Primary Cancer-associated Fibroblasts Research Articles

Tumor necrosis factor α induces myofibroblast differentiation in human tongue cancer and promotes invasiveness and angiogenesis via secretion of stromal cell-derived factor-1

Cancer-associated fibroblasts (CAFs) play an important role in tumor progression and are associated with a poor prognosis. Tumor necrosis factor α (TNFα) has been involved in growth and metastasis associated with epithelial-mesenchymal transition (EMT) in many types of cancers. However, the relationship among the TNFα, activation of CAFs and their tumor-promoting effects on tongue squamous cell carcinoma (TSCC) remain obscure. A series of matched primary CAFs and normal fibroblasts (NFs) pairs were cultured, and additionally two TSCC cell lines SCC9 and CAL27, were treated with TNFα or CAFs derived stromal cell-derived factor-1 (SDF1) respectively to study invasion and EMT in vitro. In addition, NFs were treated with TNFα to detect the expression of myofibroblast markers, invasion, induced collagen gel contraction and enhanced cancer metastasis. Finally, invasion and angiogenesis of human vein endothelial cells (HUVECs) treated with TNFα and CAFs derived SDF1 was measured. TNFα and CAFs-derived SDF1 induced TSCC cells metastasis and EMT separately. TNFα facilitated the transformation of NFs to CAFs-like fibroblasts, which was accompanied by acquisition of similar myofibroblast markers expression and malignant function to CAFs. Furthermore, TNFα and CAFs derived SDF1 also promoted invasion and angiogenesis of HUVECs. These results suggest that TNFα may not only directly but also indirectly enhance cancer metastasis, EMT and angiogenesis formation in tongue cancer through myofibroblast differentiation via SDF1 secretion.

Tumor necrosis factor α induces myofibroblast differentiation in human tongue cancer and promotes invasiveness and angiogenesis via secretion of stromal cell-derived factor-1

Summary Objectives Cancer-associated fibroblasts (CAFs) play an important role in tumor progression and are associated with a poor prognosis. Tumor necrosis factor α (TNFα) has been involved in growth and metastasis associated with epithelial–mesenchymal transition (EMT) in many types of cancers. However, the relationship among the TNFα, activation of CAFs and their tumor-promoting effects on tongue squamous cell carcinoma (TSCC) remain obscure. Materials and methods A series of matched primary CAFs and normal fibroblasts (NFs) pairs were cultured, and additionally two TSCC cell lines SCC9 and CAL27, were treated with TNFα or CAFs derived stromal cell-derived factor-1 (SDF1) respectively to study invasion and EMT in vitro . In addition, NFs were treated with TNFα to detect the expression of myofibroblast markers, invasion, induced collagen gel contraction and enhanced cancer metastasis. Finally, invasion and angiogenesis of human vein endothelial cells (HUVECs) treated with TNFα and CAFs derived SDF1 was measured. Results TNFα and CAFs-derived SDF1 induced TSCC cells metastasis and EMT separately. TNFα facilitated the transformation of NFs to CAFs-like fibroblasts, which was accompanied by acquisition of similar myofibroblast markers expression and malignant function to CAFs. Furthermore, TNFα and CAFs derived SDF1 also promoted invasion and angiogenesis of HUVECs. Conclusion These results suggest that TNFα may not only directly but also indirectly enhance cancer metastasis, EMT and angiogenesis formation in tongue cancer through myofibroblast differentiation via SDF1 secretion.

Abstract 423: CXCL12/CXCR4 activation by cancer-associated fibroblasts promotes integrin β1 clustering and invasive ability in gastric cancer

