CAF-conditioned Medium Research Articles

70P - New therapy for intrahepatic cholangiocarcinoma targeted to cancer associated fibroblasts

BackgroundThe microenvironment such as cancer associated fibroblasts (CAFs) was reported to be involved in progression of intrahepatic cholangiocarcinoma (ICC). Nintedanib (BIBF1120) is a tyrosine kinase inhibitor of PDGFR, FGFR and VEGFR which is approved for idiopathic pulmonary fibrosis. And nintedanib was reported to suppress liver fibrosis in mouse through the suppression of hepatic stellate cells activation (Ozturk Akcora B, et al. Sci Rep. 2017 Mar 14;7:44545). Therefore, we hypothesized that nintedanib can inhibit the activity of CAFs of ICC. The purpose of this study is to clarify the effect of CAFs on ICC and develop a new therapy for ICC targeted to CAFs. MethodsCAFs were established from surgically resected ICC tissues. The effect of nintedanib on proliferation and expression of alpha smooth muscle actin (αSMA) of CAFs was examined. The effect of CAF-CM (conditioned medium of CAFs) and nintedanib-CAF-CM (CM of CAFs treated with nintedanib) on proliferation and invasion of ICC cell lines (HuCCT1, RBE) was examined. The humoral factors of CAF-CM and nintedanib-CAF-CM were analyzed using cytokine array and ELISA. The effect of nintedanib and gemsitabine (GEM) treatment on xenograft model co-implanted with HuCCT1 and CAFs was examined in vivo. ResultsNintedanib(1µM) suppressed the proliferation of CAFs and expression of αSMA in CAFs. CAF-CM significantly promoted the proliferation and invasion of ICC cell lines. But in nintedanib-CAF-CM, all of those cancer promoting effects were cancelled. In cytokine array and ELISA of CAF-CMs, expression of IL-6, IL-8, VEGF, VCAM1, and Osteopontin were suppressed in nintedanib-CAF-CM compared with CAF-CM. HuCCT1 + CAFs xenograft was increased more than HuCCT1 alone in vivo. Combination treatment with nintedanib (30mg/kg) and GEM (50mg/kg) inhibited HuCCT1 + CAFs xenograft growth than nintedanib or GEM alone in vivo. The xenograft treated with nintedanib and GEM showed reduction of both αSMA-positive staining in stroma and proportion of Ki-67 positive ICC cells. ConclusionsNintedanib suppressed CAFs activity and the production of cancer-promoting cytokines produced by CAFs. Combination therapy with nintedanib and GEM may be a new promising therapy to overcome refractory ICC. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

ITGB4-mediated metabolic reprogramming of cancer-associated fibroblasts

Integrin beta 4 (ITGB4) overexpression in cancer cells contributes to cancer progression. However, the role of stromal ITGB4 expression in cancer progression remains poorly understood, despite stromal ITGB4 overexpression in malignant cancers. In our study, ITGB4-overexpressing triple negative breast cancer (TNBC) cells provided cancer-associated fibroblasts (CAFs) with ITGB4 proteins via exosomes, which induced BNIP3L-dependent mitophagy and lactate production in CAFs. In coculture assays, the ITGB4-induced mitophagy and glycolysis were suppressed in CAFs by knocking down ITGB4 or inhibiting exosome generation in MDA-MB-231, or blocking c-Jun or AMPK phosphorylation in CAFs. ITGB4-overexpressing CAF-conditioned medium promoted the proliferation, epithelial-to-mesenchymal transition, and invasion of breast cancer cells. In a co-transplant mouse model, MDA-MB-231 made a bigger tumor mass with CAFs than ITGB4 knockdown MDA-MB-231. Herein, we presented how TNBC-derived ITGB4 protein triggers glycolysis in CAFs via BNIP3L-dependent mitophagy and suggested the possibility that ITGB4-induced mitophagy could be targeted as a cancer therapy.

Role of EphA2-PI3K signaling in vasculogenic mimicry induced by cancer-associated fibroblasts in gastric cancer cells.

Although erythropoietin-producing human hepatocellular receptor A2 (EphA2) signaling serves an important role in the tumor microenvironment, its contribution to vasculogenic mimicry (VM) formation in gastric cancer cells remains unclear. The aim of the present study was to investigate the role of EphA2 in VM formation induced by cancer-associated fibroblasts (CAFs). The conditioned medium of CAFs (CAF-CM) was prepared from 12 patients with gastric adenocarcinoma. VM was evaluated by the number of tubules and intersections in gastric cancer cells following CAF-CM treatment. The role of EphA2-phosphoinositide 3-kinase (PI3K) in VM was investigated using EphA2-targeted small interfering (si)RNAs (siEphA2), EphA2 inhibitors and PI3K-inhibitors. CAF-CM-induced VM formation was significantly associated with high protein expression levels of EphA2. EphA2 inhibitor and siEphA2 manipulation significantly decreased VM formation by CAF-CM. In siEphA2 cells, decreased expression levels of VM-associated proteins were observed. CAF-CM-induced VM formation was blocked by the PI3K-inhibitor. In conclusion, CAFs facilitate VM formation via EphA2-PI3K signaling in gastric cancer cells. Thus, EphA2-PI3K signaling may be required for CAF-promoted VM formation during gastric tumorigenesis.

Abstract LB-314: Fibroblast growth factor-2, derived from cancer-associated fibroblasts, stimulate proliferation and migration of human breast cancer cells though FGFR1

Abstract Cancer-associated fibroblasts (CAFs) constitute a major compartment of the tumor microenvironment. CAFs are distinct from normal fibroblasts for their overexpression of α-smooth muscle actin (α-SMA) and fibroblast activating protein. Recent studies suggest that CAFs play an important role in proliferation and migration of cancer cells through crosstalk with them. In the present study, we investigated the role for CAFs in breast cancer progression and underlying molecular mechanisms. We found the increased expression of α-SMA in human breast tumor tissues compared with normal adjacent tissues. In particular, α-SMA was highly expressed in the most of estrogen receptor (ER)-negative tumor tissues. When ER-negative breast cancer MDA-MB-231 cells and MDA-MB-468 cells were treated with the CAF-conditioned medium (CM), the cell viability was significantly elevated. In addition, MDA-MB-231 cells exhibited elevated expression of proliferative and invasive genes when incubated with CAFs. Notably, fibroblast growth factor-2 (FGF2) expression was correlated with the presence of CAFs. We noticed that mRNA level of FGF2 in CAFs as well as the concentration of FGF2 in CAF-CM was higher than that in normal fibroblasts. FGF2 exerts its biological effects by binding to FGF receptor 1 (FGFR1). In the breast cancer tissue microarray, 42% ER-negative patients co-expressed FGF2 and FGFR1 proteins, whereas only 19% ER-positive patients co-expressed those proteins. While the expression of FGFR1 was up-regulated in breast cancer cells, another major FGF2-corresponding receptor FGFR2 was barely expressed.This prompted us to explore FGF2-FGFR1 signaling in the context of paracrine communication. CAF-stimulated MDA-MB-231 cell migration and invasion as well as FGFR1 signaling activation were abolished when FGF2-neutralizing antibody was added to the CAF-CM. In a xenograft mouse model, co-injection of shmock-MDA-MB-231 cells with activated fibroblasts expressing FGF2 dramatically enhanced tumor growth, which suggests that stromal-derived FGF2 enhances tumor formation through activation of FGFR1 in cancer cells. Furthermore, FGF2 promotes nuclear localization of FGFR1 which might be associated with cancer cell proliferation and migration. In conclusion, secretion of FGF2 by CAFs stimulates proliferation and migration of breast cancer cells through interaction with FGFR1 which may contribute to human breast cancer progression. Citation Format: Jinyoung Suh, Do-Hee Kim, Hye-Kyung Na, Young-Joon Surh. Fibroblast growth factor-2, derived from cancer-associated fibroblasts, stimulate proliferation and migration of human breast cancer cells though FGFR1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-314.

Fibroblast-Mediated Immunoregulation of Macrophage Function Is Maintained after Irradiation.

The abilities of cancer-associated fibroblasts (CAFs) to regulate immune responses in the context of radiotherapy remain largely unknown. This study was undertaken to determine whether ionizing radiation alters the CAF-mediated immunoregulatory effects on macrophages. CAFs were isolated from freshly-resected non-small cell lung cancer tumors, while monocyte-derived macrophages were prepared from peripheral blood of healthy donors. Experimental settings included both (CAF-macrophage) co-cultures and incubations of M0 and M1-macrophages in the presence of CAF-conditioned medium (CAF-CM). Functional assays to study macrophage polarization/activation included the expression of cell surface markers, production of nitric oxide, secretion of inflammatory cytokines and migratory capacity. We show that CAFs promote changes in M0-macrophages that harmonize with both M1-and M2-phenotypes. Additionally, CAFs inhibit pro-inflammatory features of M1-macrophages by reducing nitric oxide production, pro-inflammatory cytokines, migration, and M1-surface markers expression. Radiation delivered as single-high dose or in fractioned regimens did not modify the immunoregulatory features exerted by CAFs over macrophages in vitro. Protein expression analyses of CAF supernatants showed that irradiated and non-irradiated CAFs produce approximately the same protein levels of immunoregulators. Thus, CAF-derived soluble factors mediate measurable changes on uncommitted macrophages and down-regulate pro-inflammatory features of M1-polarized macrophages. Notably, ionizing radiation does not curtail the CAF-mediated immunosuppressive effects.

Snail1-dependent cancer-associated fibroblasts induce epithelial-mesenchymal transition in lung cancer cells via exosomes.

Epithelial-mesenchymal transition (EMT) is an essential component of metastasis. Our previous study demonstrated that cancer-associated fibroblasts (CAFs) induce EMT in lung cancer cells. In recent years, many studies have demonstrated that CAFs induce metastasis and drug resistance in cancer cells via exosomes. We sought to discover the mechanism underlying how CAFs induce EMT in lung cancer cells, unveiling the role of exosomes in lung cancer progression. We cultured lung cancer cell (i) with control medium, normal fibroblasts (NFs) or CAFs; (ii) with SNAI1-transfected or NC (negative control)-transfected CAFs; (iii) with exosomes extracted from NF- or CAF-conditioned medium; (iv) with exosomes released by SNAI1 or NC-transfected CAFs; (v) with CAF-conditioned medium or exosome-depleted CAF-conditioned medium. qRT-PCR was conducted to examine the expression of CDH1 (gene of E-cadherin) and VIM (gene of Vimentin), western blotting was conducted to examine E-cadherin and vimentin levels in lung cancer cells. Exosomes released by CAFs-promoted EMT in lung cancer cells. Interestingly, SNAI1 levels in exosomes secreted from CAFs were correlated with SNAI1 expression in CAFs. Furthermore, the level of SNAI1 in exosomes was crucial for inducing EMT in lung cancer cells. Finally, treatment of CAFs with GW4869, an inhibitor of exosome release, noticeably inhibited their EMT-inducing effect on recipient epithelial cells. The molecular mechanism underlying how CAFs induce EMT in cancer cells may be that CAFs deliver SNAI1 to recipient cancer cells via exosomes.

Cellular Senescence, Represented by Expression of Caveolin-1, in Cancer-Associated Fibroblasts Promotes Tumor Invasion in Pancreatic Cancer.

The role of senescence of cancer-associated fibroblasts (CAFs) in the development of cancer is controversial. In this study, we investigated whether cellular senescence of CAFs, represented by CAV1 expression, affects tumor progression in pancreatic cancers (PC). Because CAV1 plays a major role in cellular senescence, we used CAV1 expression to monitor cellular senescence. A total of 157 consecutive patients with PC who underwent curative resection were enrolled in the study. Patients were divided into two groups according to CAV1 expression in CAFs by immunohistochemistry. We investigated the relationship between the CAV1 expression in CAFs and the patients' clinicopathological characteristics, including survival. We also established ten CAFs cell lines using PC clinical samples and chose one of them to knock down CAV1 expression. Finally, we cultured a PC cell line (MIAPaCa-2) in CAF-conditioned medium (CM). Regarding patients' clinicopathological characteristics, the serum levels of carbohydrate antigen 19-9 and the rate of advanced tumor stage (pT2, 3, and 4) were significantly higher in the high-CAV1 group. The high-CAV1 group had significantly worse outcomes in both overall and disease-free survival (p < 0.01). Additionally, in co-culture assays using CAFs-CM and MIAPaCa-2 cells, we found that knockdown of CAV1 in CAFs negatively affected the invasion of PC cells. In PC, CAV1 expression in CAFs is associated with patients' poor prognosis and the downregulation of CAV1 in CAFs reduces the invasiveness of PC cells. Therefore, CAV1 of CAFs might be a new target for the treatment of PC.

Reciprocal activation of cancer-associated fibroblasts and oral squamous carcinoma cells through CXCL1

ObjectivesCrosstalk between cancer cells and carcinoma-associated fibroblasts (CAFs) is known to be involved in various aspects of tumor biology, including during invasion. Using oral squamous cell carcinoma (OSCC) cells as a model, we examined whether and how CAFs respond to inflammatory signals to influence cancer cell migration and invasion. Materials and methodsChemokine signatures within the human HNSCC datasets from The Cancer Genome Atlas (TCGA) were analyzed together with tissue assessment using immunohistochemical staining (IHC) and real-time PCR. A co-culture system was used to identify reciprocal effects exerted by CAFs and cancer cells upon one another. Recombinant CXCL1, CXCL1 neutralizing antibodies, and CXCR2 antagonist were used to confirm CXCL1/CXCR2 axis-mediated cell behaviors. ResultsAnalysis of the TCGA dataset revealed that CXCL1 is associated with poor survival, and IHC demonstrated CXCL1 is highly expressed in OSCC stromal cells. Moreover, real-time PCR showed that in addition to CXCL1, IL-1β and CXCR2 are also highly expressed in OSCC and IL-1β mRNA levels positively correlate with CXCL1 expression. Furthermore, CAFs co-cultured with SAS, a poorly differentiated OSCC cell line, or stimulated with IL-1β exhibit increased CXCL1 secretion in an NF-κB-dependent manner. Treatment of SAS cells with CAF-conditioned medium or CXCL1 increased their invasion and migration capabilities, indicating a reciprocal activation between CAFs and cancer cells. Moreover, CXCL-1 upregulated matrix metalloprotease-1 (MMP-1) expression and activity in CAFs. ConclusionThe induction of IL-1β following CXCL1 stimulation of CAFs mediates cancer cell invasion, and there is a reciprocal dependency between CAFs and cancer cells in the OSCC microenvironment.

Activated gastric cancer-associated fibroblasts contribute to the malignant phenotype and 5-FU resistance via paracrine action in gastric cancer

BackgroundCancer-associated fibroblasts (CAFs) play important roles in tumor progression. However, the behaviors of activated CAFs in gastric cancer remain to be determined. The aim of the present study was to investigate the correlations between activated gastric CAFs and the prognosis of patients with gastric cancer, and to determine the effects of activated CAFs on the malignant phenotype and 5-fluorouracil resistance in this cancer.MethodsNinety-five patients with primary gastric cancer were enrolled in this study. Activation states of gastric CAFs were evaluated by immunohistochemistry. A modified method for the primary culture of gastric CAFs was employed. Types of CAFs and activation states were identified by immunocytochemical and immunofluorescent staining. Cell co-culture and gastric CAF conditioned medium transfer models were established to investigate the paracrine effects of activated CAFs on the migration and invasion of gastric cell lines. The half maximal inhibitory concentration of 5-fluorouracil and levels of cell apoptosis were examined using cell viability assay and flow cytometry, respectively. Protein expression levels of associated molecules were measured by Western blotting.ResultsKaplan–Meier survival curves showed that activated gastric CAFs identified via fibroblast activation protein were significantly related to poorer cumulative survival in gastric cancer patients. Five strains of CAFs were successfully cultured via the modified culture method, and three gastric CAFs strains were identified as activated gastric CAFs. The migration and invasion abilities of gastric cells were significantly enhanced in both the co-culture group and the conditioned medium group. The half maximal inhibitory concentration for 5-fluorouracil in BGC-823 cells was elevated after treatment with conditioned medium, and early apoptosis was inhibited. Additionally, an obvious elevation of epithelial–mesenchymal transition level was observed in the conditioned medium group.ConclusionsActivated gastric CAFs correlate with a poor prognosis of cancer patients and may contribute to the malignant phenotype and the development of resistance to 5-fluorouracil via paracrine action in gastric cancer. Gastric CAFs with a specific activation state might be used as a tumor biomarker within the microenvironment for prognosis and as a new therapeutic target for chemoresistant gastric cancer.

Nab-paclitaxel interrupts cancer-stromal interaction through C-X-C motif chemokine 10-mediated interleukin-6 downregulation in vitro.

Cancer‐associated fibroblasts (CAF), derived from stroma of cancer tissues, interact with cancer cells and play an important role in cancer initiation, growth, and metastasis. Nab‐paclitaxel (nab‐PTX) is a 130 nm albumin‐binding paclitaxel and recommended for many types of cancer chemotherapy. The nab‐PTX stromal‐disrupting effect during pancreatic cancer treatment has been reported. The aim of the present study was to determine the role of nab‐PTX in cancer cells and CAF interaction. