell growth, invasion, and metastasis, and has been associated with resistance to chemotherapy and reduced patient survival. Here, we investigated targeted depletion of stroma for pancreatic cancer therapy via taxane nanoparticles. Cellax-DTX polymer is a conjugate of docetaxel (DTX), polyethylene glycol (PEG), and acetylated carboxymethylcellulose, a construct which condenses into well-defined 120nm particles in an aqueous solution, and is suitable for intravenous injection. We examined Cellax-DTX treatment effects in highly stromal primary patient-derived pancreatic cancer xenografts and in a metastatic PAN02 mouse model of pancreatic cancer, focusing on specific cellular interactions in the stroma, pancreatic tumor growth and metastasis. Greater than 90% of Cellax-DTX particles accumulate in smooth muscle actin (SMA) positive cancer-associated fibroblasts which results in long-term depletion of this stromal cell population, an effect not observed with Nab-paclitaxel (Nab-PTX). The reduction in stromal density leads to a >10-fold increase in tumor perfusion, reduced tumor weight and a reduction in metastasis. Consentingly, Cellax-DTX treatment increased survival when compared to treatment with gemcitabine or Nab-PTX in a metastatic PAN02 mouse model. Cellax-DTX nanoparticles interact with the tumor-associated stroma, selectively interacting with and depleting SMA positive cells and macrophage, effects of which are associated with significant changes in tumor progression and metastasis.

  18. Divergent fibroblast growth factor signaling pathways in lung fibroblast subsets: where do we go from here?

    PubMed

    Ruiz-Camp, Jordi; Morty, Rory E

    2015-10-15

    Lung fibroblasts play a key role in postnatal lung development, namely, the formation of the alveolar gas exchange units, through the process of secondary septation. Although evidence initially highlighted roles for fibroblasts in the production and remodeling of the lung extracellular matrix, more recent studies have described the presence of different fibroblast subsets in the developing lung. These subsets include myofibroblasts and lipofibroblasts and their precursors. These cells are believed to play different roles in alveologenesis and are localized to different regions of the developing septa. The precise roles played by these different fibroblast subsets remain unclear. Understanding the signaling pathways that control the discrete functions of these fibroblast subsets would help to clarify the roles and the regulation of lung fibroblasts during lung development. Here, we critically evaluate a recent report that described divergent fibroblast growth factor (FGF) signaling pathways in two different subsets of lung fibroblasts that express different levels of green fluorescent protein (GFP) driven by the platelet-derived growth factor receptor-α promoter. The GFP expression was used as a surrogate for lipofibroblasts (GFP(low)) and myofibroblasts (GFP(high)). It was suggested that Fgf10/Fgf1 and Fgf18/Fgfr3 autocrine pathways may be operative in GFP(low) and GFP(high) cells, respectively, and that these pathways might regulate the proliferation and migration of different fibroblast subsets during alveologenesis. These observations lay important groundwork for the further exploration of FGF function during normal lung development, as well as in aberrant lung development associated with bronchopulmonary dysplasia.

  19. Trisomy 8 restricted to cultured fibroblasts.

    PubMed Central

    Niss, R; Passarge, E

    1976-01-01

    In the course of re-examing cultured fibroblasts stored in liquid nitrogen from a patient with developmental retardation, solitary left kidney, and Wilms tumour, a cell line trisomic for chromosome 8 was found. Trisomy 8 was restricted to fibroblasts in the first 22 subcultures and was absent in later passages as well as in lymphocytes. A familial pericentric inversion of chromosome 2 was observed in three generations including the propositus but was though to be unrelated to the clinical problem. Multiple spontaneous chromosomal rearrangements were seen in several late subcultures. Images PMID:180293

  20. [Morphological fibroblastic changes in cytomegalovirus infection].

    PubMed

    Parkhomenko, Iu V; Solnyshkova, T G; Tishkivich, O A; Shakhgil'dian, V I; Nikonova, E A

    2006-01-01

    Cytomegalovirus (CMV) infection is widely spread among population. While immunocompetent patients suffer rarely from this virus, it can lead to a lethal outcome in immunocompromised patients. An electron microscopic study has detected fibroblastic morphological changes of a definite cytodestructive character. The nuclei of some fibroblasts have chromatine condensation. A clear zone arising due to vacuolization near this inclusion may reflect nuclear rearrangement leading to further CMV metamorphosis of the cell. This metamorphosis is characteristic of the changes developing in the cells of different parenchymatous organs.

  1. Mesenchymal Stem Cell Transition to Tumor-Associated Fibroblasts Contributes to Fibrovascular Network Expansion and Tumor Progression

    PubMed Central

    Sasser, A. Kate; Watson, Keri; Klopp, Ann; Hall, Brett; Andreeff, Michael; Marini, Frank

    2009-01-01

    Background Tumor associated fibroblasts (TAF), are essential for tumor progression providing both a functional and structural supportive environment. TAF, known as activated fibroblasts, have an established biological impact on tumorigenesis as matrix synthesizing or matrix degrading cells, contractile cells, and even blood vessel associated cells. The production of growth factors, cytokines, chemokines, matrix-degrading enzymes, and immunomodulatory mechanisms by these cells augment tumor progression by providing a suitable environment. There are several suggested origins of the TAF including tissue-resident, circulating, and epithelial-to-mesenchymal-transitioned cells. Methodology/Principal Findings We provide evidence that TAF are derived from mesenchymal stem cells (MSC) that acquire a TAF phenotype following exposure to or systemic recruitment into adenocarcinoma xenograft models including breast, pancreatic, and ovarian. We define the MSC derived TAF in a xenograft ovarian carcinoma model by the immunohistochemical presence of 1) fibroblast specific protein and fibroblast activated protein; 2) markers phenotypically associated with aggressiveness, including tenascin-c, thrombospondin-1, and stromelysin-1; 3) production of pro-tumorigenic growth factors including hepatocyte growth factor, epidermal growth factor, and interleukin-6; and 4) factors indicative of vascularization, including alpha-smooth muscle actin, desmin, and vascular endothelial growth factor. We demonstrate that under long-term tumor conditioning in vitro, MSC express TAF–like proteins. Additionally, human MSC but not murine MSC stimulated tumor growth primarily through the paracrine production of secreted IL6. Conclusions/Significance Our results suggest the dependence of in vitro Skov-3 tumor cell proliferation is due to the presence of tumor-stimulated MSC secreted IL6. The subsequent TAF phenotype arises from the MSC which ultimately promotes tumor growth through the contribution of

  2. Evaluation of adipose-derived stromal vascular fraction from the lateral tailhead, inguinal region, and mesentery of horses

    PubMed Central

    Metcalf, Garrett L.; McClure, Scott R.; Hostetter, Jesse M.; Martinez, Rudy F.; Wang, Chong

    2016-01-01

    Use of mesenchymal stem cells (MSCs) found in the stromal vascular fraction (SVF) of equine adipose tissue has promising applications for regenerative therapies. The most commonly used source of equine adipose tissue is the subcutaneous tailhead. The objective of this study was to compare 3 adipose depot sites in horses and determine the viability and cellular yield, capillary density, gene expression for selected markers, and colony-forming unit fibroblasts (CFU-Fs) in adipose tissue taken from these sites. Adipose tissue was excised from the area lateral to the tailhead, the inguinal region, and the small colon mesentery of 6 horses. Lipoaspirate was also collected from the area lateral to the tailhead. Stromal vascular fraction (SVF) was prepared in duplicate from the 3 different adipose tissue depots. The total nucleated and dead cell counts was determined manually using a hemocytometer and percent viability was calculated. Mass and volume of adipose were determined in order to calculate density and factor-VIII immunohistochemical staining was used to determine vascular density in the excisional adipose tissue samples from each horse. Quantitative polymerase chain reaction (qPCR) was used to quantify gene expression for selected cellular markers from each site. There were significant differences in viability, yield of nucleated cells/gram of adipose tissue, vascular density, gene expression, and CFU-Fs among adipose depots. Adipose from the mesentery yielded the highest number of nucleated cells/gram of tissue and the highest vascular density and percentage of CFU-Fs. In the horse, both the anatomical site of collection and the method of tissue collection significantly impact the yield and composition of cells in the SVF. Further study is needed to assess whether one adipose source is superior for harvesting mesenchymal stem cells (MSCs) and whether the differences among sources are clinically relevant for in-vivo treatment of musculoskeletal injuries in horses

  3. Evaluation of adipose-derived stromal vascular fraction from the lateral tailhead, inguinal region, and mesentery of horses.

    PubMed

    Metcalf, Garrett L; McClure, Scott R; Hostetter, Jesse M; Martinez, Rudy F; Wang, Chong

    2016-10-01

    Use of mesenchymal stem cells (MSCs) found in the stromal vascular fraction (SVF) of equine adipose tissue has promising applications for regenerative therapies. The most commonly used source of equine adipose tissue is the subcutaneous tailhead. The objective of this study was to compare 3 adipose depot sites in horses and determine the viability and cellular yield, capillary density, gene expression for selected markers, and colony-forming unit fibroblasts (CFU-Fs) in adipose tissue taken from these sites. Adipose tissue was excised from the area lateral to the tailhead, the inguinal region, and the small colon mesentery of 6 horses. Lipoaspirate was also collected from the area lateral to the tailhead. Stromal vascular fraction (SVF) was prepared in duplicate from the 3 different adipose tissue depots. The total nucleated and dead cell counts was determined manually using a hemocytometer and percent viability was calculated. Mass and volume of adipose were determined in order to calculate density and factor-VIII immunohistochemical staining was used to determine vascular density in the excisional adipose tissue samples from each horse. Quantitative polymerase chain reaction (qPCR) was used to quantify gene expression for selected cellular markers from each site. There were significant differences in viability, yield of nucleated cells/gram of adipose tissue, vascular density, gene expression, and CFU-Fs among adipose depots. Adipose from the mesentery yielded the highest number of nucleated cells/gram of tissue and the highest vascular density and percentage of CFU-Fs. In the horse, both the anatomical site of collection and the method of tissue collection significantly impact the yield and composition of cells in the SVF. Further study is needed to assess whether one adipose source is superior for harvesting mesenchymal stem cells (MSCs) and whether the differences among sources are clinically relevant for in-vivo treatment of musculoskeletal injuries in horses.

  4. Stem and stromal cell reconstitution of lethally irradiated mice following transplantation of hematopoietic tissue from donors of various ages

    SciTech Connect

    Schmidt, C.M.; Doran, G.A.; Crouse, D.A.; Sharp, J.G.

    1987-10-01

    If the limited life span of hematopoietic tissues in vitro is due to a finite proliferative capacity of individual stem cells, one might expect tissues of young donors to possess a greater proliferative capacity and to contain a larger population of primitive stem cells than those of older donors. To test this hypothesis, we used 12- and 8-day spleen colony formation (CFU-s) to assay more and less primitive stem cell subpopulations of three murine hematopoietic tissues: fetal liver (FL) and weanling (WBM) and adult (ABM) bone marrow. Subsequently, the same assays and a stromal cell assay were performed on the bone marrow from groups of lethally irradiated mice reconstituted with these tissues. Comparison of the CFU-s content of the donor tissues revealed that FL contained a significantly greater proportion of primitive stem cells as evidenced by a (Day 12):(Day 8) CFU-s ratio of 3.0 +/- 1.0 as compared to 0.9 +/- 0.1 for WBM and ABM. In addition, at 21 weeks post-transplantation the CFU-s/femur values of the FL reconstituted group were significantly greater than those of the ABM and WBM reconstituted groups. These results suggest that fetal hematopoietic tissue contains a greater proportion of primitive stem cells and has a greater proliferative potential than hematopoietic tissue from older donors. No differences were seen in stromal cell reconstitution of the three experimental groups. In all cases, assayable fibroblast colony forming cells (CFU-f) remained at 20-40% of control values, even at 21 weeks postreconstitution.

  5. Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer

    PubMed Central

    Handly-Santana, Abram; Biffi, Giulia; Elyada, Ela; Ponz-Sarvise, Mariano; Lee, Eun Jung; Chio, Iok In Christine; Tiriac, Hervé; Feig, Christine; Kultti, Anne; Crawford, James M.; Clevers, Hans; Park, Youngkyu

    2017-01-01

    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. We recapitulated this finding in co-cultures of murine PSCs and PDA organoids, and demonstrated that organoid-activated CAFs produced desmoplastic stroma. The co-cultures showed cooperative interactions and revealed another distinct subpopulation of CAFs, located more distantly from neoplastic cells, which lacked elevated αSMA expression and instead secreted IL6 and additional inflammatory mediators. These findings were corroborated in mouse and human PDA tissue, providing direct evidence for CAF heterogeneity in PDA tumor biology with implications for disease etiology and therapeutic development. PMID:28232471

  6. Direct contacts with colon cancer cells regulate the differentiation of bone marrow mesenchymal stem cells into tumor associated fibroblasts.

    PubMed

    Peng, Yanan; Li, Zongwei; Yang, Peng; Newton, Ian P; Ren, Hua; Zhang, Lichao; Wu, Haili; Li, Zhuoyu

    2014-08-15

    Tumor-stroma interactions are referred to as essential events in tumor progression. There has been growing attention that bone marrow-derived mesenchymal stem cells (BMSCs) can travel to tumor stroma, where they differentiate into tumor-associated fibroblast (TAF)-like cells, a predominant tumor-promoting stromal cell. However, little is definitively known about the contributors for this transition. Here, using an in vitro direct co-culture model of colon cancer cells and BMSCs, we identify that colon cancer cells can induce adjoining BMSCs to exhibit the typical characteristic of TAFs, with increased expression of α-smooth muscle actin (α-SMA). Importantly, the present data also reveals that activated Notch signaling mediates transformation of BMSCs to TAFs through the downstream TGF-β/Smad signaling pathway.

  7. Cancer-associated fibroblast-secreted CXCL16 attracts monocytes to promote stroma activation in triple-negative breast cancers

    PubMed Central

    Allaoui, Roni; Bergenfelz, Caroline; Mohlin, Sofie; Hagerling, Catharina; Salari, Kiarash; Werb, Zena; Anderson, Robin L.; Ethier, Stephen P.; Jirström, Karin; Påhlman, Sven; Bexell, Daniel; Tahin, Balázs; Johansson, Martin E.; Larsson, Christer; Leandersson, Karin

    2016-01-01

    Triple-negative (TN) breast cancers (ER−PR−HER2−) are highly metastatic and associated with poor prognosis. Within this subtype, invasive, stroma-rich tumours with infiltration of inflammatory cells are even more aggressive. The effect of myeloid cells on reactive stroma formation in TN breast cancer is largely unknown. Here, we show that primary human monocytes have a survival advantage, proliferate in vivo and develop into immunosuppressive myeloid cells expressing the myeloid-derived suppressor cell marker S100A9 only in a TN breast cancer environment. This results in activation of cancer-associated fibroblasts and expression of CXCL16, which we show to be a monocyte chemoattractant. We propose that this migratory feedback loop amplifies the formation of a reactive stroma, contributing to the aggressive phenotype of TN breast tumours. These insights could help select more suitable therapies targeting the stromal component of these tumours, and could aid prediction of drug resistance. PMID:27725631

  8. No evidence of clonal somatic genetic alterations in cancer-associated fibroblasts from human breast and ovarian carcinomas.

    PubMed

    Qiu, Wen; Hu, Min; Sridhar, Anita; Opeskin, Ken; Fox, Stephen; Shipitsin, Michail; Trivett, Melanie; Thompson, Ella R; Ramakrishna, Manasa; Gorringe, Kylie L; Polyak, Kornelia; Haviv, Izhak; Campbell, Ian G

    2008-05-01

    There is increasing evidence showing that the stromal cells surrounding cancer epithelial cells, rather than being passive bystanders, might have a role in modifying tumor outgrowth. The molecular basis of this aspect of carcinoma etiology is controversial. Some studies have reported a high frequency of genetic aberrations in carcinoma-associated fibroblasts (CAFs), whereas other studies have reported very low or zero mutation rates. Resolution of this contentious area is of critical importance in terms of understanding both the basic biology of cancer as well as the potential clinical implications of CAF somatic alterations. We undertook genome-wide copy number and loss of heterozygosity (LOH) analysis of CAFs derived from breast and ovarian carcinomas using a 500K SNP array platform. Our data show conclusively that LOH and copy number alterations are extremely rare in CAFs and cannot be the basis of the carcinoma-promoting phenotypes of breast and ovarian CAFs.

  9. No evidence of clonal somatic genetic alterations in cancer-associated fibroblasts from human breast and ovarian carcinomas

    PubMed Central

    Qiu, Wen; Hu, Min; Sridhar, Anita; Opeskin, Ken; Fox, Stephen; Shipitsin, Michail; Trivett, Melanie; Thompson, Ella R; Ramakrishna, Manasa; Gorringe, Kylie L; Polyak, Kornelia; Haviv, Izhak; Campbell, Ian G

    2013-01-01

    There is increasing evidence showing that the stromal cells surrounding cancer epithelial cells, rather than being passive bystanders, might have a role in modifying tumor outgrowth. The molecular basis of this aspect of carcinoma etiology is controversial. Some studies have reported a high frequency of genetic aberrations in carcinoma-associated fibroblasts (CAFs), whereas other studies have reported very low or zero mutation rates. Resolution of this contentious area is of critical importance in terms of understanding both the basic biology of cancer as well as the potential clinical implications of CAF somatic alterations. We undertook genome-wide copy number and loss of heterozygosity (LOH) analysis of CAFs derived from breast and ovarian carcinomas using a 500K SNP array platform. Our data show conclusively that LOH and copy number alterations are extremely rare in CAFs and cannot be the basis of the carcinoma-promoting phenotypes of breast and ovarian CAFs. PMID:18408720

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

    SciTech Connect

    Eppley, B.L.; Connolly, D.T.; Winkelmann, T.; Sadove, A.M.; Heuvelman, D.; Feder, J. )

    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 in 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.

  11. Learning Non-Adjacent Regularities at Age 0 ; 7

    ERIC Educational Resources Information Center

    Gervain, Judit; Werker, Janet F.

    2013-01-01

    One important mechanism suggested to underlie the acquisition of grammar is rule learning. Indeed, infants aged 0 ; 7 are able to learn rules based on simple identity relations (adjacent repetitions, ABB: "wo fe fe" and non-adjacent repetitions, ABA: "wo fe wo", respectively; Marcus et al., 1999). One unexplored issue is…

  12. View of north side from exterior stairs of adjacent building, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    View of north side from exterior stairs of adjacent building, bottom cut off by fringed buildings, view facing south-southwest - U.S. Naval Base, Pearl Harbor, Industrial X-Ray Building, Off Sixth Street, adjacent to and south of Facility No. 11, Pearl City, Honolulu County, HI

  13. A Study of the Pronunciation of Words Containing Adjacent Vowels.

    ERIC Educational Resources Information Center

    Greif, Ivo P.

    To determine the usefulness of the commonly taught phonics rule, "only pronounce the first vowel in words that contain adjacent vowels" (the VV rule, with the first "v" pronounced with the long vowel sound), two new studies applied it to words with adjacent vowels in several lists and dictionaries. The first study analyzed words containing…

  14. 47 CFR 90.221 - Adjacent channel power limits.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 47 Telecommunication 5 2013-10-01 2013-10-01 false Adjacent channel power limits. 90.221 Section 90.221 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) SAFETY AND SPECIAL RADIO SERVICES PRIVATE LAND MOBILE RADIO SERVICES General Technical Standards § 90.221 Adjacent channel...

  15. 47 CFR 90.221 - Adjacent channel power limits.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 5 2014-10-01 2014-10-01 false Adjacent channel power limits. 90.221 Section 90.221 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) SAFETY AND SPECIAL RADIO SERVICES PRIVATE LAND MOBILE RADIO SERVICES General Technical Standards § 90.221 Adjacent channel...

  16. 30 CFR 57.14109 - Unguarded conveyors with adjacent travelways.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Unguarded conveyors with adjacent travelways... conveyors with adjacent travelways. Unguarded conveyors next to travelways shall be equipped with— (a) Emergency stop devices which are located so that a person falling on or against the conveyor can...

  17. 30 CFR 56.14109 - Unguarded conveyors with adjacent travelways.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Unguarded conveyors with adjacent travelways... MINES Machinery and Equipment Safety Devices and Maintenance Requirements § 56.14109 Unguarded conveyors with adjacent travelways. Unguarded conveyors next to the travelways shall be equipped with—...