Abstract Purpose: Gastric cancer (GC) is the fourth most commonly diagnosed cancer and the third leading cause of cancer mortality worldwide. The range of therapeutic strategies available for the treatment of GC has improved in recent decades. However, the prognosis of patients with advanced GC remains poor even after curative resection, mainly because of recurrence, such as peritoneal dissemination and liver metastases. Previous studies have shown that the interaction between cancer cells and their surrounding stroma plays an important role in tumor development. Cancer-associated fibroblasts (CAFs) have been reported to be involved in invasion and metastasis in cancers, including gastric cancer (GC), via their stimulation of CXCL12/CXCR4 signaling. However, the mechanisms underlying the tumor-promoting effects and the potential of developing therapeutic targets using CXCL12/CXCR4 signaling activated by CAFs are not fully understood. Experimental Design: We first analyzed CXCL12 expression in resected GC tissues of 110 patients by immunohistochemistry (IHC). We next established primary normal fibroblasts (NFs) and CAFs from GC tissues and examined the functional differences between these primary fibroblasts using co-culture assays with GC cell lines. Furthermore, we evaluated the efficacy of a CXCR4 antagonist (AMD3100) and a FAK inhibitor (PF-573,228) on the invasive ability of GC cells. Results: We determined that high CXCL12 expression was significantly associated with larger tumor size, deeper tumor depth, lymphatic invasion and poor prognosis in GC. We established new primary CAFs and their adjacent NFs from resected stomach tissue in GC patients. We evaluated the change in mobility during the direct co-culture of GC cells and fibroblasts using real-time imaging. The motility of GC cells significantly increased upon direct co-culture with CAFs more than it did in cells with NFs compared with control RPMI during observation for 24 hours. The motility of GC cells with CAFs was effectively suppressed by AMD 3100 compared to GC cells with NFs. CXCL12/CXCR4 activation by CAFs mediated the clustering of integrin β1 on the cell surface and promoted the invasive ability of GC cells. Notably, AMD3100 was more efficient at inhibiting GC cell invasion through the suppression of integrin β1/FAK signaling compared with PF-573,228. Conclusions: These results suggest that CXCL12 derived from CAFs promotes invasive ability by enhancing the clustering of integrin β1 in GC cells and results in GC progression. The current data provide evidence that CXCL12/CXCR4 inhibition by AMD3100 is more efficient in suppressing GC cell invasion by not only inhibiting intracellular signaling but also by attenuating the interaction between cancer cells and the extracellular matrix compared with PF-573,228. Our present study represents the rationale for establishing a therapeutic strategy targeting CXCL12/CXCR4 signaling. Citation Format: Daisuke Izumi, Takatsugu Ishimoto, Hidetaka Sugihara, Eto Kojiro, Hiroshi Sawayama, Keisuke Miyake, Yuki Kiyozumi, Keisuke Kosumi, Ryuma Tokunaga, Kazuto Harada, Junji Kurashige, Masaaki Iwatsuki, Shiro Iwagami, Yoshifumi Baba, Yasuo Sakamoto, Yuji Miyamoto, Naoya Yoshida, Masayuki Watanabe, Hideo Baba. CXCL12/CXCR4 activation by cancer-associated fibroblasts promotes integrin β1 clustering and invasive ability in gastric cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 423. doi:10.1158/1538-7445.AM2015-423

Abstract 5071: Inhibition of Porcupine reduces Wnt-dependent colony formation and/or proliferation in both autocrine and paracrine cell models

Abstract Aberrant activation of the pro-survival Wnt signalling pathway is a key determinant in numerous cancers (Nusse and Varmus, 2012) and therefore constituents of this pathway are potentially promising therapeutic targets. The membrane-bound o-acyltransferase porcupine (PORCN) is required for post-translational modification of all Wnts, and is vital for Wnt secretion (Herr et. al., 2012). Due to the complex and varied oncogenic roles of the known Wnt ligands, prevention of active ligand secretion provides an effective mechanism for inhibiting canonical and non-canonical Wnt signalling in both paracrine and autocrine systems. Here we show that a novel inhibitor of porcupine potently inhibits both autocrine and paracrine models of Wnt/β-catenin signalling. Treatment of the pancreatic cancer cell line CAPAN-2 with REDX05562 resulted in a significant decrease of axin 2 mRNA expression by qPCR (10.4 fold, 10nM). This reduction in expression was partially reversed by the addition of recombinant human Wnt-3A (100ng.ml−1). The decrease in axin 2 expression was concomitant with an inhibition of CAPAN-2 proliferation (IC50 1.4nM +/- 0.8nM). Conversely treatment of MCF-7 mammospheres with REDX05562 did not inhibit sphere forming efficiency (SFE). However, when stimulated with recombinant Wnt-3A an increase in anoikis resistance was observed suggesting a potential paracrine influence. In vivo the presence of cancer associated fibroblasts (CAFs) are a likely source of paracrine Wnt (Green et. al., 2013). Indeed, conditioned media taken from primary CAFs (isolated from fresh mastectomies) increased anoikis resistance in MCF-7 cells without any discernible effect on CAF proliferation. This effect was significantly inhibited when MCF-7 cells were incubated with conditioned media taken from primary CAFs treated with REDX05562. Moreover, immunofluorescent labelling of PORCN revealed that PORCN expression is markedly higher in CAFs than in MCF-7s. This is consistent with the hypothesis that different mechanisms underpin Wnt-signalling in breast and pancreatic cancer. The above data show that the novel PORCN inhibitor REDX05562 effectively reduces the impact of pro-survival Wnt ligand-mediated signalling in both paracrine and autocrine models. This further emphasises the broad therapeutic potential of PORCN inhibition in Wnt-dependent cancers. Citation Format: Ben A. Thompson, Kate J. Messenger, Emily Linnane, Sarah E. Coupland, Peter J. Calcraft. Inhibition of Porcupine reduces Wnt-dependent colony formation and/or proliferation in both autocrine and paracrine cell models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5071. doi:10.1158/1538-7445.AM2015-5071

