Cancer-associated Fibroblasts Cells Research Articles

Abstract 5895: Circulating CAF and cCAF circulating tumor cell co clusters are associated with metastatic breast cancer

Abstract Background: Breast cancer metastasis is the cause of breast cancer-related mortality. The tumor microenvironment (TME) plays a critical role in governing tumor initiation, progression and metastasis. In breast cancer, cancer associated fibroblasts (CAFs) are master regulators of the TME. Cancer metastasis occurs in part by transport of circulating tumor cells (CTCs) in the circulation. CTCs in clusters, rather than individual CTCs, have a greater capacity to establish metastases. We recently demonstrated that CAFs also circulate (cCAFs), both in clusters with CTCs and individually, in the blood of the majority of breast cancer patients with metastases, some patients without overt metastases, and in no patients with no evidence of disease. Our lab has also established primary breast cancer and primary CAF cell lines from dissociated breast tumors of different molecular subtypes; these are ideal models to dissect tumor-stromal interactions, both in vitro and in vivo. We hypothesize that cCAFs originate from the primary tumor, and further, that cCAFs cluster with CTCs to facilitate metastatic seeding. Methods: We used separately labeled CAFs and primary breast cancer cells (DT28) co-injected into NSG mice and followed by a novel 2-color IVIS. We also modeled the ability of CAFs and breast cancer cells to form clusters in culture and used our microfluidic filter technology to evaluate the composition of clusters resulting from admixed CAFs and BC cells of differing metastatic capacity – metastatic DT28 and non-metastatic MCF-7. Results: Through sequential dual-color IVIS we demonstrate that we can monitor the presence of CAFs and breast cancer cells at orthotopic tumor injection sites and to sites of distant metastasis, indicating that cCAFs originate from the primary tumor. Evaluation of cCAFs and CTCs in tumor bearing mice indicates that breast cancer cells with high metastatic potential mobilize greater numbers of cCAFs. CAFs formed robust clusters with metastatic DT28 cells, while non-metastatic MCF-7 cells clustered with each other but not with CAFs suggesting that the ability of breast cancer cells to form clusters with CAFs, both in vitro and in vivo, is reflective of the metastatic capability of the breast cancer cell line. Conclusions: The intrinsic metastatic capabilities of breast cancer cells are augmented by contact and clustering with cCAFs. In turn, the ability of CAFs to mobilize into circulation is in part conferred by properties of aggressive breast cancer cells. These data corroborate observations from our pilot clinical study that indicated the presence of cCAFs is overwhelmingly associated with the presence of metastatic breast cancer. Citation Format: Utsav Sharma, Philip Miller, Kelsie Medina-Saenz, Pedro Ferrer, Svetlana Speransky, Toni Yeasky, Dorraya El-Ashry. Circulating CAF and cCAF circulating tumor cell co clusters are associated with metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5895. doi:10.1158/1538-7445.AM2017-5895

Abstract 3840: The National Cancer Institute’s patient-derived models repository (PDMR)

Abstract The National Cancer Institute (NCI) is developing a Patient-Derived Models Repository (PDMR) comprised of quality-controlled early-passage clinically-annotated patient-derived xenografts (PDXs) and in vitro patient-derived cell cultures (PDCs), including tumor cell and cancer-associated fibroblast cell cultures, to serve as a resource for public-private partnerships and for academic drug discovery efforts. These PDMs will be clinically-annotated with molecular information (whole exome sequence, RNASeq) available in a publicly accessible database and will be available to the extramural community for research use. The PDMR was established by NCI at the Frederick National Laboratory for Cancer Research (FNLCR) in direct response to discussions with academia and industry; the oncology community’s highest priority need is better preclinical models that more faithfully reflect the patient’s tumor and are associated with the patient’s treatment history. NCI has focused on collecting specimens from patients with cancer that are under-represented in many other PDX collections such as head and neck, pancreatic, bladder, ovarian and small cell lung cancers, melanomas and sarcomas. In addition, NCI is increasing its focus on creating PDXs from minority/underserved populations and will soon be expanding to include pediatric cancers. The PDMR generates the majority of its PDXs by subcutaneous implantation; however certain histologies have better take-rates in either orthotopic or alternate implant sites. All SOPs and quality-control standards developed by the PDMR as well as those shared by collaborators will be posted to the public web site that houses the PDMR database. The overall goal of NCI is to create a long-term home for at least 1000 models such that sufficient biological and clinical diversity is represented to allow researchers to ask questions such as: what is the impact of tumor heterogeneity on target qualification or clinical response; do PDXs more faithfully represent the human tumor for pharmacodynamic assay and predictive marker development; or can an adequately powered preclinical PDX clinical trial lead to better evaluation of therapies for future clinical use? Grant Support: This project has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. Citation Format: Yvonne A. Evrard, Michelle Ahalt-Gottholm, Sergio Alcoser, Carrie Bonomi, Suzanne Borgel, John Carter, Biswajit Das, Vivekananda Datta, Cheryl Davis, Kelly Dougherty, Michelle Eugeni, Marion Gibson, Catherine Karangwa, Jason Lih, Dianne Newton, Han Si, Shivaani Kummar, Larry Rubinstein, Alice Chen, P. Mickey Williams, Melinda G. Hollingshead, James H. Doroshow. The National Cancer Institute’s patient-derived models repository (PDMR) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3840. doi:10.1158/1538-7445.AM2017-3840

Abstract 958: Blocking CD70+ cancer associated fibroblasts: Are we paving the way towards immunotherapy in colorectal cancer

Abstract Introduction: Numerous studies have reported that tumor progression and invasiveness are determined not only by the malignant cancer cells themselves but also by the surrounding tumor microenvironment, including cancer-associated fibroblasts (CAFs). Although CAFs are implicated in tumor progression, their total depletion of CAFs has been demonstrated to induce more aggressive tumors, indicating that different CAF subpopulations have opposing tumor-promoting or tumor-inhibitory roles. Unfortunately, specific markers to target these subsets of CAFs are lacking. Expression of the immune checkpoint CD70 is normally tightly regulated and limited to cells of the lymphoid lineage only. Instead, tumors hijack CD70 to facilitate immune evasion by increasing the amount of suppressive regulatory T cells (Tregs), inducing T cell apoptosis and skewing T cells towards T cell exhaustion. Recently, a lot of clinical successes have been generated by the blockade of immune checkpoints. However, in colorectal cancer (CRC) the efficacy remains limited to a small subset of patients with mismatch repair-deficient (MSI) tumors which might be caused by the intense dialogue between stroma and malignant cells. Therefore, we have explored the expression patterns of the immune checkpoint molecule CD70 in CRC, with a particular focus on CAFs. Methods: The prognostic value of CD70 was analyzed by immunohistochemistry on 51 CRC specimens. In addition, the relationship with Tregs and microsatellite instability was explored. Furthermore, primary CAF cell lines were successfully cultured from 20 different primary resection specimens. These cell lines were used to study the effect of CD70 on the tumor microenvironment in vitro. Results: We revealed expression of CD70, not just on the malignant cells but on the majority of CAFs in invasive CRC specimens. Thereby, CD70-expression was significantly correlated with negative clinicopathological parameters such as metastasis (P=0.007), differentiation (P=0.053) and advanced stage (P=0.001). Moreover, CD70-positive CAFs proved to be a poor prognostic marker by univariate as well as multivariate analysis. We have also detected a significant association between elevated Treg amounts and CD70-expressing CAFs (P=0.012). In vitro data on the effects of CD70 on CRC behavior and immune escape are currently being analyzed. Conclusion: We have identified a new targetable CAF subpopulation, marked by the expression of CD70 and equipped with strong tumor-promoting properties. Thereby, we have found evidence of a potential cross talk between CD70+ CAFs and Treg, paving the way towards immune escape. The lack of association of CD70 expression and MSI-status, which highlights the potential of this target in CRC subsets that do not benefit from immune checkpoint blockade. We believe that targeting CD70 holds great potential in CRC, especially in light of the limited immunotherapeutic options available. Citation Format: Julie Jacobs, Vanessa Deschoolmeester, Karen Zwaenepoel, Christophe Hermans, Christian Rolfo, Marc Peeters, Filip Lardon, Vasiliki Siozopoulou, Evelien Smits, Patrick Pauwels. Blocking CD70+ cancer associated fibroblasts: Are we paving the way towards immunotherapy in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 958. doi:10.1158/1538-7445.AM2017-958

Activation of mutated TRPA1 ion channel by resveratrol in human prostate cancer associated fibroblasts (CAF).

Previous studies showed the effects of resveratrol (RES) on several cancer cells, including prostate cancer (PCa) cell apoptosis without taking into consideration the impact of the tumor microenvironment (TME). The TME is composed of cancer cells, endothelial cells, blood cells, and cancer-associated fibroblasts (CAF), the main source of growth factors. The latter cells might modify in the TME the impact of RES on tumor cells via secreted factors. Recent data clearly show the impact of CAF on cancer cells apoptosis resistance via secreted factors. However, the effects of RES on PCa CAF have not been studied so far. We have investigated here for the first time the effects of RES on the physiology of PCa CAF in the context of TME. Using a prostate cancer CAF cell line and primary cultures of CAF from prostate cancers, we show that RES activates the N-terminal mutated Transient Receptor Potential Ankyrin 1 (TRPA1) channel leading to an increase in intracellular calcium concentration and the expression and secretion of growth factors (HGF and VEGF) without inducing apoptosis in these cells. Interestingly, in the present work, we also show that when the prostate cancer cells were co-cultured with CAF, the RES-induced cancer cell apoptosis was reduced by 40%, an apoptosis reduction canceled in the presence of the TRPA1 channel inhibitors. The present work highlights CAF TRPA1 ion channels as a target for RES and the importance of the channel in the epithelial-stromal crosstalk in the TME leading to resistance to the RES-induced apoptosis.

MP87-13 ESTROGEN RECEPTOR α IN CANCER ASSOCIATED FIBROBLASTS SUPPRESSES PROSTATE CANCER INVASION VIA REDUCING CCL5, IL6 AND MACROPHAGE INFILTRATION IN THE TUMOR MICROENVIRONMENT.

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology III1 Apr 2017MP87-13 ESTROGEN RECEPTOR α IN CANCER ASSOCIATED FIBROBLASTS SUPPRESSES PROSTATE CANCER INVASION VIA REDUCING CCL5, IL6 AND MACROPHAGE INFILTRATION IN THE TUMOR MICROENVIRONMENT. Yang Yang, Chiuan-Ren Yeh, Spencer Slavin, Jie Luo, Fu-Ju Chou, Keliang Wang, Matthew Truong, Chawnshang Chang, Edward M. Messing, and Shuyuan Yeh Yang YangYang Yang More articles by this author , Chiuan-Ren YehChiuan-Ren Yeh More articles by this author , Spencer SlavinSpencer Slavin More articles by this author , Jie LuoJie Luo More articles by this author , Fu-Ju ChouFu-Ju Chou More articles by this author , Keliang WangKeliang Wang More articles by this author , Matthew TruongMatthew Truong More articles by this author , Chawnshang ChangChawnshang Chang More articles by this author , Edward M. MessingEdward M. Messing More articles by this author , and Shuyuan YehShuyuan Yeh More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2717AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Cancer associated fibroblasts (CAF) play important roles in tumor growth that involves inflammation and epithelial cell differentiation. Early studies suggested that estrogen receptor alpha (ERα) was expressed in stromal cells in normal prostates and prostate cancer (PCa), but the detailed functions of stromal ERα in the PCa remain to be further elucidated. METHODS In vitro study, we applied migration and invasion assays to demonstrated whether the levels of ERα in CAF cells (CAF.ERα(+)) could suppress PCa invasion via influencing the infiltration of tumor associated macrophages. Q-PCR, Elisa assay and luciferase assay were used to detect which cytokines may mediate the CAF.ERα(+) suppressed macrophage infiltration and PCa invasion. In vivo study, nude mice were orthotopically implanted with CAF.ERα(+) or CAF.ERα(-) mixed with CWR22Rv1 cells. Then, IHC was used to verify the data in vitro study. We further examined the ERα, CD206, CCL5 and IL6 expressions in 14 human PCa tissue specimens by IHC staining. RESULTS Both in vitro and in vivo mouse PCa model studies demonstrated CAF.ERα(+) led to a reduced macrophage migration toward PCa via inhibiting CAF cells secreted chemokine CCL5 (Figure1 and 3). This CAF.ERα(+) suppressed macrophage infiltration affected the neighboring PCa cells invasion and the reduced invasiveness of PCa cells are at least partly due to reduced IL6 expression in the macrophages and CAF. (Figure 2). In human PCa tissues, our results showed a positive correlation between ERα, M2 macrophages, CCL5 and IL-6. In high stromal ERα expression samples, the expression levels of CD206 (M2 macrophage marker), CCL5 and IL6 were higher than in samples with low stromal ERα expression (Figure 4). CONCLUSIONS Our data suggest that CAF ERα could be applied as a prognostic marker to predict the recurrence-free survival, and targeting CCL5 and IL6 may be applied as an alternative therapeutic approach to reduce M2 type macrophages and PCa invasion in PCa patients with low or little ERα expression in CAF cells. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1170-e1171 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Yang Yang More articles by this author Chiuan-Ren Yeh More articles by this author Spencer Slavin More articles by this author Jie Luo More articles by this author Fu-Ju Chou More articles by this author Keliang Wang More articles by this author Matthew Truong More articles by this author Chawnshang Chang More articles by this author Edward M. Messing More articles by this author Shuyuan Yeh More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Androgen receptor in cancer-associated fibroblasts influences stemness in cancer cells.

Androgen receptor (AR) regulation pathways are essential for supporting the growth and survival of prostate cancer cells. Recently, sub-populations of prostate cancer cells have been identified with stem cell features and are associated with the emergence of treatment-resistant prostate cancer. Here, we explored the function of AR in prostate cancer-associated fibroblasts (CAFs) relative to growth and stem cell-associated characteristics. CAFs were isolated from the murine cPten-/-L prostate cancer model and cultured with human prostate cancer epithelial (hPCa) cells. A murine-specific AR antisense oligonucleotide (ASO) was used to suppress the expression of AR in the CAF cells. CAFs express low, but significant levels of AR relative to fibroblasts derived from non-malignant tissue. CAFs promoted growth and colony formation of hPCa cells, which was attenuated by the suppression of AR expression. Surprisingly, AR-depleted CAFs promoted increased stem cell marker expression in hPCa cells. Interferon gamma (IFN-γ) and macrophage colony-stimulating factor (M-CSF) were increased in AR-depleted CAF cells and exhibited similar effects on stem cell marker expression as seen in the CAF co-culture systems. Clinically, elevated IFN-γ expression was found to correlate with histologic grade in primary prostate cancer samples. In summary, AR and androgen-dependent signaling are active in CAFs and exert significant effects on prostate cancer cells. IFN-γ and M-CSF are AR-regulated factors secreted by CAF cells, which promote the expression of stem cell markers in prostate cancer epithelial cells. Understanding how CAFs and other constituents of stromal tissue react to anti-cancer therapies may provide insight into the development and progression of prostate cancer.

Abstract P4-03-18: A hierarchy of cancer associated fibroblasts in situ and in circulation promote breast cancer metastasis