  18. 30 CFR 56.14109 - Unguarded conveyors with adjacent travelways.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Unguarded conveyors with adjacent travelways... MINES Machinery and Equipment Safety Devices and Maintenance Requirements § 56.14109 Unguarded conveyors with adjacent travelways. Unguarded conveyors next to the travelways shall be equipped with—...

  19. 30 CFR 57.14109 - Unguarded conveyors with adjacent travelways.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Unguarded conveyors with adjacent travelways... conveyors with adjacent travelways. Unguarded conveyors next to travelways shall be equipped with— (a) Emergency stop devices which are located so that a person falling on or against the conveyor can...

  20. 30 CFR 56.14109 - Unguarded conveyors with adjacent travelways.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Unguarded conveyors with adjacent travelways... MINES Machinery and Equipment Safety Devices and Maintenance Requirements § 56.14109 Unguarded conveyors with adjacent travelways. Unguarded conveyors next to the travelways shall be equipped with—...

  1. 30 CFR 57.14109 - Unguarded conveyors with adjacent travelways.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Unguarded conveyors with adjacent travelways... conveyors with adjacent travelways. Unguarded conveyors next to travelways shall be equipped with— (a) Emergency stop devices which are located so that a person falling on or against the conveyor can...

  2. 30 CFR 56.14109 - Unguarded conveyors with adjacent travelways.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Unguarded conveyors with adjacent travelways... MINES Machinery and Equipment Safety Devices and Maintenance Requirements § 56.14109 Unguarded conveyors with adjacent travelways. Unguarded conveyors next to the travelways shall be equipped with—...

  3. 30 CFR 56.14109 - Unguarded conveyors with adjacent travelways.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Unguarded conveyors with adjacent travelways... MINES Machinery and Equipment Safety Devices and Maintenance Requirements § 56.14109 Unguarded conveyors with adjacent travelways. Unguarded conveyors next to the travelways shall be equipped with—...

  4. 30 CFR 57.14109 - Unguarded conveyors with adjacent travelways.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Unguarded conveyors with adjacent travelways... conveyors with adjacent travelways. Unguarded conveyors next to travelways shall be equipped with— (a) Emergency stop devices which are located so that a person falling on or against the conveyor can...

  5. 30 CFR 57.14109 - Unguarded conveyors with adjacent travelways.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Unguarded conveyors with adjacent travelways... conveyors with adjacent travelways. Unguarded conveyors next to travelways shall be equipped with— (a) Emergency stop devices which are located so that a person falling on or against the conveyor can...

  6. Distinct phenotype and therapeutic potential of gingival fibroblasts.

    PubMed

    Häkkinen, Lari; Larjava, Hannu; Fournier, Benjamin P J

    2014-09-01

    Gingiva of the oral mucosa provides a practical source to isolate fibroblasts for therapeutic purposes because the tissue is easily accessible, tissue discards are common during routine clinical procedures and wound healing after biopsy is fast and results in complete wound regeneration with very little morbidity or scarring. In addition, gingival fibroblasts have unique traits, including neural crest origin, distinct gene expression and synthetic properties and potent immunomodulatory functions. These characteristics may provide advantages for certain therapeutic approaches over other more commonly used cells, including skin fibroblasts, both in intraoral and extra-oral sites. However, identity and phenotype of gingival fibroblasts, like other fibroblasts, are still not completely understood. Gingival fibroblasts are phenotypically heterogeneous, and these…fibroblast subpopulations may play different roles in tissue maintenance, regeneration and pathologies. The purpose of this review is to summarize what is currently known about gingival fibroblasts, their distinct potential for tissue regeneration and their potential therapeutic uses in the future.

  7. Unique transcriptome, pathways, and networks in the human endometrial fibroblast response to progesterone in endometriosis.

    PubMed

    Aghajanova, L; Tatsumi, K; Horcajadas, J A; Zamah, A M; Esteban, F J; Herndon, C N; Conti, M; Giudice, L C

    2011-04-01

    Eutopic endometrium in endometriosis has molecular evidence of resistance to progesterone (P(4)) and activation of the PKA pathway in the stromal compartment. To investigate global and temporal responses of eutopic endometrium to P(4), we compared early (6-h), intermediate (48-h), and late (14-Day) transcriptomes, signaling pathways, and networks of human endometrial stromal fibroblasts (hESF) from women with endometriosis (hESF(endo)) with hESF from women without endometriosis (hESF(nonendo)). Endometrial biopsy samples were obtained from subjects with and without mild peritoneal endometriosis (n = 4 per group), and hESF were isolated and treated with P(4) (1 μM) plus estradiol (E(2)) (10 nM), E(2) alone (10 nM), or vehicle for up to 14 days. Total RNA was subjected to microarray analysis using a Gene 1.0 ST (Affymetrix) platform and analyzed by using bioinformatic algorithms, and data were validated by quantitative real-time PCR and ELISA. Results revealed unique kinetic expression of specific genes and unique pathways, distinct biological and molecular processes, and signaling pathways and networks during the early, intermediate, and late responses to P(4) in both hESF(nonendo) and hESF(endo), although a blunted response to P(4) was observed in the latter. The normal response of hESF to P(4) involves a tightly regulated kinetic cascade involving key components in the P(4) receptor and MAPK signaling pathways that results in inhibition of E(2)-mediated proliferation and eventual differentiation to the decidual phenotype, but this was not established in the hESF(endo) early response to P(4). The abnormal response of this cell type to P(4) may contribute to compromised embryonic implantation and infertility in women with endometriosis.

  8. ANT2-defective fibroblasts exhibit normal mitochondrial bioenergetics

    PubMed Central

    Prabhu, Dolly; Goldstein, Amy C.; El-Khoury, Riyad; Rak, Malgorzata; Edmunds, Lia; Rustin, Pierre; Vockley, Jerry; Schiff, Manuel

    2015-01-01

    Adenine nucleotide translocase 2 (ANT2) transports glycolytic ATP across the inner mitochondrial membrane. Patients with ANT2 deletion were recently reported. We aimed at characterizing mitochondrial functions in ANT2-defective fibroblasts. In spite of ANT2 expression in fibroblasts, we observed no difference between ANT2-defective and control fibroblasts for mitochondrial respiration, respiratory chain activities, mitochondrial membrane potential and intracellular ATP levels. This indicates that ANT2 insufficiency does not alter fibroblast basal mitochondrial bioenergetics. PMID:26000237

  9. Electric Cell-Substrate Impedance Sensing (ECIS) with Microelectrode Arrays for Investigation of Cancer Cell – Fibroblasts Interaction

    PubMed Central

    Tran, Trong Binh; Baek, Changyoon; Min, Junhong

    2016-01-01

    The tumor microenvironment, including stromal cells, surrounding blood vessels and extracellular matrix components, has been defined as a crucial factor that influences the proliferation, drug-resistance, invasion and metastasis of malignant epithelial cells. Among other factors, the communications and interaction between cancer cells and stromal cells have been reported to play pivotal roles in cancer promotion and progression. To investigate these relationships, an on-chip co-culture model was developed to study the cellular interaction between A549—human lung carcinoma cells and MRC-5—human lung epithelial cells in both normal proliferation and treatment conditions. In brief, a co-culture device consisting of 2 individual fluidic chambers in parallel, which were separated by a 100 μm fence was utilized for cell patterning. Microelectrodes arrays were installed within each chamber including electrodes at various distances away from the confrontation line for the electrochemical impedimetric sensing assessment of cell-to-cell influence. After the fence was removed and cell-to-cell contact occurred, by evaluating the impedance signal responses representing cell condition and behavior, both direct and indirect cell-to-cell interactions through conditioned media were investigated. The impact of specific distances that lead to different influences of fibroblast cells on cancer cells in the co-culture environment was also defined. PMID:27088611

  10. Electric Cell-Substrate Impedance Sensing (ECIS) with Microelectrode Arrays for Investigation of Cancer Cell-Fibroblasts Interaction.

    PubMed

    Tran, Trong Binh; Baek, Changyoon; Min, Junhong

    2016-01-01

    The tumor microenvironment, including stromal cells, surrounding blood vessels and extracellular matrix components, has been defined as a crucial factor that influences the proliferation, drug-resistance, invasion and metastasis of malignant epithelial cells. Among other factors, the communications and interaction between cancer cells and stromal cells have been reported to play pivotal roles in cancer promotion and progression. To investigate these relationships, an on-chip co-culture model was developed to study the cellular interaction between A549-human lung carcinoma cells and MRC-5-human lung epithelial cells in both normal proliferation and treatment conditions. In brief, a co-culture device consisting of 2 individual fluidic chambers in parallel, which were separated by a 100 μm fence was utilized for cell patterning. Microelectrodes arrays were installed within each chamber including electrodes at various distances away from the confrontation line for the electrochemical impedimetric sensing assessment of cell-to-cell influence. After the fence was removed and cell-to-cell contact occurred, by evaluating the impedance signal responses representing cell condition and behavior, both direct and indirect cell-to-cell interactions through conditioned media were investigated. The impact of specific distances that lead to different influences of fibroblast cells on cancer cells in the co-culture environment was also defined.