Cancer-Associated Fibroblasts from Lung Tumors Maintain Their Immunosuppressive Abilities after High-Dose Irradiation

Accumulating evidence supports the notion that high-dose (>5 Gy) radiotherapy (RT) regimens are triggering stronger pro-immunogenic effects than standard low-dose (2 Gy) regimens. However, the effects of RT on certain immunoregulatory elements in tumors remain unexplored. In this study, we have investigated the effects of high-dose radiotherapy (HD-RT) on the immunomodulating functions of cancer-associated fibroblasts (CAFs). Primary CAF cultures were established from lung cancer specimens derived from patients diagnosed for non-small cell lung cancer. Irradiated and non-irradiated CAFs were examined for immunomodulation in experiments with peripheral blood mononuclear cells from random, healthy donors. Regulation of lymphocytes behavior was checked by lymphocyte proliferation assays, lymphocyte migration assays, and T-cell cytokine production. Additionally, CAF-secreted immunoregulatory factors were studied by multiplex protein arrays, ELISAs, and by LC-MS/MS proteomics. In all functional assays, we observed a powerful immunosuppressive effect exerted by CAF-conditioned medium on activated T-cells (p > 0.001), and this effect was sustained after a single radiation dose of 18 Gy. Relevant immunosuppressive molecules such as prostaglandin E2, interleukin-6, and -10, or transforming growth factor-β were found in CAF-conditioned medium, but their secretion was unchanged after irradiation. Finally, immunogenic cell death responses in CAFs were studied by exploring the release of high motility group box-1 and ATP. Both alarmins remained undetectable before and after irradiation. In conclusion, CAFs play a powerful immunosuppressive effect over activated T-cells, and this effect remains unchanged after HD-RT. Importantly, CAFs do not switch on immunogenic cell death responses after exposure to HD-RT.

Cancer-associated fibroblasts from invasive breast cancer have an attenuated capacity to secrete collagens.

Normal fibroblasts produce extracellular matrix(ECM) components that form the structural framework of tissues. Cancer-associated fibroblasts(CAFs) with an activated phenotype mainly contribute to ECM deposition and construction of cancer masses. However, the stroma of breast cancer tissues has been shown to be more complicated, and the mechanisms through which CAFs influence ECM deposition remain elusive. In this study, we found that the activated fibroblast marker α-smooth muscle actin(α-SMA) was only present in the stroma of breast cancer tissue, and the CAFs isolated from invasive breast cancer sample remained to be activated and proliferative in passages. To further assess the difference between CAFs and normal breast fibroblasts(NFs), MALDITOF/TOF‑MS was used to analyze the secretory proteins of primary CAFs and NFs. In total, 2,903 and 3,023 proteins were identified. Mass spectrum quantitative assay and data analysis for extracellular proteins indicated that the CAFs produce less collagens and matrix-degrading enzymes compared with NFs. This finding was confirmed by western blot analysis. Furthermore, we discovered that reduced collagen deposition was present in the stroma of invasive breast cancer. These studies showed that although CAFs from invasive breast cancer possess an activated phenotype, they secreted less collagen and induced less ECM deposition in cancer stroma. In cancer tissue, the remodeling of stromal structure and tumor microenvironment might, therefore, be attributed to the biological changes in CAFs including their protein expression profile.

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Podoplanin expression in cancer-associated fibroblasts enhances tumor progression of invasive ductal carcinoma of the pancreas

BackgroundInteractions between cancer cells and surrounding cancer-associated fibroblasts (CAFs) play an important role in cancer progression. Invasive ductal carcinoma (IDC) of the pancreas is characterized by abundant fibrous connective tissue called desmoplasia. Podoplanin (PDPN) is a lymphatic vessel marker (D2-40), and expression of PDPN by stromal CAFs has been reported to be a prognostic indicator in various types of cancer.MethodsExpression of PDPN in pancreatic IDCs was assessed by immunohistochemical examination in 105 patients who underwent pancreatic resection. Primary CAFs were established from pancreatic cancer tissue obtained by surgery. Quantitative reverse transcription-polymerase chain reaction and flow cytometric analysis were performed to investigate PDPN expression in CAFs. We sorted CAFs according to PDPN expression, and analyzed the functional differences between PDPN+ CAFs and PDPN– CAFs using indirect co-culture with pancreatic cancer cell lines. We also investigated the culture conditions to regulate PDPN expression in CAFs.ResultsPDPN expression in stromal fibroblasts was associated with lymphatic vessel invasion (P = 0.0461), vascular invasion (P = 0.0101), tumor size ≥3 cm (P = 0.0038), histological grade (P = 0.0344), Union for International Cancer Control classification T stage (P = 0.029), and shorter survival time (P < 0.0001). Primary CAFs showed heterogeneous PDPN expression in vitro. Moreover, migration and invasion of pancreatic cancer cell lines (PANC-1 and SUIT-2) were associated with PDPN expression in CAFs (P < 0.01) and expression of CD10, matrix metalloproteinase (MMP) 2, and MMP3. In cultured CAFs, PDPN positivity changed over time under several conditions including co-culture with cancer cells, different culture media, and addition of growth factor.ConclusionsPDPN-expressing CAFs enhance the progression of pancreatic IDC, and a high ratio of PDPN-expressing CAFs is an independent predictor of poor outcome. Understanding the regulation of the tumor microenvironment is an important step towards developing new therapeutic strategies.