Abstract Background: Metastasis is the primary cause of breast cancer mortality. Interactions between cancer cells and non-cancer cells of the tumor microenvironment (TME) are pivotal in governing tumor initiation, progression and metastasis, and cancer associated fibroblasts (CAFs) are critical orchestrators of these interactions. We recently identified circulating CAFs (cCAFs) as a novel circulating biomarker associated with metastatic breast cancer. We established CAF cell lines from dissociated luminal A, ER- Her-2 amplified, and triple-negative/basal-like (TN) breast tumors. We demonstrated that “aggressive” CAFs differentially secrete miRNAs that contribute to ER-negativity, activated growth factor signaling, and induction of EMT in breast cancers compared to “indolent” CAFs. We hypothesized that a hierarchy exists within CAFs regarding their ability to facilitate tumor progression and metastasis. Here we demonstrate that CAFs derived from aggressive TN breast tumors differ from those derived from more indolent Luminal A breast tumors in secretion of cytokines and chemokines that can confer differential effects on the behavior of breast cancer cells. We also demonstrate that “aggressive” CAFs more potently facilitate tumor progression and metastasis than “indolent” CAFs. We additionally evaluated if “aggressive” and “indolent” CAFs differ in their ability to mobilize CTCs and circulating CAFs into circulation. Methods: Conditioned media (CM) from “aggressive” and “indolent” CAFs was analyzed for chemokine/cytokine expression. Luminal A breast cancer cells (MCF-7) or primary tumor cells from an aggressive TN tumor (DT28) were injected into the mammary fat pad of 6-8 week old female NSG mice, either alone or in combination with CAF19-I or CAF23-A. Tumor progression was monitored and mice were examined for metastasis at necropsy. Tissues were harvested for histology and blood was collected by cardiac puncture. Plasma was analyzed for cytokine/chemokine expression and blood was processed for enumeration of circulating tumor cells (CTCs) and cCAFs. Results: “Aggressive” CAF CM had significantly higher levels of a number of factors, including IL-8, SDF-1, and CXCL1, compared to “indolent” CAF CM. MCF-7 cells co-injected with “aggressive” CAFs formed tumors much faster than those co-injected with the “indolent” CAFs or without CAFs. While DT28 cells readily form tumors and metastasize in the NSG model, fewer DT28 cells do not form metastases in the timeframe that this same lower number of DT28 cells co-injected with “aggressive” CAFs demonstrated robust tumor growth and developed metastases in liver and pancreas. DT28 cells co-injected with “indolent” CAFs did not exhibit metastases. Conclusion: The data presented here further demonstrate that there is a hierarchy within CAFs regarding their ability to facilitate tumor growth and metastasis, and that this may largely be mediated by secreted soluble factors. “Aggressive” CAFs may retain their programmed role in circulation and accelerate metastasis more than “indolent” CAFs. We suggest that targeting CAFs in situ and in circulation and disrupting their interactions with breast cancer cells could provide novel strategies to combat breast cancer and breast cancer metastasis. Citation Format: Sharma U, Miller P, Speransky S, Medina-Saenz K, Ferrer P, Lippman M, El-Ashry D. A hierarchy of cancer associated fibroblasts in situ and in circulation promote breast cancer metastasis [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-03-18.

Brain derived neutrophic factor (BDNF) coordinates lympho-vascular metastasis through a fibroblast-governed paracrine axis in the tumor microenvironment.

It has long been known that the tumor microenvironment contributes to the proliferation and survival of neoplasms through the constant interaction with the stromal and immune compartments. In this investigation, we explored the role of cancer-associated fibroblasts (CAFs) in the regulation of the tumor microenvironment in head and neck squamous cell carcinoma (HNSCC) though a complex intercellular BDNF-TrkB signaling system. Our studies show that conditioned media derived from patient-derived CAFs promoted HNSCC cell proliferation, in vitro cell migration, cell invasion and chemotherapy resistance, compared to normal fibroblasts. Furthermore, examination of the in vivo impact of CAF pathophysiology in the tumor microenvironment in animal xenograft models revealed that HNSCC cell lines in combination with CAFs promoted tumor growth and increased incidence of lymphovascular metastasis as compared to injection of tumor cells or CAF cells alone. Using pharmacological and genetic alterations, we mechanistically demonstrate the critical importance of BDNF-TrkB signaling in the tumor microenvironment. These investigations further support the rationale for BDNF/TRKB targeted therapy against in the treatment of HNSCC.

Rapid determination of the tumour stroma ratio in squamous cell carcinomas with desorption electrospray ionization mass spectrometry (DESI-MS): a proof-of-concept demonstration

Squamous cell carcinomas constitute a major class of head & neck cancers, where the tumour stroma ratio (TSR) carries prognostic information. Patients affected by stroma-rich tumours exhibit a poor prognosis and a higher chance of relapse. As such, there is a need for a technology platform that allows rapid determination of the tumour stroma ratio. In this work, we provide a proof-of-principle demonstration that Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) can be used to determine tumour stroma ratios. Slices from three independent mouse xenograft tumours from the human FaDu cell line were subjected to DESI-MS imaging, staining and detailed analysis using digital pathology methods. Using multivariate statistical methods we compared the MS profiles with those of isolated stromal cells. We found that m/z 773.53 [PG(18:1)(18:1) - H]-, m/z 835.53 [PI(34:1) - H]- and m/z 863.56 [PI(18:1)(18:0) - H]- are biomarker ions that can distinguish FaDu cancer from cancer associated fibroblast (CAF) cells. A comparison with DESI-MS analysis of controlled mixtures of the CAF and FaDu cells showed that the abundance of the biomarker ions above can be used to determine, with an error margin of close to 5% compared with quantitative pathology estimates, TSR values. This proof-of-principle demonstration is encouraging and must be further validated using human samples and a larger sample base. At maturity, DESI-MS thus may become a stand-alone molecular pathology tool providing an alternative rapid cancer assessment without the need for time-consuming staining and microscopy methods, potentially further conserving human resources.

Invasion of low passage oral carcinoma cells in organotypic models is influenced by the source of both the cancer and cancer-associated fibroblast cells

Invasion of low passage oral carcinoma cells in organotypic models is influenced by the source of both the cancer and cancer-associated fibroblast cells

A Multiscale Approach to the Migration of Cancer Stem Cells: Mathematical Modelling and Simulations.

We propose a multiscale model for the invasion of the extracellular matrix by two types of cancer cells, the differentiated cancer cells and the cancer stem cells. We investigate the epithelial mesenchymal-like transition between them being driven primarily by the epidermal growth factors. We moreover take into account the transdifferentiation program of the cancer stem cells towards the cancer-associated fibroblast cells as well as the fibroblast-driven remodelling of the extracellular matrix. The proposed haptotaxis model combines the macroscopic phenomenon of the invasion of the extracellular matrix by both types of cancer cells with the microscopic dynamics of the epidermal growth factors. We analyse our model in a component-wise manner and compare our findings with the literature. We investigate pathological situations regarding the epidermal growth factors and accordingly propose "mathematical-treatment" scenarios to control the aggressiveness of the tumour.

Prometastatic mechanisms of CAF-mediated EMT regulation in pancreatic cancer cells.

Tumor metastasis are accompanied by the EMT (epithelial-mesenchymal transition)-MET (mesenchymal-epithelial transition) two-step process. In this study, we investigated the importance of cancer associated fibroblasts (CAF) in the process. First, the primary cultures of isolated pancreatic CAF, fibroblasts of normal pancreatic tissues(NF), and normal hepatic stellate cells (HSF) were identified and verified via the expression of α-SMA and vimentin. Using an indirect three-dimensional co-culture model, the morphological changes were observed by light microscopy and laser scanning confocal microscopy. The invasive and migration capacity of pancreatic cancer cells was determined by Transwell chamber migration assay or scratch assay. The mRNA and protein expression levels of E-cadherin, vimentin, and Gli1 were determined by RT-PCR and western blotting. Primary cultures of isolated CAF, NF, HSF showed satisfactory growth with active proliferation. Indirect co-culture with CAF, BxPc-3 and Panc-1 cells showed significant partial EMT, reduced E-cadherin expression, and enhanced vimentin expression as compared with the single culture and NF/HSF co-culture groups, with corresponding increases in migratory and invasive capacities. PCR and western blotting results showed that mRNA and protein expression levels of Gli1 in CAF and Snail in cancer cells were increased. This process could be reversed by inhibition of hedgehog (HH) signaling in CAF. In the tumor microenvironment, activation of CAF is the key event in mediating partial EMT, and its mechanism may be associated with paracrine action after activation of HH signaling in CAF.

Growth differentiation factor 15 contributes to cancer-associated fibroblasts-mediated chemo-protection of AML cells.

BackgroundChemo-resistance is still a major obstacle in efforts to overcome acute myeloid leukemia (AML). An emerging concept has proposed that interactions between the bone marrow (BM) microenvironment and leukemia cells reduce the sensitivity of the leukemia cells to chemotherapy. As an important element of the tumor microenvironment, the cancer-associated fibroblasts (CAFs) are considered to be activated modulators in the chemo-resistance of many solid tumors. But their contribution to AML has yet to be fully understood. Here we report a critical role for CAFs which were thought to be a survival and chemo-protective factor for leukemia cells.MethodsA retrospective study on the BM biopsies from 63 primary AML patients and 59 normal controls was applied to quantitative analysis the fiber stroma in the BM sections. Then immunohistochemistry on the BM biopsies were used to detect the makers of the CAFs. Their effects on drug resistance of leukemia cells were further to be assessed by co-cultured experiments in vitro. Moreover, the possible mechanisms involved in CAF-mediated chemo-protection of AML cells was investigated by antibody neutralization and siRNA knockdown experiments, with particular emphasis on the role of GDF15.ResultsIn our study, excessive reticular fibers in the BM led to higher frequency of relapse and mortality in primary AML patients, bringing the inspiration for us to investigate the functional roles of the fiber-devied cells. We declared that the CAF cells which expressed higher levels of FSP1, α-SMA or FAP protein were widely distributed in the marrow of AML. Then in vitro co-cultured tests showed that these CAFs could protect leukemia cell lines (THP-1/K562) from chemotherapy. Interestingly, this effect could be decreased by either treatment with a neutralizing anti-GDF15 antibody or knockdown GDF15 (with siGDF15) in CAFs. Furthermore, we also confirmed that the GDF15+ cells mainly co-localized with FAP, which was identified as the typical phenotype of CAFs in the BM stroma.ConclusionsWe firstly demonstrate that the functional CAFs are widespread within the BM of AML patients and should be a critical chemo-protective element for AML cells by producing amount of GDF15.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0405-0) contains supplementary material, which is available to authorized users.

Reduced Contractility and Motility of Prostatic Cancer-Associated Fibroblasts after Inhibition of Heat Shock Protein 90

Background: Prostate cancer-associated fibroblasts (CAF) can stimulate malignant progression and invasion of prostatic tumour cells via several mechanisms including those active in extracellular matrix; Methods: We isolated CAF from prostate cancer patients of Gleason Score 6–10 and confirmed their cancer-promoting activity using an in vivo tumour reconstitution assay comprised of CAF and BPH1 cells. We tested the effects of heat shock protein 90 (HSP90) inhibitors upon reconstituted tumour growth in vivo. Additionally, CAF contractility was measured in a 3D collagen contraction assay and migration was measured by scratch assay; Results: HSP90 inhibitors dipalmitoyl-radicicol and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) reduced tumour size and proliferation in CAF/BPH1 reconstituted tumours in vivo. We observed that the most contractile CAF were derived from patients with lower Gleason Score and of younger age compared with the least contractile CAF. HSP90 inhibitors radicicol and 17-DMAG inhibited contractility and reduced the migration of CAF in scratch assays. Intracellular levels of HSP70 and HSP90 were upregulated upon treatment with HSP90 inhibitors. Inhibition of HSP90 also led to a specific increase in transforming growth factor beta 2 (TGFβ2) levels in CAF; Conclusions: We suggest that HSP90 inhibitors act not only upon tumour cells, but also on CAF in the tumour microenvironment.

Abstract IA12: NCI's patient-derived cancer models repository

Abstract National Cancer Institute is developing a national repository of patient-derived cancer models (PDMs) comprised of clinically-annotated patient-derived xenografts (PDXs); patient-derived tumor cell cultures (PDCs, including conditionally-reprogrammed tumor cell cultures) prepared from primary and metastatic tumors, circulating tumor cells (CTCs), and/or PDXs; tumor cell lysates, DNA, and RNA; and cancer-associated fibroblast cell lines (CAFs, autologous when possible) to serve as a resource for academic discovery efforts and public-private partnerships for drug discovery. NCI will provide a long-term home for >1000 PDX and PDC models, each produced from tissues and blood supplied by NCI-designated Cancer Centers and NCI-supported clinical trials networks. The effort is targeting the collection of tumors that are less prevalent in current resources, such as: small cell lung cancer, prostate cancer, bladder cancer, pancreatic cancer, head and neck cancers, as well as sarcomas and melanomas. The goals of the project are: (1) to develop a minimum of ~50 unique patient models (both PDXs and PDCs) per disease such that the size of each molecularly-characterized subgroup is useful for subsequent validation and/or efficacy studies; (2) to perform comprehensive pre-competitive molecular characterization of patient samples and earliest passage PDXs and PDCs that includes the NCI-MPACT mutation panel, WES, RNASeq, copy number determination, histology, growth curves, and pilot proteomic/phospho-proteomic studies; and (3) to make all models and associated pre-clinical and clinical data available through a publicly available website. To date, over 1700 specimens from 1100 patients have been received for the development of PDMs; the overall take rate for PDXs originating from solid tumors is 70% with >170 assessable models and another 270 early passage tumors currently in evaluation. As expected, based on collection priorities, tumors of genitourinary, digestive, head and neck, musculoskeletal, respiratory, and skin origin are the major histological sites of origin for our PDX models. In addition, over 90 conditionally-reprogrammed cell lines have been expanded from both 18-gauge needle biopsies and surgical resections, and have passed initial quality control procedures; many of these cell cultures have a matched PDX. Over 150 CAF lines have been developed following repeated (>10) purification steps using flow cytometry and are in the process of quality control procedures that demonstrate complete lack of growth in NOD-SCID gamma IL2 receptor null (NSG) mice; of these CAFs, we have developed matched pairs of PDCs and CAFs from the same patient in 16 cases. To evaluate the potential utility of the NCI PDM Repository, we have prospectively “entered” 22 models in a pre-clinical trial for which eligibility (based on actionable mutations) and treatment arms are identical to those in the NCI-MPACT study (NCT01827384). Multiple objective responses (significant improvement in overall survival) have been observed in all arms of the study and in a variety of models. WES and RNASeq analysis have proven essential to explain the therapeutic responses that we have observed. We are also evaluating the relationship of in vitro and in vivo activity for the NCI-MPACT drug panel in the models where concurrent PDCs and PDXs have been produced. A web site has been developed that will provide annotated information on the models (such as DNA sequence, gene expression, prior therapy) to investigators to assist in the distribution of the contents of the repository to the research community. We expect to be able to begin distribution in late spring of 2016. Citation Format: James H. Doroshow. NCI's patient-derived cancer models repository. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr IA12.

Abstract A32: Il-6 initiates a self-sustaining signaling loop to promote nurturing tumor microenvironment

Abstract Desmoplasia, or deposition of connective tissue proper in the stroma, is a key feature of esophageal squamous cell carcinoma (ESCC), a common malignancy worldwide. The cellular component of connective tissue proper is comprised of fibroblasts, and cancer-associated fibroblasts (CAFs) are widely recognized as a major constituent of the tumor microenvironment (TME). The purpose of this study was to investigate the cross-talk between CAFs and tumor cells in ESCC, as well as identify factors that mediate this cross-talk and evaluate these factors as therapeutic targets in ESCC and potentially other squamous cell carcinomas. We have observed that co-culture with CAFs prompts ESCC cells to acquire a more mesenchymal phenotype and express potent mediators of cell migration and invasion, such as matricellular proteins (periostin, fibrillin, osteonectin) and growth factors (EGF, HGF, VEGF). Furthermore, co-culture of ESCC cells with CAFs promotes tumor cell migration in our 3D organotypic culture, while co-transplantation of ESCC cells with fibroblasts leads to enhanced tumor growth in vivo, compared to tumors transplanted without fibroblasts. In order to identify potential mediators of ESCC-CAF cross-talk, we have performed a comprehensive cytokine array and found that interleukin 6 (IL-6) is significantly overexpressed in conditioned media from co-culture of esophageal CAFs and ESCC cells, compared to mono-culture. IL-6 is known to play important roles in the development of multiple types of cancer, mostly via activation of the STAT3 signaling pathway. To confirm relevance of our in vitro findings, we have performed immunohistochemical staining of tissue samples from ESCC patients and found that, compared to normal esophagus, expression of IL-6 is enhanced in ESCC in both epithelial cells and fibroblasts. Interestingly, knockdown of IL-6 resulted in altered morphology of ESCC cells in 3D organotypic culture. This was accompanied by restored membrane localization of E-cadherin and reduced activation of STAT3 signaling. In order to investigate the importance of STAT3 and MEK/ERK signaling (the latter being another pathway activated by IL-6 in cancer) on ESCC cell biology, we have treated 3D organotypic cultures with stattic and trametinib (inhibitors of STAT3 and MEK, respectively). This treatment resulted in impaired invasion, reduced proliferation and enhanced apoptosis of ESCC cells, accompanied by decreased expression of the basal cell marker p63. Furthermore, treatment with stattic and trametinib leads to reduced secretion of IL-6 by these cultures. These effects were more pronounced upon treatment with combination of stattic and trametinib. Since we found IL-6 to be important for ESCC progression in vitro, we have conducted a therapeutic study utilizing tocilizumab (an FDA-approved anti-human IL-6R neutralizing antibody). Subcutaneous xenograft tumors comprised of human ESCC and CAF cell lines, were treated with tocilizumab, which resulted in suppressed tumor growth, accompanied by decreased activation of STAT3 and ERK within the tumor, compared to control IgG-treated tumors. Furthermore, tocilizumab-treated animals had reduced numbers of splenic monocytic immature myeloid cells (CD11b+Ly-6C+), which is in agreement with previously published findings regarding MDSCs in murine models of ESCC. In summary, our findings indicate that IL-6, at least in part, is responsible for migration, proliferation, survival and poor differentiation of ESCC cells, promoted by CAFs in the TME. We also report that these effects of IL-6 are mediated by STAT3 and ERK signaling. Finally, we demonstrate that inhibition of IL-6 signaling suppresses ESCC tumor growth in vivo. This is the first report of anti-tumorigenic activity of tocilizumab in ESCC, which makes it a promising candidate for ESCC therapy. Supported by NCI P01 CA098101. Citation Format: Tatiana A. Karakasheva, Monica Soni, Todd J. Waldron, Eric W. Lin, Philip D. Hicks, Andres Klein-Szanto, Kwok K. Wong, Adam J. Bass, Anil K. Rustgi. Il-6 initiates a self-sustaining signaling loop to promote nurturing tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A32.

Abstract 3026: Galunisertib inhibits ovarian cancer cell fibrosis and metastasis in vitro

Abstract Background: In ovarian cancer (OC), the intraperitoneal fibrosis that accompanies metastasis is a common scenario and associated with significant morbidity and gut dysfunction. Accumulating evidences indicate that Transforming Growth Factor β (TGF-β) signaling pathway plays a key role in cancer associated fibrosis and metastasis. Several TGF-β inhibitors, including Galunisertib (LY2157299) which is a TGF-β receptor I inhibitor, are currently in clinical trials for fibrosis-related diseases and cancer. Our hypothesis is that targeted anti-cancer therapies can block the growth and spread of cancer or specifically address the fibrosis accompanying metastatic peritoneal cancer. In this study, we investigated the efficacy of Galunisertib in inhibiting OC fibrosis and metastasis in vitro. Methods: Five OC cell lines, including SKOV3ip, HeyA8, OVCAR8, OV2008, and PEO4, and one cancer-associated fibroblast (CAF) cell line, TRS3, were tested in this study. We utilized a co-culture system that has successfully been used to study the interactions between cancer cells and local environment, including CAFs. Twenty-three candidate TGF-β signaling- and fibrosis-related genes were selected based on previous studies. RNA and protein from both fibroblast cells and OC cells were collected for RT-qPCR and western blot analysis. MTT assay, Wound healing assay and Matrigel invasion assay were used to study OC cell proliferation, migration and invasion. Chou-Talalay assays were performed to study the interactions between Galunisertib and Carboplatin and Paclitaxel. Results: RT-qPCR results showed that Galunisertib treatment suppresses the expression of both TGF-β receptor I and TGF-β1 which is the most abundant isoform of TGF-β in OC. Western blot results showed that Galunisertib treatment reverses the TGF-β1-induced SMAD2/3 phosphorylation, confirming the efficacy of Galunisertib in TGF-β signaling pathway blockage. Intriguingly, Galunisertib treatment inhibits the expression of candidate fibrosis-related genes, like ACTA2, TIMP3, COL5A1, COL11A1, and VCAN in both OC and CAF cells. Would healing and Transwell invasion assay also demonstrated that Galunisertib treatment effectively reduces both basal and TGF-β1-induced cell migration and invasion (p<0.05). MTT assay showed that Galunisertib is able to inhibit the proliferation of both OC and CAFs cells with IC50 values range from 100-400 μm. Additionally, Galunisertib shows strong synergistic effects with both Carboplatin and Paclitaxel in Chou-Talalay assays. Conclusion: Galunisertib treatment suppresses the expression of TGF-β-dependent fibrosis-related genes from OC cell lines and CAFs. Coincident with this inhibition of fibrosis related genes, galunisrtib reduces cell proliferation, migration, and invasion. These data suggest that targeting the TGF-β pathway is a viable therapeutic approach to reduce both symptoms related to OC-related fibrosis and cancer metastasis in OC. Citation Format: Qing Zhang, William A. Cliby. Galunisertib inhibits ovarian cancer cell fibrosis and metastasis in vitro. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3026.

Abstract 299: Patient-derived tumor microenvironment models uncover non-autonomous TKI resistance mechanisms in NSCLC

Abstract Background: Tyrosine kinase inhibitors (TKI) have yielded great responses in non-small-cell lung cancer (NSCLC) with EGFR mutations and ALK translocations, however these and other targeted therapies are limited by intrinsic and acquired drug resistance. Previous studies looking into relapsed samples largely expanded our understanding of tumor autonomous resistance mechanisms. In this study, we aimed to decipher the non-autonomous mechanisms in vitro by recapitulating the in vivo tumor microenvironment from patient-derived models. Method: Cancer-associated fibroblast cells are isolated directly from EGFR mutant and ALK translocated NSCLC biopsies. Over 20 patient-derived fibroblast (PDF) models have been established for studying the tumor microenvironment's effect on TKI response. Result: We found that many, but not all, PDF cells exert significant resistance to TKIs. Furthermore, there is considerable variability in the degree and secreted factor / mechanism by which they are protecting the tumor cells. These variations are further correlated with patient in vivo response. Conclusion: Together, our results indicate that PDFs are clinically relevant models for deciphering non-autonomous resistance mechanisms, that they are heterogeneous in protecting cancer cells from TKI treatment, and that the resistance mediated by PDFs can be overcome by specific therapeutic combinations. Citation Format: Haichuan Hu, Matthew Niederst, Jeffrey Engelman. Patient-derived tumor microenvironment models uncover non-autonomous TKI resistance mechanisms in NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 299.

Abstract 764: LOX and LOXL2 inhibition as a treatment for ovarian cancer

Abstract Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy in women. Despite advances in our understanding of the molecular biology of EOC, patient survival has not significantly improved in decades and new treatments are needed. There is increased interest in therapeutic targeting of the tumor microenvironment (TME). The lysyl oxidase (LOX) family of enzymes are involved in extracellular matrix remodelling through crosslinking of collagen and elastin. They have been shown to play an important role in the TME of other cancers and are regulated by hypoxia, however little is known about their role in EOC. Therefore we aimed to investigate the role of LOX and LOXL2 in EOC using in vitro models. Tumor epithelial (n = 2) and matched cancer-associated fibroblast cell lines (n = 2) derived from an SV40-induced mouse model of EOC were generated. MTT assays were performed on all cell lines to assess the effect of LOX and LOXL2 inhibition on cell viability. The effect of LOX and LOXL2 inhibition on cancer cell migration and invasion towards cancer-associated fibroblast conditioned media was also investigated using transwell assays. RNA and protein were isolated and analysed from cells cultured in normoxic and hypoxic conditions to assess whether LOX and LOXL2 gene and protein expression is regulated by hypoxia. Inhibition of LOX and LOXL2 did not affect cell viability. LOX and LOXL2 inhibition significantly reduced the migration and invasion of EOC cells towards cancer-associated fibroblast conditioned media in vitro (p<0.05). Gene expression analysis comparing Lox and Loxl2 expression in all cell lines demonstrated a significant increase in Lox and Loxl2 expression in cells cultured in hypoxia (p<0.05). Similarly, cellular and secreted LOX and LOXL2 protein expression was increased in cells cultured in hypoxic conditions, supporting hypoxia as a regulator of LOX and LOXL2 expression. This study demonstrates a role for LOX and LOXL2 in promoting migration and invasion of EOC cells in vitro. The upregulation of LOX and LOXL2 in hypoxia suggests a role in facilitating tumor growth and progression within hypoxic tumors. These results provide support for the utility of LOX and LOXL2 inhibition as promising and novel therapeutic strategies in EOC. Citation Format: Angela Cho, Amanda L. Hudson, Samuel Yuen, Nham Tran, Viive M. Howell, Emily K. Colvin. LOX and LOXL2 inhibition as a treatment for ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 764.

Abstract LB-268: Androgen signaling in cancer-associated fibroblasts contributes to progression of prostate cancer

Abstract Interactions between epithelial and stromal cells are important in the development of prostate cancer (PCa). Cancer-associated fibroblasts (CAFs) have been to support tumor progression, metastasis, and differentiation. Androgen receptor (AR) and related pathways are known to support the growth and survival of prostate epithelial cancer cells, the roles of AR-dependent processes in cancerous stroma are less clear. We sought to investigate if AR-dependent pathways present in CAF cells influence the growth and tumorogencity of epithelial cancer cells in relation to androgen-deprivation therapy in prostate cancer. Murine CAFs were isolated from a well-described PTEN-dependent cancer mouse model. A co-culture system was developed based on multiple lines of murine CAFs grown along with human prostate cancer epithelial cells, and a murine-specific anti-sense oligonucleotide (ASO) against murine AR was used to specifically suppress AR expression in murine CAFs in this system. Using this co-culture system, we found that murine CAFs promoted cell proliferation and colony formation in several human prostate cancer cell lines. Further, these processes were decreased by suppression of AR-expression in CAFs. Expression of genes related to tumorogenicity in epithelial cells were investigated by real-time quantitative PCR. Markers associated with epithelial-mesenchymal transition (EMT) and stemness were increased in human prostate cancer cells grown with low-AR CAFs. Our data indicates that suppression of AR in CAFs results in down-regulation in the growth and tumorogenecity of prostate cancer cells through pathways related to EMT and “cell reprograming”. As such, development of therapies which inhibit the tumor-promoting pathways present in stromal cells may be one approach to improve the treatment of prostate cancer. Citation Format: Chun-Peng Liao, Leng-Ying Chen, Andrea Luethy, Youngsoo Kim, Robert MacLeod, Mitchell Gross. Androgen signaling in cancer-associated fibroblasts contributes to progression of prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-268.