  11. Bone marrow regeneration promoted by biophysically sorted osteoprogenitors from mesenchymal stromal cells.

    PubMed

    Poon, Zhiyong; Lee, Wong Cheng; Guan, Guofeng; Nyan, Lin Myint; Lim, Chwee Teck; Han, Jongyoon; Van Vliet, Krystyn J

    2015-01-01

    Human tissue repair deficiencies can be supplemented through strategies to isolate, expand in vitro, and reimplant regenerative cells that supplant damaged cells or stimulate endogenous repair mechanisms. Bone marrow-derived mesenchymal stromal cells (MSCs), a subset of which is described as mesenchymal stem cells, are leading candidates for cell-mediated bone repair and wound healing, with hundreds of ongoing clinical trials worldwide. An outstanding key challenge for successful clinical translation of MSCs is the capacity to produce large quantities of cells in vitro with uniform and relevant therapeutic properties. By leveraging biophysical traits of MSC subpopulations and label-free microfluidic cell sorting, we hypothesized and experimentally verified that MSCs of large diameter within expanded MSC cultures were osteoprogenitors that exhibited significantly greater efficacy over other MSC subpopulations in bone marrow repair. Systemic administration of osteoprogenitor MSCs significantly improved survival rates (>80%) as compared with other MSC subpopulations (0%) for preclinical murine bone marrow injury models. Osteoprogenitor MSCs also exerted potent therapeutic effects as "cell factories" that secreted high levels of regenerative factors such as interleukin-6 (IL-6), interleukin-8 (IL-8), vascular endothelial growth factor A, bone morphogenetic protein 2, epidermal growth factor, fibroblast growth factor 1, and angiopoietin-1; this resulted in increased cell proliferation, vessel formation, and reduced apoptosis in bone marrow. This MSC subpopulation mediated rescue of damaged marrow tissue via restoration of the hematopoiesis-supporting stroma, as well as subsequent hematopoiesis. Together, the capabilities described herein for label-freeisolation of regenerative osteoprogenitor MSCs can markedly improve the efficacy of MSC-based therapies.

  12. Adipose-derived stromal cells inhibit prostate cancer cell proliferation inducing apoptosis

    SciTech Connect

    Takahara, Kiyoshi; Ii, Masaaki; Inamoto, Teruo; Komura, Kazumasa; Ibuki, Naokazu; Minami, Koichiro; Uehara, Hirofumi; Hirano, Hajime; Nomi, Hayahito; Kiyama, Satoshi; Asahi, Michio; Azuma, Haruhito

    2014-04-18

    Highlights: • AdSC transplantation exhibits inhibitory effect on tumor progressions of PCa cells. • AdSC-induced PCa cell apoptosis may occur via the TGF-β signaling pathway. • High expression of the TGF-β1 gene in AdSCs. - Abstract: Mesenchymal stem cells (MSCs) have generated a great deal of interest in the field of regenerative medicine. Adipose-derived stromal cells (AdSCs) are known to exhibit extensive proliferation potential and can undergo multilineage differentiation, sharing similar characteristics to bone marrow-derived MSCs. However, as the effect of AdSCs on tumor growth has not been studied sufficiently, we assessed the degree to which AdSCs affect the proliferation of prostate cancer (PCa) cell. Human AdSCs exerted an inhibitory effect on the proliferation of androgen-responsive (LNCaP) and androgen-nonresponsive (PC3) human PCa cells, while normal human dermal fibroblasts (NHDFs) did not, and in fact promoted PCa cell proliferation to a degree. Moreover, AdSCs induced apoptosis of LNCaP cells and PC3 cells, activating the caspase3/7 signaling pathway. cDNA microarray analysis suggested that AdSC-induced apoptosis in both LNCaP and PC3 cells was related to the TGF-β signaling pathway. Consistent with our in vitro observations, local transplantation of AdSCs delayed the growth of tumors derived from both LNCaP- and PC3-xenografts in immunodeficient mice. This is the first preclinical study to have directly demonstrated that AdSC-induced PCa cell apoptosis may occur via the TGF-β signaling pathway, irrespective of androgen-responsiveness. Since autologous AdSCs can be easily isolated from adipose tissue without any ethical concerns, we suggest that therapy with these cells could be a novel approach for patients with PCa.

  13. Isolation and Characterization of Multipotential Mesenchymal Stromal Cells from Congenital Pseudoarthrosis of the Tibia: Case Report.

    PubMed

    Diaz-Solano, Dylana; Wittig, Olga; Mota, Jose D; Cardier, Jose E

    2015-10-01

    Congenital pseudoarthrosis of the tibia (CPT) is an uncommon disease whose etiology and pathogenesis is unknown. Several evidences suggest that decreased osteogenic capacities, impaired local vascularization, and microenvironment alterations may play a role in the pathogenesis of CPT. Additionally, it is not clear if the pathogenesis of this disease is related to the absence of cells with osteogenic capacity of differentiation. In this work, a two-year-old patient diagnosed with CPT underwent an orthopedic surgery to promote bone union in a pseudoarthrosis lesion. Tissue from CPT lesion was excised, and histological evaluation and tissue culture were performed. Histologic analysis of the soft CPT lesion showed the presence of highly cellular fibrous tissue, vascularization, and abundant extracellular matrix. Fusiform cells of mesenchymal appearance were observed but osteoblasts, osteoclasts, chondrocytes, and adipose cells were not found. There was no evidence of osteogenesis. CPT tissue cultured as explants showed, after one month of culture, evidence of osteogenesis, chondrogenesis, and adipogenesis. Cells isolated from explants of CPT tissue showed a fibroblast-like morphology and expressed the mesenchymal stromal cell (MSC) markers: CD105, CD73, and CD90 (CPT-MSC). Functional analysis showed that CPT-MSC differentiate, in vitro, into osteogenic, chondrogenic, and adipocytic cells. CPT-MSC expressed osteocalcin and agrecan. CPT-MSC produced collagen in the presence of ascorbic acid. MSC from BM of normal individuals were used as control. In summary, our results indicate that CPT tissue contains MSC with osteogenic capacity of differentiation. It is possible that CPT microenvironment may contribute to impair the osteogenic capacity of differentiation of CPT-MSC.

  14. High abundance of CD271+ multipotential stromal cells (MSCs) in intramedullary cavities of long bones

    PubMed Central

    Cox, George; Boxall, Sally A.; Giannoudis, Peter V.; Buckley, Conor T.; Roshdy, Tarek; Churchman, Sarah M.; McGonagle, Dennis; Jones, Elena

    2012-01-01

    Aspiration of iliac crest bone marrow (ICBM) remains the most frequent technique used in harvesting multipotential stromal cells (MSCs) for bone regeneration. Although this tissue type is easily accessed by a surgeon, it has a low frequency of MSCs, which is significant given the high cell numbers required for bone regeneration strategies. Lipoaspirates possess higher MSC frequencies, albeit cells with a differentiation profile less suited to orthopaedic interventions. Intra-medullary cavities of long bones have previously been shown to harbour MSCs in animals, however evaluation of their frequency, differentiation capacity and phenotype in humans had not previously been performed. Long bone fatty bone marrow (LBFBM) was collected prior to harvesting bone graft. Basic cellular compositions of donor-matched LBFBM and ICBM aspirates, including the numbers of CD34+ hematopoietic stem cells and CD31+ endothelial cells, were similar. MSCs were enumerated using colony-forming-unit-fibroblast assays and flow cytometry for the presence of a resident LBFBM CD45−/low CD271+ MSC population and revealed a trend for higher MSC numbers (average 5 fold, n = 6) per millilitre of LBFBM compared to donor-matched ICBM. Functional characteristics of resident MSCs, including their growth rates, differentiation potentials and surface phenotypes (CD73+CD105+CD90+) before and after culture-amplification, were similar. Enhanced numbers of MSCs could be recovered following brief enzymatic treatment of solid fragments of LBFBM. Our findings therefore reveal that the intramedullary cavity of the human femur is a depot of MSCs, which, although closely associated with fat, have a differentiation profile equivalent to ICBM. This anatomical site is frequently accessed by the orthopaedic/trauma surgeon and aspiration of the intramedullary cavity represents a ‘low-tech’ method of harvesting potentially large numbers of MSCs for regenerative therapies and research. This article is part of a

  15. Modes of Antigen Presentation by Lymph Node Stromal Cells and Their Immunological Implications.

    PubMed

    Hirosue, Sachiko; Dubrot, Juan

    2015-01-01

    Antigen presentation is no longer the exclusive domain of cells of hematopoietic origin. Recent works have demonstrated that lymph node stromal cell (LNSC) populations, such as fibroblastic reticular cells, lymphatic and blood endothelial cells, not only provide a scaffold for lymphocyte interactions but also exhibit active immunomodulatory roles that are critical to mounting and resolving effective immune responses. Importantly, LNSCs possess the ability to present antigens and establish antigen-specific interactions with T cells. One example is the expression of peripheral tissue antigens, which are presented on major histocompatibility complex (MHC)-I molecules with tolerogenic consequences on T cells. Additionally, exogenous antigens, including self and tumor antigens, can be processed and presented on MHC-I complexes, which result in dysfunctional activation of antigen-specific CD8(+) T cells. While MHC-I is widely expressed on cells of both hematopoietic and non-hematopoietic origins, antigen presentation via MHC-II is more precisely regulated. Nevertheless, LNSCs are capable of endogenously expressing, or alternatively, acquiring MHC-II molecules. Transfer of antigen between LNSC and dendritic cells in both directions has been recently suggested to promote tolerogenic roles of LNSCs on the CD4(+) T cell compartment. Thus, antigen presentation by LNSCs is thought to be a mechanism that promotes the maintenance of peripheral tolerance as well as generates a pool of diverse antigen-experienced T cells for protective immunity. This review aims to integrate the current and emerging literature to highlight the importance of LNSCs in immune responses, and emphasize their role in antigen trafficking, retention, and presentation.

  16. Stromal remodeling by the BET bromodomain inhibitor JQ1 suppresses the progression of human pancreatic cancer

    PubMed Central

    Yamamoto, Keisuke; Tateishi, Keisuke; Kudo, Yotaro; Hoshikawa, Mayumi; Tanaka, Mariko; Nakatsuka, Takuma; Fujiwara, Hiroaki; Miyabayashi, Koji; Takahashi, Ryota; Tanaka, Yasuo; Ijichi, Hideaki; Nakai, Yousuke; Isayama, Hiroyuki; Morishita, Yasuyuki; Aoki, Taku; Sakamoto, Yoshihiro; Hasegawa, Kiyoshi; Kokudo, Norihiro; Fukayama, Masashi; Koike, Kazuhiko

    2016-01-01

    Inhibitors of bromodomain and extraterminal domain (BET) proteins, a family of chromatin reader proteins, have therapeutic efficacy against various malignancies. However, the detailed mechanisms underlying the anti-tumor effects in distinct tumor types remain elusive. Here, we show a novel antitumor mechanism of BET inhibition in pancreatic ductal adenocarcinoma (PDAC). We found that JQ1, a BET inhibitor, decreased desmoplastic stroma, a hallmark of PDAC, and suppressed the growth of patient-derived tumor xenografts (PDX) of PDACs. In vivo antitumor effects of JQ1 were not always associated with the JQ1 sensitivity of respective PDAC cells, and were rather dependent on the suppression of tumor-promoting activity in cancer-associated fibroblasts (CAFs). JQ1 inhibited Hedgehog and TGF-β pathways as potent regulators of CAF activation and suppressed the expression of α-SMA, extracellular matrix, cytokines, and growth factors in human primary CAFs. Consistently, conditioned media (CM) from CAFs promoted the proliferation of PDAC cells along with the activation of ERK, AKT, and STAT3 pathways, though these effects were suppressed when CM from JQ1-treated CAFs was used. Mechanistically, chromatin immunoprecipitation experiments revealed that JQ1 reduced TGF-β–dependent gene expression by disrupting the recruitment of the transcriptional machinery containing BET proteins. Finally, combination therapy with gemcitabine plus JQ1 showed greater efficacy than gemcitabine monotherapy against PDAC in vivo. Thus, our results reveal BET proteins as the critical regulators of CAF-activation and also provide evidence that stromal remodeling by epigenetic modulators can be a novel therapeutic option for PDAC. PMID:27528027

  17. Gene expression in normal-appearing tissue adjacent to prostate cancers are predictive of clinical outcome: evidence for a biologically meaningful field effect

    PubMed Central

    Magi-Galluzzi, Cristina; Maddala, Tara; Falzarano, Sara Moscovita; Cherbavaz, Diana B.; Zhang, Nan; Knezevic, Dejan; Febbo, Phillip G.; Lee, Mark; Lawrence, Hugh Jeffrey; Klein, Eric A.

    2016-01-01

    Purpose We evaluated gene expression in histologically normal-appearing tissue (NT) adjacent to prostate tumor in radical prostatectomy specimens, assessing for biological significance based on prediction of clinical recurrence (cR - metastatic disease or local recurrence). Results A total of 410 evaluable patients had paired tumor and NT. Fortysix genes, representing diverse biological pathways (androgen signaling, stromal response, stress response, cellular organization, proliferation, cell adhesion, and chromatin remodeling) were associated with cR in NT (FDR < 20%), of which 39 concordantly predicted cR in tumor (FDR < 20%). Overall GPS and its stromal response and androgen-signaling gene group components also significantly predicted time to cR in NT (RM-corrected HR/20 units = 1.25; 95% CI: 1.01-1.56; P = 0.024). Experimental Design Expression of 732 genes was measured by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) separately in tumor and adjacent NT specimens from 127 patients with and 374 without cR following radical prostatectomy for T1/T2 prostate cancer. A 17-gene expression signature (Genomic Prostate Score [GPS]), previously validated to predict aggressive prostate cancer when measured in tumor tissue, was also assessed using pre-specified genes and algorithms. Analysis used Cox proportional hazards models, Storey's false discovery rate (FDR) control, and regression to the mean (RM) correction. Conclusions Gene expression profiles, including GPS, from NT adjacent to tumor can predict prostate cancer outcome. These findings suggest that there is a biologically significant field effect in primary prostate cancer that is a marker for aggressive disease. PMID:27121323

  18. Gaining myocytes or losing fibroblasts: Challenges in cardiac fibroblast reprogramming for infarct repair.

    PubMed

    Nagalingam, Raghu S; Safi, Hamza A; Czubryt, Michael P

    2016-04-01

    Unlike most somatic tissues, the heart possesses a very limited inherent ability to repair itself following damage. Attempts to therapeutically salvage the myocardium after infarction, either by sparing surviving myocytes or by injection of exogenous cells of varied provenance, have met with limited success. Cardiac fibroblasts are numerous, resistant to hypoxia, and amenable to phenotype reprogramming to cardiomyocytes - a potential panacea to an intractable problem. However, the long-term effects of mass conversion of fibroblasts are as-yet unknown. Since fibroblasts play key roles in normal cardiac function, treating these cells as a ready source of replacements for myocytes may have the effect of swapping one problem for another. This review briefly examines the roles of cardiac fibroblasts, recaps the strides made so far in their reprogramming to cardiomyocytes both in vitro and in vivo, and discusses the potential ramifications of large-scale cellular identity swapping. While such therapy offers great promise, the potential repercussions require consideration and careful study.

  19. Activated human mesenchymal stem/stromal cells suppress metastatic features of MDA-MB-231 cells by secreting IFN-β.

    PubMed

    Yoon, N; Park, M S; Shigemoto, T; Peltier, G; Lee, R H

    2016-04-14

    Our recent study showed that human mesenchymal stem/stromal cells (hMSCs) are activated to express tumor necrosis factor (TNF)-α-related apoptosis-inducing ligand (TRAIL) by exposure to TNF-α and these activated hMSCs effectively induce apoptosis in triple-negative breast cancer MDA-MB-231 (MDA) cells in vitro and in vivo. Here, we further demonstrated that activated hMSCs not only induced apoptosis of MDA cells but also reduced metastatic features in MDA cells. These activated hMSC-exposed MDA cells showed reduced tumorigenicity and suppressed formation of lung metastasis when implanted in the mammary fat pad. Surprisingly, the activated hMSC-exposed MDA cells increased TRAIL expression, resulting in apoptosis in MDA cells. Interestingly, upregulation of TRAIL in MDA cells was mediated by interferon-beta (IFN-β) secreted from activated hMSCs. Furthermore, IFN-β in activated hMSCs was induced by RNA and DNA released from apoptotic MDA cells in absent in melanoma 2 (AIM2) and IFN induced with helicase C domain 1 (IFIH1)-dependent manners. These observations were only seen in the TRAIL-sensitive breast cancer cell lines but not in the TRAIL-resistant breast cancer cell lines. Consistent with these results, Kaplan-Meier survival analysis also showed that lack of innate sensors detecting DNA or RNA is strongly associated with poor survival in estrogen receptor-negative breast cancer patients. In addition, cancer-associated fibroblasts (CAF) isolated from a breast cancer patient were also able to express TRAIL and IFN-β upon DNA and RNA stimulation. Therefore, our results suggest that the crosstalk between TRAIL-sensitive cancer cells and stromal cells creates a tumor-suppressive microenvironment and further provide a novel therapeutic approach to target stromal cells within cancer microenvironment for TRAIL sensitive cancer treatment.

  20. Co-culture of mesenchymal-like stromal cells derived from human foreskin permits long term propagation and differentiation of human embryonic stem cells.

    PubMed

    Mamidi, Murali Krishna; Pal, Rajarshi; Mori, Nor Azah Binti; Arumugam, Greetha; Thrichelvam, Saratha Thevi; Noor, Puteri J; Abdullah, Hj Mohamad Farouk; Gupta, Pawan Kumar; Das, Anjan Kumar; Zakaria, Zubaidah; Bhonde, Ramesh

    2011-05-01

    Among the different parameters governing the successful derivation and expansion of human embryonic stem cells (hESC), feeder layers play the most important role. Human feeders in form of human mesenchymal stromal cells (hMSCs) and human foreskin fibroblasts (HFFs) lay the foundation for eradication of animal-derived hESC culture system. In this study we explored the potential of human foreskin derived mesenchymal like stromal cells (HF-MSCs) to support self renewal and pluripotency of hESC. The MSCs isolated from human foreskin were found to be resistant to standard concentrations and duration of mitomycin-C treatment. Growth pattern, gene profiling (Oct-4, Nanog, Sox-2, Rex-1), cytoskeletal protein expression (vimentin, nestin) and tri-lineage differentiation potential into adipocytes, chondrocytes and osteocytes confirmed their mesenchymal stromal cell status. Further, the HF-MSCs were positive for CD105, CD166, CD73, CD44, CD90, SSEA-4, and negative for CD34, CD45, HLA-DR cell-surface markers and were found to exhibit BM-MSC-like characteristics. hESC lines co-cultured with HF-MSC feeders showed expression of expected pluripotent transcription factors Oct-4, Nanog, Sox-2, GDF-3, Rex-1, STELLAR, ABCG2, Dppa5, hTERT; surface markers SSEA-4, TRA-1-81 and maintained their cytogenetic stability during long term passaging. These novel feeders also improved the formation of embryoid bodies (EBs) from hESC which produced cell types representing three germ layers. This culture system has the potential to aid the development of clinical-grade hESCs for regenerative medicine and drug screening. Further, we envisage foreskin can serve as a valuable source of alternative MSCs for specific therapeutic applications.

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

    PubMed

    Nakamura, Ryosuke; Kayamori, Kou; Oue, Erika; Sakamoto, Kei; Harada, Kiyoshi; Yamaguchi, Akira

    2015-03-20

    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 the 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.

  2. Stromal Modulators of TGF-β in Cancer

    PubMed Central

    Costanza, Brunella; Umelo, Ijeoma Adaku; Bellier, Justine; Castronovo, Vincent; Turtoi, Andrei

    2017-01-01

    Transforming growth factor-β (TGF-β) is an intriguing cytokine exhibiting dual activities in malignant disease. It is an important mediator of cancer invasion, metastasis and angiogenesis, on the one hand, while it exhibits anti-tumor functions on the other hand. Elucidating the precise role of TGF-β in malignant development and progression requires a better understanding of the molecular mechanisms involved in its tumor suppressor to tumor promoter switch. One important aspect of TGF-β function is its interaction with proteins within the tumor microenvironment. Several stromal proteins have the natural ability to interact and modulate TGF-β function. Understanding the complex interplay between the TGF-β signaling network and these stromal proteins may provide greater insight into the development of novel therapeutic strategies that target the TGF-β axis. The present review highlights our present understanding of how stroma modulates TGF-β activity in human cancers. PMID:28067804

  3. Stromal Modulators of TGF-β in Cancer.

    PubMed

    Costanza, Brunella; Umelo, Ijeoma Adaku; Bellier, Justine; Castronovo, Vincent; Turtoi, Andrei

    2017-01-06

    Transforming growth factor-β (TGF-β) is an intriguing cytokine exhibiting dual activities in malignant disease. It is an important mediator of cancer invasion, metastasis and angiogenesis, on the one hand, while it exhibits anti-tumor functions on the other hand. Elucidating the precise role of TGF-β in malignant development and progression requires a better understanding of the molecular mechanisms involved in its tumor suppressor to tumor promoter switch. One important aspect of TGF-β function is its interaction with proteins within the tumor microenvironment. Several stromal proteins have the natural ability to interact and modulate TGF-β function. Understanding the complex interplay between the TGF-β signaling network and these stromal proteins may provide greater insight into the development of novel therapeutic strategies that target the TGF-β axis. The present review highlights our present understanding of how stroma modulates TGF-β activity in human cancers.

  4. Molecular diagnostics in soft tissue sarcomas and gastrointestinal stromal tumors.

    PubMed

    Smith, Stephen M; Coleman, Joshua; Bridge, Julia A; Iwenofu, O Hans

    2015-04-01

    Soft tissue sarcomas are rare malignant heterogenous tumors of mesenchymal origin with over fifty subtypes. The use of hematoxylin and eosin stained sections (and immunohistochemistry) in the morphologic assessment of these tumors has been the bane of clinical diagnosis until recently. The last decade has witnessed considerable progress in the understanding and application of molecular techniques in refining the current understanding of soft tissue sarcomas and gastrointestinal stromal tumors beyond the limits of traditional approaches. Indeed, the identification of reciprocal chromosomal translocations and fusion genes in some subsets of sarcomas with potential implications in the pathogenesis, diagnosis and treatment has been revolutionary. The era of molecular targeted therapy presents a platform that continues to drive biomarker discovery and personalized medicine in soft tissue sarcomas and gastrointestinal stromal tumors. In this review, we highlight how the different molecular techniques have enhanced the diagnosis of these tumors with prognostic and therapeutic implications.

  5. [Effect of continuous gamma-radiation at low doses on clonogenic hemopoietic (CFU-S) and stromal (CFU-F) bone marrow cells ].

    PubMed

    Domaratskaia, E I; Starostin, V I; Tsetlin, V V; Butorina, N N; Bueverova, E I; Bragina, E V; Khrushchov, N G

    2002-01-01

    We studied the effects of low doses of continuous gamma-irradiation (Co60, 10 days, mean daily dose power 1.5-2.0 mGy, total dose 15 mGy) on hemopoietic and stromal progenitor cells of murine bone marrow. The content of hemopoietic clonogenic cells representing a "younger" (CFU-S-11) and more "mature" (CFU-S-7) categories in the compartment of stem cells was determined in the bone marrow. The state of bone marrow stroma was estimated by the method of in vitro cloning according to the number of progenitor cells that form colonies of fibroblasts (CFU-F) and by the method of ectopic transplantation according to the capacity of stroma of organizing and building new hemopoietic territories. Continuous gamma-irradiation at low doses, that were by one order of magnitude lower than those inducing hermesis, exerted a stimulating effect on both hemopoietic (CFU-S) and stromal (CFU-F) progenitor cells. The number of CFU-S in the compartment of stem cells of the bone marrow markedly increased and they formed larger hemopoietic territories but these cells appeared to create a qualitatively different microenvironment, which stimulated the proliferation of CFU-S.

  6. Cancer-associated fibroblasts enhance pancreatic cancer cell invasion by remodeling the metabolic conversion mechanism.

    PubMed

    Shan, Tao; Chen, Shuo; Chen, Xi; Lin, Wan Run; Li, Wei; Ma, Jiancang; Wu, Tao; Cui, Xijuan; Ji, Hong; Li, Yiming; Kang, Ya'an

    2017-04-01

    We investigated the mechanism of cancer-associated fibroblasts (CAFs) in promoting the invasion and metastasis of pancreatic cancer cells in a non-vascular manner. We verified the original generation of isolated cultured CAFs and normal fibroblasts (NFs) based on the expression of α-SMA and vimentin, and we examined the cell glycolysis level through glucose consumption and lactate production experiments. The mRNA and protein expression of CAF glycolytic enzymes, lactate dehydrogenase and pyruvate kinase m2, were examined by RT-PCR and western blotting, respectively. In vitro culture first-generation pancreatic CAFs were collected and cultured together with pancreas cancer BxPc-3 and Panc-1 cells. Cell invasion and migration were assessed using a Transwell assay and scratch test, respectively. Mitochondrial activity was assessed by experimentally determining oxidative phosphorylation (OP) activity. The aerobic oxidation index of cancer cells was also examined. Succinate dehydrogenase, fumarate hydratase (FH), and monocarboxylate transporter 1 (MCT1) expression were examined using an MCT1-specific inhibitor to remove 'tumor-stromal' metabolic coupling to observe the influence of cell interstices on pancreas cancer progression. First-generation isolated cultured CAFs and NFs both grew well, and showed active proliferation. Glucose absorption and lactate production were significantly enhanced in CAFs compared with that in NFs. PCR and western blotting showed that the lactate dehydrogenase and pyruvate kinase m2 mRNA and protein expression levels were increased in the CAFs. After indirect co-culture, OP was increased in the BxPc-3 and Panc-1 cells; correspondingly, succinate dehydrogenase, FH and MCT expression were increased. After the MCT1-specific inhibitor removed 'tumor-stromal' metabolic coupling, the migration and invasion abilities of the pancreatic cancer cells were decreased. Pancreatic CAFs can alter metabolism as well as communicate with and respond to

  7. Effect of Stromal Adipokines on Breast Cancer Development

    DTIC Science & Technology

    2009-09-01

    menopausal women. The degree of risk increases proportionally with an increase in adiposity. There is mounting evidence that stromal cells in the tumor...the risk of breast cancer in post- menopausal women. The degree of risk increases proportionally with an increase in adiposity. There is mounting...cells has not been resolved. It is apparent that concomitant changes at the organismic level (e.g. degree of adiposity and/or menopausal status) and

  8. Giant rectal gastrointestinal stromal tumours: a diagnostic and therapeutic challenge

    PubMed Central

    Alder, L.S.; Elver, G.; Foo, F.J.; Dobson, M.

    2013-01-01

    Gastrointestinal stromal tumour (GIST are the most common mesenchymal tumours; however, rectal GISTs account for <5%. In the pelvis they represent a diagnostic challenge with giant GISTs likely to be malignant. They may present with urological, gynaecological or rectal symptoms. Sphincter-preserving surgery can be aided by neoadjuvant therapy. We present an uncommon case of giant rectal GIST masquerading as acute urinary retention. PMID:24968434

  9. Looking older: Fibroblast Collapse and Therapeutic Implications

    PubMed Central

    Fisher, Gary J.; Varani, James; Voorhees, John J.

    2010-01-01

    Background Skin appearance is a primary indicator of age. During the last decade, substantial progress has been made towards understanding underlying mechanisms of human skin aging. This understanding provides the basis for current use and new development of anti-aging treatments. Objective To present state of the art knowledge pertaining to mechanisms involved in skin aging, with specific focus on the dermal collagen matrix. Results A major feature of aged skin is fragmentation of the dermal collagen matrix. Fragmentation results from actions of specific enzymes (matrix metalloproteinases), and impairs the structural integrity of the dermis. Fibroblasts that produce and organize the collagen matrix cannot attach to fragmented collagen. Loss of attachment prevents fibroblasts from receiving mechanical information from their support and they collapse. Stretch is critical for normal balanced production of collagen and collagen-degrading enzymes. In aged skin, collapsed fibroblasts produce low levels of collagen and high levels of collagen–degrading enzymes. This imbalance advances the aging process, in a self-perpetuating, never-ending deleterious cycle. Clinically-proven anti-aging treatments such as topical retinoic acid, CO2 laser resurfacing, and intradermal injection of cross-linked hyaluronic acid stimulate production of new undamaged collagen. Attachment of fibroblasts to this new collagen allows stretch, which in turn balances collagen production/degradation and thereby slows the aging process. Conclusion Collagen fragmentation is responsible for loss of structural integrity and impairment of fibroblast function in aged human skin. Treatments that stimulate production of new, non-fragmented collagen should provide substantial improvement to the appearance and health of aged skin. PMID:18490597

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

    PubMed

    Zhu, Zhong Xin; Sun, Cong Cong; Ting Zhu, Yu; Wang, Ying; Wang, Tao; Chi, Li Sha; Cai, Wan Hui; Zheng, Jia Yong; Zhou, Xuan; Cong, Wei Tao; Li, Xiao Kun; Jin, Li Tai

    2017-03-28

    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. Western 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.

  11. 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.

  12. Interstitial stromal progenitors during kidney development: here, there and everywhere.

    PubMed

    Fanni, Daniela; Gerosa, Clara; Vinci, Laura; Ambu, Rossano; Dessì, Angelica; Eyken, Peter Van; Fanos, Vassilios; Faa, Gavino

    2016-12-01

    In recent years, the renal interstitium has been identified as the site of multiple cell types, giving rise to multiple contiguous cellular networks with multiple fundamental structural and functional roles. Few studies have been carried out on the morphological and functional properties of the stromal/interstitial renal cells during the intrauterine life. This work was aimed at reviewing the peculiar features of renal interstitial stem/progenitor cells involved in kidney development. The origin of the renal interstitial progenitor cells remains unknown. During kidney development, besides the Six2 + cells of the cap mesenchyme, a self-renewing progenitor population, characterized by the expression of Foxd1, represents the first actor of the non-nephrogenic lineage. Foxd1 + interstitial progenitors originate the cortical and the renal medullary interstitial progenitors. Here, the most important stromal/interstitial compartments present in the developing human kidney will be analyzed: capsular stromal cells, cortical interstitial cells, medullary interstitial cells, the interstitium inside the renal stem cell niche, Hilar interstitial cells and Ureteric interstitial cells. Data reported here indicate that the different interstitial compartments of the developing kidney are formed by different cell types that characterize the different renal areas. Further studies are needed to better characterize the different pools of renal interstitial progenitors and their role in human nephrogenesis.

  13. Engineering epithelial-stromal interactions in vitro for toxicology assessment.

    PubMed

    Belair, David G; Abbott, Barbara D

    2017-03-08

    Crosstalk between epithelial and stromal cells drives the morphogenesis of ectodermal organs during development and promotes normal mature adult epithelial tissue homeostasis. Epithelial-stromal interactions (ESIs) have historically been examined using mammalian models and ex vivo tissue recombination. Although these approaches have elucidated signaling mechanisms underlying embryonic morphogenesis processes and adult mammalian epithelial tissue function, they are limited by the availability of tissue, low throughput, and human developmental or physiological relevance. In this review, we describe how bioengineered ESIs, using either human stem cells or co-cultures of human primary epithelial and stromal cells, have enabled the development of human in vitro epithelial tissue models that recapitulate the architecture, phenotype, and function of adult human epithelial tissues. We discuss how the strategies used to engineer mature epithelial tissue models in vitro could be extrapolated to instruct the design of organotypic culture models that can recapitulate the structure of embryonic ectodermal tissues and enable the in vitro assessment of events critical to organ/tissue morphogenesis. Given the importance of ESIs towards normal epithelial tissue development and function, such models present a unique opportunity for toxicological screening assays to incorporate ESIs to assess the impact of chemicals on mature and developing epidermal tissues.

  14. Stromal Targets for Fluorescent-Guided Oncologic Surgery

    PubMed Central

    Boonstra, Martin C.; Prakash, Jai; Van De Velde, Cornelis J. H.; Mesker, Wilma E.; Kuppen, Peter J. K.; Vahrmeijer, Alexander L.; Sier, Cornelis F. M.

    2015-01-01

    Pre-operative imaging techniques are essential for tumor detection and diagnosis, but offer limited help during surgery. Recently, the applicability of imaging during oncologic surgery has been recognized, using near-infrared fluorescent dyes conjugated to targeting antibodies, peptides, or other vehicles. Image-guided oncologic surgery (IGOS) assists the surgeFon to distinguish tumor from normal tissue during operation, and can aid in recognizing vital structures. IGOS relies on an optimized combination of a dedicated fluorescent camera system and specific probes for targeting. IGOS probes for clinical use are not widely available yet, but numerous pre-clinical studies have been published and clinical trials are being established or prepared. Most of the investigated probes are based on antibodies or peptides against proteins on the membranes of malignant cells, whereas others are directed against stromal cells. Targeting stroma cells for IGOS has several advantages. Besides the high stromal content in more aggressive tumor types, the stroma is often primarily located at the periphery/invasive front of the tumor, which makes stromal targets particularly suited for imaging purposes. Moreover, because stroma up-regulation is a physiological reaction, most proteins to be targeted on these cells are “universal” and not derived from a specific genetic variation, as is the case with many upregulated proteins on malignant cancer cells. PMID:26636036

  15. [Extragastrointestinal stromal tumor (EGIST)--a case review].

    PubMed

    Kolarík, J; Drápela, J

    2012-04-01

    Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the gastrointestinal tract. Due to the presence of thyrosine kinase receptors within the tumor tissue, GIST is thought to originate from gastrointestinal pacemaker cells, the intersticial cells of Cajal. Tumors with the same morphological and imunohistochemical characteristics detected outside the gastrointestinal tract, are called extragastrointestinal stromal tumors (EGIST). Biological characteristics of these tumors is uncertain and the malignancy rates are difficult to predict. Surgical R0 resection in resecable tumors is the only option with the potential for complete cure. Nevertheless, the recurrence rates are high. Adjuvant biological treatment with imatinib, a thyrosine kinase inhibitor, reduces the risk of relapses. Imatinib administration is also the principal treatment method in metastatic GIST disorders. The article offers a short and complex overview of gastrointestinal stromal tumor (GIST) problematics and presents a case report of a patient suffering from EGIST of mesocolon transversum treated by R0 resection which was performed under multidisciplinary cooperation, with a specialist follow up.

  16. Extragastrointestinal Stromal Tumour of The Abdominal Wall - A Case Report

    PubMed Central

    Kumar, A. Sathish Selva; Padmini, R; Veena, G; Murugesan, N

    2013-01-01

    Stromal tumours occurring in areas other than the GastroIntestinal Tract (GIT) are known as Extra GastroIntestinal Stromal Tumours (EGISTs). They usually arise in the mesentery, omentum or retroperitoneum, while EGISTs which occur in the abdominal wall are very rare. Both gastrointestinal stromal tumours (GISTs) and EGISTs are histologically and immunophenotypically similar. We are reporting a case of EGIST, which occurred in the anterior abdominal wall in a twenty five-year-old female patient. The tumour was present in the right loin and imaging studies suggested that it was a desmoid tumour. It was surgically excised by doing an abdominal wall mesh repair. The histological examinations revealed a tumour with spindle cell morphology, with <2 mitoses per 50 High Power Field (HPF) and no necrosis, with tumour free margins. Immunohistochemistry was strongly positive for CD117 and Smooth Muscle Actin (SMA), while it was negative for β-catenin and S100. The patient is well post operatively and is on close follow up. EGISTs should be considered in the differential diagnosis of mesenchymal tumours which occur in the abdominal wall, inspite of their rarity, as the high risk patients may need Imatinib chemotherapy. PMID:24551695

  17. Characterization of bone marrow mesenchymal stromal cells in aplastic anaemia.

    PubMed

    Hamzic, Edita; Whiting, Karen; Gordon Smith, Edward; Pettengell, Ruth

    2015-06-01

    In aplastic anaemia (AA), haemopoietic activity is significantly reduced and generally attributed to failure of haemopoietic stem cells (HSC) within the bone marrow (BM). The regulation of haemopoiesis depends on the interaction between HSC and various cells of the BM microenvironment, including mesenchymal stromal cells (MSC). MSC involvement in the functional restriction of HSC in AA is largely unknown and therefore, the physical and functional properties of AA MSC were studied in vitro. MSC were characterized by their phenotype and ability to form adherent stromal layers. The functional properties of AA MSC were assessed through proliferative, clonogenic and cross-over culture assays. Results indicate that although AA MSC presented typical morphology and distinctive mesenchymal markers, stromal formation was reduced, with 50% of BM samples failing to produce adherent layers. Furthermore, their proliferative and clonogenic capacity was markedly decreased (P = 0·03 and P = 0·04 respectively) and the ability to sustain haemopoiesis was significantly reduced, as assessed by total cell proliferation (P = 0·032 and P = 0·019 at Week 5 and 6, respectively) and clonogenic potential of HSC (P = 0·02 at Week 6). It was concluded that the biological characteristics of AA MSC are different from those of control MSC and their in vitro haemopoiesis-supporting ability is significantly reduced.

  18. Targeted Proapoptotic Peptides Depleting Adipose Stromal Cells Inhibit Tumor Growth

    PubMed Central

    Daquinag, Alexes C; Tseng, Chieh; Zhang, Yan; Amaya-Manzanares, Felipe; Florez, Fernando; Dadbin, Ali; Zhang, Tao; Kolonin, Mikhail G

    2016-01-01

    Progression of many cancers is associated with tumor infiltration by mesenchymal stromal cells (MSC). Adipose stromal cells (ASC) are MSC that serve as adipocyte progenitors and endothelium-supporting cells in white adipose tissue (WAT). Clinical and animal model studies indicate that ASC mobilized from WAT are recruited by tumors. Direct evidence for ASC function in tumor microenvironment has been lacking due to unavailability of approaches to specifically inactivate these cells. Here, we investigate the effects of a proteolysis-resistant targeted hunter-killer peptide D-WAT composed of a cyclic domain CSWKYWFGEC homing to ASC and of a proapoptotic domain KLAKLAK2. Using mouse bone marrow transplantation models, we show that D-WAT treatment specifically depletes tumor stromal and perivascular cells without directly killing malignant cells or tumor-infiltrating leukocytes. In several mouse carcinoma models, targeted ASC cytoablation reduced tumor vascularity and cell proliferation resulting in hemorrhaging, necrosis, and suppressed tumor growth. We also validated a D-WAT derivative with a proapoptotic domain KFAKFAK2 that was found to have an improved cytoablative activity. Our results for the first time demonstrate that ASC, recruited as a component of tumor microenvironment, support cancer progression. We propose that drugs targeting ASC can be developed as a combination therapy complementing conventional cancer treatments. PMID:26316391

  19. Stromal and hematopoietic cells in secondary lymphoid organs: partners in immunity.

    PubMed

    Malhotra, Deepali; Fletcher, Anne L; Turley, Shannon J

    2013-01-01

    Secondary lymphoid organs (SLOs), including lymph nodes, Peyer's patches, and the spleen, have evolved to bring cells of the immune system together. In these collaborative environments, lymphocytes scan the surfaces of antigen-presenting cells for cognate antigens, while moving along stromal networks. The cell-cell interactions between stromal and hematopoietic cells in SLOs are therefore integral to the normal functioning of these tissues. Not only do stromal cells physically construct SLO architecture but they are essential for regulating hematopoietic populations within these domains. Stromal cells interact closely with lymphocytes and dendritic cells, providing scaffolds on which these cells migrate, and recruiting them into niches by secreting chemokines. Within lymph nodes, stromal cell-ensheathed conduit networks transport small antigens deep into the SLO parenchyma. More recently, stromal cells have been found to induce peripheral CD8(+) T-cell tolerance and control the extent to which newly activated T cells proliferate within lymph nodes. Thus, stromal-hematopoietic crosstalk has important consequences for regulating immune cell function within SLOs. In addition, stromal cell interactions with hematopoietic cells, other stroma, and the inflammatory milieu have profound effects on key stromal functions. Here, we examine ways in which these interactions within the lymph node environment influence the adaptive immune response.

  20. Stromal and hematopoietic cells in secondary lymphoid organs: partners in immunity

    PubMed Central

    Malhotra, Deepali; Fletcher, Anne L.; Turley, Shannon J.

    2012-01-01

    Summary Secondary lymphoid organs (SLOs), including lymph nodes, Peyer's patches, and the spleen, have evolved to bring cells of the immune system together. In these collaborative environments, lymphocytes scan the surfaces of antigen-presenting cells for cognate antigens, while moving along stromal networks. The cell-cell interactions between stromal and hematopoietic cells in SLOs are therefore integral to the normal functioning of these tissues. Not only do stromal cells physically construct SLO architecture, but they are essential for regulating hematopoietic populations within these domains. Stromal cells interact closely with lymphocytes and dendritic cells, providing scaffolds on which these cells migrate, and recruiting them into niches by secreting chemokines. Within lymph nodes, stromal cell-ensheathed conduit networks transport small antigens deep into the SLO parenchyma. More recently, stromal cells have been found to induce peripheral CD8+ T-cell tolerance and control the extent to which newly activated T cells proliferate within lymph nodes. Thus, stromal-hematopoietic crosstalk has important consequences for regulating immune cell function within SLOs. In addition, stromal cell interactions with hematopoietic cells, other stroma, and the inflammatory milieu have profound effects on key stromal functions. Here, we examine ways in which these interactions within the lymph node environment influence the adaptive immune response. PMID:23278748

  1. PIXE analysis of elements in gastric cancer and adjacent mucosa

    NASA Astrophysics Data System (ADS)

    Liu, Qixin; Zhong, Ming; Zhang, Xiaofeng; Yan, Lingnuo; Xu, Yongling; Ye, Simao

    1990-04-01

    The elemental regional distributions in 20 resected human stomach tissues were obtained using PIXE analysis. The samples were pathologically divided into four types: normal, adjacent mucosa A, adjacent mucosa B and cancer. The targets for PIXE analysis were prepared by wet digestion with a pressure bomb system. P, K, Fe, Cu, Zn and Se were measured and statistically analysed. We found significantly higher concentrations of P, K, Cu, Zn and a higher ratio of Cu compared to Zn in cancer tissue as compared with normal tissue, but statistically no significant difference between adjacent mucosa and cancer tissue was found.

  2. Thermoelastic response of thin metal films and their adjacent materials

    SciTech Connect

    Kang, S.; Yoon, Y.; Kim, J.; Kim, W.

    2013-01-14

    A pulsed laser beam applied to a thin metal film is capable of launching an acoustic wave due to thermal expansion. Heat transfer from the thin metal film to adjacent materials can also induce thermal expansion; thus, the properties of these adjacent materials (as well as the thin metal film) should be considered for a complete description of the thermoelastic response. Here, we show that adjacent materials with a small specific heat and large thermal expansion coefficient can generate an enhanced acoustic wave and we demonstrate a three-fold increase in the peak pressure of the generated acoustic wave on substitution of parylene for polydimethylsiloxane.

  3. Epigenetic silencing of microRNA-149 in cancer-associated fibroblasts mediates prostaglandin E2/interleukin-6 signaling in the tumor microenvironment

    PubMed Central

    Li, Pu; Shan, Jing-Xuan; Chen, Xue-Hua; Zhang, Di; Su, Li-Ping; Huang, Xiu-Ying; Yu, Bei-Qin; Zhi, Qiao-Ming; Li, Cheng-Long; Wang, Ya-Qing; Tomei, Sara; Cai, Qu; Ji, Jun; Li, Jian-Fang; Chouchane, Lotfi; Yu, Ying-Yan; Sun, Fang-Zhen; Xu, Zhi-Heng; Liu, Bing-Ya; Zhu, Zheng-Gang

    2015-01-01

    Tumor initiation and growth depend on its microenvironment in which cancer-associated fibroblasts (CAFs) in tumor stroma play an important role. Prostaglandin E2 (PGE2) and interleukin (IL)-6 signal pathways are involved in the crosstalk between tumor and stromal cells. However, how PGE2-mediated signaling modulates this crosstalk remains unclear. Here, we show that microRNA (miR)-149 links PGE2 and IL-6 signaling in mediating the crosstalk between tumor cells and CAFs in gastric cancer (GC). miR-149 inhibited fibroblast activation by targeting IL-6 and miR-149 expression was substantially suppressed in the CAFs of GC. miR-149 negatively regulated CAFs and their effect on GC development both in vitro and in vivo. CAFs enhanced epithelial-to-mesenchymal transition (EMT) and the stem-like properties of GC cells in a miR-149-IL-6-dependent manner. In addition to IL-6, PGE2 receptor 2 (PTGER2/EP2) was revealed as another potential target of miR-149 in fibroblasts. Furthermore, H. pylori infection, a leading cause of human GC, was able to induce cyclooxygenase-2 (COX-2)/PGE2 signaling and to enhance PGE2 production, resulting in the hypermethylation of miR-149 in CAFs and increased IL-6 secretion. Our findings indicate that miR-149 mediates the crosstalk between tumor cells and CAFs in GC and highlight the potential of interfering miRNAs in stromal cells to improve cancer therapy. PMID:25916550

  4. A model of electrical conduction in cardiac tissue including fibroblasts.

    PubMed

    Sachse, Frank B; Moreno, A P; Seemann, G; Abildskov, J A

    2009-05-01

    Fibroblasts are abundant in cardiac tissue. Experimental studies suggested that fibroblasts are electrically coupled to myocytes and this coupling can impact cardiac electrophysiology. In this work, we present a novel approach for mathematical modeling of electrical conduction in cardiac tissue composed of myocytes, fibroblasts, and the extracellular space. The model is an extension of established cardiac bidomain models, which include a description of intra-myocyte and extracellular conductivities, currents and potentials in addition to transmembrane voltages of myocytes. Our extension added a description of fibroblasts, which are electrically coupled with each other and with myocytes. We applied the extended model in exemplary computational simulations of plane waves and conduction in a thin tissue slice assuming an isotropic conductivity of the intra-fibroblast domain. In simulations of plane waves, increased myocyte-fibroblast coupling and fibroblast-myocyte ratio reduced peak voltage and maximal upstroke velocity of myocytes as well as amplitudes and maximal downstroke velocity of extracellular potentials. Simulations with the thin tissue slice showed that inter-fibroblast coupling affected rather transversal than longitudinal conduction velocity. Our results suggest that fibroblast coupling becomes relevant for small intra-myocyte and/or large intra-fibroblast conductivity. In summary, the study demonstrated the feasibility of the extended bidomain model and supports the hypothesis that fibroblasts contribute to cardiac electrophysiology in various manners.

  5. Increased infiltrated macrophages in benign prostatic hyperplasia (BPH): role of stromal androgen receptor in macrophage-induced prostate stromal cell proliferation.

    PubMed

    Wang, Xiaohai; Lin, Wen-Jye; Izumi, Kouji; Jiang, Qi; Lai, Kuo-Pao; Xu, Defeng; Fang, Lei-Ya; Lu, Tianjing; Li, Lei; Xia, Shujie; Chang, Chawnshang

    2012-05-25

    Infiltrated macrophages may play important roles in the development and progression of benign prostatic hyperplasia (BPH), but the underlying mechanisms remain largely unknown. We found increased macrophages infiltration in human and mouse BPH tissues. By establishing a co-culture transwell system, we found increased migration of macrophages and proliferation of prostate stromal cells during co-culture. Importantly, stromal androgen receptor (AR) could enhance the migration of macrophages and macrophage-mediated stromal cell proliferation. We identified CCL3 as an AR downstream player, and found CCL3 levels were notably increased in human and mouse BPH prostates. Ablation of prostate stromal AR in a mouse BPH model significantly reduced CCL3 expression levels in prostates. Consistently, targeting AR via an AR degradation enhancer, ASC-J9®, or neutralization of CCL3 with an antibody, resulted in suppression of macrophage migration and prostate stromal cell growth. Our study provides mechanistic insights on the regulation of prostate stromal cells by macrophages via stromal AR/CCL3 signaling pathways, which could potentially allow the development of therapeutic approaches for battling BPH with persistent inflammation.

  6. Evidence for the involvement of fibroblast growth factor 10 in lipofibroblast formation during embryonic lung development.

    PubMed

    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

    2015-12-01

    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.

  7. 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

  8. Overall view of tower and adjacent aircraft shelters on flight ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Overall view of tower and adjacent aircraft shelters on flight line. View to east. - Plattsburgh Air Force Base, Security Guard Tower, Florida Street at Aircraft Shelters Area, Plattsburgh, Clinton County, NY

  9. Interior building details of Building A, dungeon cell adjacent to ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Interior building details of Building A, dungeon cell adjacent to northwest cell: granite and brick threshold, poured concrete floors, plastered finished walls, vaulted veiling; northwesterly view - San Quentin State Prison, Building 22, Point San Quentin, San Quentin, Marin County, CA

  10. 10. View north from the adjacent B & O railroad ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    10. View north from the adjacent B & O railroad bridge of portion of the Main truss span over the reservoir of the Augustine Paper Mills, National Register Site. - Augustine Bridge, Brandywine River,Augustine Cutoff, Wilmington, New Castle County, DE

  11. Lock 4 View east of lock wall and adjacent ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Lock 4 - View east of lock wall and adjacent roadway built atop tow path. The gate pocket can be seen at center. - Savannah & Ogeechee Barge Canal, Between Ogeechee & Savannah Rivers, Savannah, Chatham County, GA

  12. 1. A BRICK AND CONCRETE FAN HOUSING ADJACENT TO ONE ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    1. A BRICK AND CONCRETE FAN HOUSING ADJACENT TO ONE OF THE ADIT OPENINGS (VIEW TO THE NORTH). - Foster Gulch Mine, Fan Housing, Bear Creek 1 mile Southwest of Town of Bear Creek, Red Lodge, Carbon County, MT

  13. 1. VIEW FROM ROOFTOP OF BUILDING (MOTEL) ADJACENT TO TECHWOOD ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    1. VIEW FROM ROOFTOP OF BUILDING (MOTEL) ADJACENT TO TECHWOOD HOMES, LOOKING SOUTH. GARAGE TO EXTREME LEFT, BUILDING 1 TO EXTREME RIGHT. - Techwood Homes (Public Housing), Bounded by North Avenue, Parker Street, William Street & Lovejoy Street, Atlanta, Fulton County, GA

  14. VIEW FROM ROOFTOP OF BUILDING (MOTEL) ADJACENT TO TECHWOOD HOMES, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    VIEW FROM ROOFTOP OF BUILDING (MOTEL) ADJACENT TO TECHWOOD HOMES, LOOKING SOUTH. GARAGE TO EXTREME LEFT, BUILDING 1 TO EXTREME RIGHT. - Techwood Homes, Building No. 16, 488-514 Techwood Drive, Atlanta, Fulton County, GA

  15. View from water showing south facade and adjacent boat slips ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    View from water showing south facade and adjacent boat slips (Facility Nos. S375 & S376) - U.S. Naval Base, Pearl Harbor, Boat House, Hornet Avenue at Independence Street, Pearl City, Honolulu County, HI

  16. Cement Leakage into Adjacent Vertebral Body Following Percutaneous Vertebroplasty.

    PubMed

    Park, Jae Hoo; Kim, Hyeun Sung; Kim, Seok Won