Functional Heterogeneity of Cancer-Associated Fibroblasts from Human Colon Tumors Shows Specific Prognostic Gene Expression Signature

Cancer-associated fibroblasts (CAF) actively participate in reciprocal communication with tumor cells and with other cell types in the microenvironment, contributing to a tumor-permissive neighborhood and promoting tumor progression. The aim of this study is the characterization of how CAFs from primary human colon tumors promote migration of colon cancer cells. Primary CAF cultures from 15 primary human colon tumors were established. Their enrichment in CAFs was evaluated by the expression of various epithelial and myofibroblast specific markers. Coculture assays of primary CAFs with different colon tumor cells were performed to evaluate promigratory CAF-derived effects on cancer cells. Gene expression profiles were developed to further investigate CAF characteristics. Coculture assays showed significant differences in fibroblast-derived paracrine promigratory effects on cancer cells. Moreover, the association between CAFs' promigratory effects on cancer cells and classic fibroblast activation or stemness markers was observed. CAF gene expression profiles were analyzed by microarray to identify deregulated genes in different promigratory CAFs. The gene expression signature, derived from the most protumorogenic CAFs, was identified. Interestingly, this "CAF signature" showed a remarkable prognostic value for the clinical outcome of patients with colon cancer. Moreover, this prognostic value was validated in an independent series of 142 patients with colon cancer, by quantitative real-time PCR (qRT-PCR), with a set of four genes included in the "CAF signature." In summary, these studies show for the first time the heterogeneity of primary CAFs' effect on colon cancer cell migration. A CAF gene expression signature able to classify patients with colon cancer into high- and low-risk groups was identified.

Abstract 1504: Pancreatic cancer associated fibroblasts are characterized by widespread epigenetic reprogramming that leads to aberrant expression of druggable target CXCR4.

Abstract The microenvironment where a tumor originates plays an important role in its initiation, growth, progression and metastatic capability. Since in most cancers the microenvironment is not derived from the malignant clone and does not contain oncogenic mutations, it is likely that the tumor microenvironment is reprogrammed epigenetically to support the growth of the tumor. To test this hypothesis, Fibroblasts associated with primary pancreatic adenocarcinomas (N=7) &amp; healthy fibroblast controls (N=4) were analyzed for genome wide alterations in DNA cytosine methylation by the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR). Methylome profiling revealed widespread aberrant cytosine methylation in Pancreatic cancer associated fibroblasts (CAFs) with demethylation of many important gene promoters as the predominant epigenetic event. In addition to loss of methylation, aberrant hypermethylation of GALNTS, JAZF1, MTCH1, SP8, GRB14 was also seen in CAFs. In addition to epigenetic differences, pancreatic CAFs were also found to have widespread transcriptmoic differences as seen by parallel gene expression profiling. Next, we analyzed tumor mediated reprogramming of microenvironment at a higher resolution by a Massive parallel sequencing based methylation analysis (HELP-Tagging) on CAFs differentiated from mesenchymal stem cells (MSCs) in the presence of pancreatic cancer cell conditioned medium. HELP-tagging showed widespread epigenetic reprogramming with 11,100 hypomethylated and 1709 hypermethylated loci. Comparison of in-vitro reprogramed loci with the aberrantly methylated loci from primary CAFs showed a core set of 140 loci that were commonly differentially methylated in both samples. The chemokine receptor CXCR4 was observed to overexpressed &amp; demethylated in both cohorts &amp; was found to be expressed on the surface of primary pancreatic CAFs by immunohistochemistry. Functional studies demonstrated that co-culture of pancreatic cancer cells with CAFs (from MSCs) led to significant increase in malignant cell invasion when compared to co-culture with naïve MSCs. This increased invasion was abrogated by blockade of CXCR4 by AMD-3100 and by knockdown of CXCR4 by siRNAs in orthotopically derived CAFs; demonstrating a critical role for this receptor in regulating tumor promoting abilities of the microenvironment. Thus our results reveal for the first time that pancreatic CAFs are characterized by widespread epigenomic reprogramming that includes loss of methylation at many important loci. Validation of an aberrantly demethylated target, CXCR4, shows that inhibition of this receptor can abrogate the ability of CAFs in promoting cancer cell invasion. These results also provide a preclinical rationale for the use of clinically relevant CXCR4 antagonist AMD-3100 (plerixafor) in pancreatic cancer. Citation Format: Tushar D. Bhagat, Yanique Rattigan, Brijesh Patel, Strepell Mirte, Yiting Yu, Davendra Sohal, Matthias Bartenstein, Orsolya Giricz, Shanisha AK Gordon, Nishanth Vallumsetla, Rahul Polineni, Meher Walia, Paraic Kenny, John Greally, Debabrata Banerjee, Anirban Maitra, Amit Verma. Pancreatic cancer associated fibroblasts are characterized by widespread epigenetic reprogramming that leads to aberrant expression of druggable target CXCR4. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1504. doi:10.1158/1538-7445.AM2013-1504

Inhibition of Platelet-Derived Growth Factor Receptorαby MEDI-575 Reduces Tumor Growth and Stromal Fibroblast Content in a Model of Non-Small Cell Lung Cancer

Platelet-derived growth factor receptor α (PDGFRα) is a receptor tyrosine kinase that promotes cell survival and is expressed in both the tumor and the stromal components of human cancers. We have developed a fully human monoclonal antibody, MEDI-575, that selectively binds to human PDGFRα with high affinity, with no observable affinity for murine PDGFRα. To more fully characterize the role of PDGFRα in the regulation of tumor stroma, we evaluated the in vivo antitumor effects of MEDI-575 in tumor-bearing severe combined immunodeficient (SCID) mice and in genetically altered SCID mice expressing human PDGFRα in place of murine PDGFRα. We used the Calu-6 non-small cell lung cancer model because it lacks an in vitro proliferative response to PDGFRα activation. Antitumor activity was observed when the study was performed in mice expressing the human receptor, but no activity was observed in the mice expressing the murine receptor. Immunohistologic analysis of the tumors from mice expressing human PDGFRα showed a highly significant reduction in stromal fibroblast content and only minor changes in tumor proliferative index in tumors exposed to MEDI-575 compared with the results seen in vehicle-treated tumors or in tumors from mice expressing murine PDGFRα. Additional in vitro studies indicated that exposure of primary cancer-associated fibroblasts to MEDI-575 can directly affect proliferation and key signaling pathways in these cells. These results highlight the potential for observing antitumor activity with MEDI-575 through modulation of the stromal component of tumors and confirm that the PDGFRα pathway can play a role in maintaining a tumor microenvironment conducive to tumor growth.

Impact of Tumor Microenvironment and Epithelial Phenotypes on Metabolism in Breast Cancer

Cancer cells have altered metabolism, with increased glucose uptake, glycolysis, and biomass production. This study conducted genomic and metabolomic analyses to elucidate how tumor and stromal genomic characteristics influence tumor metabolism. Thirty-three breast tumors and six normal breast tissues were analyzed by gene expression microarray and by mass spectrometry for metabolites. Gene expression data and clinical characteristics were evaluated in association with metabolic phenotype. To evaluate the role of stromal interactions in altered metabolism, cocultures were conducted using breast cancer cells and primary cancer-associated fibroblasts (CAF). Across all metabolites, unsupervised clustering resulted in two main sample clusters. Normal breast tissue and a subset of tumors with less aggressive clinical characteristics had lower levels of nucleic and amino acids and glycolysis byproducts, whereas more aggressive tumors had higher levels of these Warburg-associated metabolites. While tumor-intrinsic subtype did not predict metabolic phenotype, metabolic cluster was significantly associated with expression of a wound response signature. In cocultures, CAFs from basal-like breast cancers increased glucose uptake and basal-like epithelial cells increased glucose oxidation and glycogen synthesis, suggesting interplay of stromal and epithelial phenotypes on metabolism. Cytokine arrays identified hepatocyte growth factor (HGF) as a potential mediator of stromal-epithelial interaction and antibody neutralization of HGF resulted in reduced expression of glucose transporter 1 (GLUT1) and decreased glucose uptake by epithelium. Both tumor/epithelial and stromal characteristics play important roles in metabolism. Warburg-like metabolism is influenced by changes in stromal-epithelial interactions, including altered expression of HGF/Met pathway and GLUT1 expression.

Tumorigenic Responses of Cancer-Associated Stromal Fibroblasts after Ablative Radiotherapy: A Transcriptome-Profiling Study

Cancer-associated fibroblasts (CAFs) are key elements in the progression of cancer and thereby represent important targets for cancer therapies. Increased attention has been given to ablative radiotherapy in the clinics. Therefore, in this study we have aimed at identifying the transcriptional responses occurring in primary CAFs exposed to high-dose irradiation. Established primary CAFs obtained from non-small-cell lung cancer (NSCLC) patient material were irradiated with a single dose of 18 Gy and total RNA was isolated 24 hrs after treatment. Radiation-induced transcriptional alterations were investigated by gene expression analysis using genome-wide microarrays. Obtained results were verified by qRT-PCR of relevant genes. Confirmation of gene expression outcomes was achieved by diverse functional and expression assays including DNA damage response, measurements of reactive oxygen species (ROS) by flow cytometry and senescence-associated β-galactosidase. Irradiation resulted in differential expression of 680 genes of which 557 were up- and 127 down-regulated. Of those, 153 genes were differentially expressed with a fold-change greater than 1.0 and an adjusted p-value less than 0.05 across different comparisons (non-irradiated vs. irradiated). Expression patterns revealed profound changes in biological functions and processes involved in DNA repair, apoptosis, p53 pathway, autophagy, senescence, ROS production and immune response. CAFs display pro- and anti-tumorigenic effects after having received a single high-dose radiation. The measured effects will have an impact on the tumor microenvironment in respect to tumor growth and metastasis.

Human breast cancer associated fibroblasts exhibit subtype specific gene expression profiles

BackgroundBreast cancer is a heterogeneous disease for which prognosis and treatment strategies are largely governed by the receptor status (estrogen, progesterone and Her2) of the tumor cells. Gene expression profiling of whole breast tumors further stratifies breast cancer into several molecular subtypes which also co-segregate with the receptor status of the tumor cells. We postulated that cancer associated fibroblasts (CAFs) within the tumor stroma may exhibit subtype specific gene expression profiles and thus contribute to the biology of the disease in a subtype specific manner. Several studies have reported gene expression profile differences between CAFs and normal breast fibroblasts but in none of these studies were the results stratified based on tumor subtypes.MethodsTo address whether gene expression in breast cancer associated fibroblasts varies between breast cancer subtypes, we compared the gene expression profiles of early passage primary CAFs isolated from twenty human breast cancer samples representing three main subtypes; seven ER+, seven triple negative (TNBC) and six Her2+.ResultsWe observed significant expression differences between CAFs derived from Her2+ breast cancer and CAFs from TNBC and ER + cancers, particularly in pathways associated with cytoskeleton and integrin signaling. In the case of Her2+ breast cancer, the signaling pathways found to be selectively up regulated in CAFs likely contribute to the enhanced migration of breast cancer cells in transwell assays and may contribute to the unfavorable prognosis of Her2+ breast cancer.ConclusionsThese data demonstrate that in addition to the distinct molecular profiles that characterize the neoplastic cells, CAF gene expression is also differentially regulated in distinct subtypes of breast cancer.

CCL2 Mediates Cross-talk between Cancer Cells and Stromal Fibroblasts That Regulates Breast Cancer Stem Cells

Cancer stem cells (CSC) play critical roles in cancer initiation, progression, and therapeutic refractoriness. Although many studies have focused on the genes and pathways involved in stemness, characterization of the factors in the tumor microenvironment that regulate CSCs is lacking. In this study, we investigated the effects of stromal fibroblasts on breast cancer stem cells. We found that compared with normal fibroblasts, primary cancer-associated fibroblasts (CAF) and fibroblasts activated by cocultured breast cancer cells produce higher levels of chemokine (C-C motif) ligand 2 (CCL2), which stimulates the stem cell-specific, sphere-forming phenotype in breast cancer cells and CSC self-renewal. Increased CCL2 expression in activated fibroblasts required STAT3 activation by diverse breast cancer-secreted cytokines, and in turn, induced NOTCH1 expression and the CSC features in breast cancer cells, constituting a cancer-stroma-cancer signaling circuit. In a xenograft model of paired fibroblasts and breast cancer tumor cells, loss of CCL2 significantly inhibited tumorigenesis and NOTCH1 expression. In addition, upregulation of both NOTCH1 and CCL2 was associated with poor differentiation in primary breast cancers, further supporting the observation that NOTCH1 is regulated by CCL2. Our findings therefore suggest that CCL2 represents a potential therapeutic target that can block the cancer-host communication that prompts CSC-mediated disease progression.

OC-121 Fibroblast activation in the tumour microenvironment promotes tumour cell invasion and resistance to chemotherapy in oesophageal adenocarcinoma

IntroductionStromal and other non-malignant cells have the potential to undergo modifications that can synergistically create a supportive microenvironment for tumour growth, invasion and metastasis. Oesophageal adenocarcinoma (EAC) is characterised by...

Targeting of distinct signaling cascades and cancer-associated fibroblasts define the efficacy of Sorafenib against prostate cancer cells

Sorafenib, a multi-tyrosine kinase inhibitor, kills more effectively the non-metastatic prostate cancer cell line 22Rv1 than the highly metastatic prostate cancer cell line PC3. In 22Rv1 cells, constitutively active STAT3 and ERK are targeted by sorafenib, contrasting with PC3 cells, in which these kinases are not active. Notably, overexpression of a constitutively active MEK construct in 22Rv1 cells stimulates the sustained phosphorylation of Bad and protects from sorafenib-induced cell death. In PC3 cells, Src and AKT are constitutively activated and targeted by sorafenib, leading to an increase in Bim protein levels. Overexpression of constitutively active AKT or knockdown of Bim protects PC3 cells from sorafenib-induced killing. In both PC3 and 22Rv1 cells, Mcl-1 depletion is required for the induction of cell death by sorafenib as transient overexpression of Mcl-1 is protective. Interestingly, co-culturing of primary cancer-associated fibroblasts (CAFs) with 22Rv1 or PC3 cells protected the cancer cells from sorafenib-induced cell death, and this protection was largely overcome by co-administration of the Bcl-2 antagonist, ABT737. In summary, the differential tyrosine kinase profile of prostate cancer cells defines the cytotoxic efficacy of sorafenib and this profile is modulated by CAFs to promote resistance. The combination of sorafenib with Bcl-2 antagonists, such as ABT737, may constitute a promising therapeutic strategy against prostate cancer.

Abstract C235: Interleukin-6 secreted by cancer-associated fibroblasts mediates gemcitabine resistance in pancreatic cancer.

Abstract Introduction: Pancreatic ductal adenocarcinoma is characterized by a dense stroma containing an abundance of cancer-associated fibroblasts (CAFs). It is also notably resistant to gemcitabine. We hypothesize that the large population of CAFs in pancreatic tumors secrete elevated levels of multiple soluble factors, which promote survival of pancreatic cancer cells and reduce their sensitivity to gemcitabine. Previously, we found that growth of MiaPaCa-2 and BxPC-3 tumors was suppressed with gemcitabine treatment, but that of tumors from MiaPaCa-2 and BxPC-3 co-injected with primary pancreatic CAFs was not. This suggests that the presence of CAFs causes pancreatic cancer cells to become more resistant to gemcitabine. Subsequent Affymetrix analyses showed that interleukin-6 (IL-6) is one of 382 genes that are differentially upregulated by &amp;gt;2-fold (P&amp;lt;0.05) in primary pancreatic CAFs as compared to normal fibroblasts. The objectives of the current study are to validate the expression/secretion of IL-6 as well as other components of the IL-6 signaling pathway, and to determine how IL-6 affects the sensitivity of pancreatic cancer cells to gemcitabine. Methods: Sixteen primary CAF lines were established in culture from resected pancreatic ductal adenocarcinoma tumor tissues. As well, eleven primary orthotopic tumor xenograft lines were established in SCID mice from the resected tissues. Subcutaneous tumors were also grown by injecting 5×106 MiaPaCa-2, BxPC-3, or Panc-1 pancreatic cancer cells into SCID mice. Using ELISA, the levels of IL-6, soluble IL-6 receptor (sIL-6R) and the soluble form of gp130 were evaluated in the primary CAFs and orthotopic tumor xenografts, as well as in several pancreatic cancer cell lines. Levels of mouse IL-6 in the serum of mice bearing orthotopic tumors and subcutaneous cell line xenografts were also assayed. Results: The levels of human IL-6 protein were 1.1- to 79-fold higher in the CAF lines than in normal human fibroblasts. However, human IL-6 protein was not detected in pancreatic cancer cell lines PK8, MiaPaCa-2, Panc-1, Capan-2, BxPC-3, or in normal human pancreatic ductal epithelial cells (HPDEs). The levels of mouse IL-6 protein were 1.2- to 94-fold higher in the serum of mice bearing orthotopic tumors as compared to the serum of normal healthy mice. Interestingly, no mouse IL-6 protein was detected in the serum of mice bearing subcutaneous cancer cell line tumors. Furthermore, high levels of mouse IL-6 (12–100 pg/mg) but not human IL-6 were evident in the lysates of the orthotopic tumors. Human sIL-6Rprotein levels were found to be high (24–65 pg/mg) in pancreatic cancer cell lines, but present only at low levels (∼12 pg/mg) in four of the sixteen CAF lines. It was also detected at 13–66 pg/mg in all of the orthotopic tumors. Soluble gp130 protein was observed at higher levels in the CAFs than in the pancreatic cancer cell lines. It was also detected in all of the orthotopic tumors. Conclusion: Our results demonstrate that CAFs are the major producers of IL-6 in pancreatic tumors. It is likely that IL-6 secreted by CAFs binds to IL-6 receptors on pancreatic cancer cells, resulting in the activation of downstream cell survival proteins. Co-culture experiments are underway to identify these proteins and to determine if inhibition of IL-6 signaling may increase the sensitivity of pancreatic cancer cells to gemcitabine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C235.

Targeting ErbB3-mediated stromal–epithelial interactions in pancreatic ductal adenocarcinoma

Background:We sought to investigate the role of ErbB3-mediated signalling on the interaction between pancreatic cancer-associated fibroblasts (CAF) and carcinoma cells in an effort to disrupt tumourigenic pancreatic ductal adenocarcinoma (PDAC) stromal–epithelial cross-communication.Methods:Primary CAF cultures were established from human PDAC surgical specimens. AsPC-1 pancreatic cancer cell murine subcutaneous xenografts were developed in the presence and absence of CAF and were subsequently treated with epidermal growth factor receptor (EGFR) inhibitors (erlotinib) and ErbB3 inhibitors (MM-121, monoclonal ErbB3 antibody).Results:Cancer-associated fibroblasts were found to secrete neuregulin-1 (NRG-1), which promoted proliferation via phosphorylation of ErbB3 and AKT in AsPC-1 PDAC cells. This signalling cascade was effectively inhibited both in vitro and in vivo by specific ErbB3 blockade with MM-121, with greater degree of tumourigenesis inhibition when combined with erlotinib. The CAF–AsPC-1 pancreatic cancer xenografts reached significantly greater tumour volume than those xenografts lacking CAF and were resistant to the anti-tumour effects of EGFR inhibition with erlotinib.Conclusion:Cancer-associated fibroblasts-derived NRG-1 promote PDAC tumourigenesis via ErbB3-AKT signalling and overcomes single-agent EGFR inhibition. Disruption of this stromally mediated tumourigenic mechanism is best obtained through combined EGFR-ErbB3 inhibition with both erlotinib and MM-121. We have identified the NRG-1/ErbB3 axis as an attractive molecular target for the interruption of tumourigenic stromal–epithelial interactions within the PDAC microenvironment.

Abstract 527: Loss of TGF-β responsiveness in stromal cells of the prostate facilitates early prostate cancer bone lesion development

Abstract The death of late stage prostate cancer patients is commonly due to incurable bone metastasis. Prostate cancer associated fibroblasts (CAF) play a role in cancer progression at the primary site, however it is unclear whether they are significant players at the bone metastatic site. We hypothesize that the primary CAF facilitate prostate tumor cell establishment in the bone environment and initial bone lesion development. Around two thirds of human prostate cancer tissues show loss of stromal TGFbeta type II receptor (Tgfbr2) expression at the primary site. We observed Tgfbr2 expression was also lost in bone marrow adjacent to prostate cancer cells. In addition, prostate cancer bone metastases expressed androgen receptor (AR) in the cancer cells and alpha-smooth muscle actin (SMA) in the adjacent CAF. Neither AR nor SMA was seen in the bone marrow environment when no prostate cancer cells were present. A mouse model of conditional Tgfbr2 knock-out in the stromal cells was used in this study. Since there is no evidence, so far, of CAF metastasis along with cancer cells, we focused on the paracrine effects of CAFs from primary site on prostate cancer bone metastasis. GFP labeled prostate cancer C4-2B cells, were incubated with conditioned medium from primary cultured Tgfbr2-flox or Tgfbr2-KO mouse prostate stromal cells for 48hrs. The C4-2B cells were then injected into the tibiae of SCID mice. Bone lesion was monitored weekly and the tumor growth was imaged by Maestro when the tibiae were harvested. It was found that the conditioned medium from Tgfbr2-KO mice prostate stromal cells promoted prostate cancer growth in the bone and mixed bone lesion development early at week 4 and week 5. The incidence and area of the bone lesions were not significant different between week 6 and 10. The tumor growth in the tibia was not different week 10. After 48 hr of incubation with the conditioned medium from Tgfbr2-KO mice prostate stromal cells, the adhesion of C4-2B cells to collagen I was increased by 5.8 fold compared to the cells without previous conditioned media incubation, and 1.5 fold compared to incubation with stromal conditioned medium from Tgfbr2-Flox mice. To examine the secreted factors from the Tgfbr2-KO prostate stromal cells we performed cytokine antibody array analysis. GCSF, KC(Cxcl1), Cxcl16, LIX(Cxcl5) and Decorin were found to be increased more than 2 fold in the medium conditioned by prostate stromal cells from Tgfbr2-KO mice compared to that conditioned by Tgfbr2-flox mice. In conclusion, loss of TGF-β responsiveness in prostate stromal cells resulted in up-regulation of some cytokines. These changes are proposed to increase prostate cancer cell adhesion in the bone microenvironment promoting early establishment of prostate cancer cells in the bone and subsequent bone lesion development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 527. doi:10.1158/1538-7445.AM2011-527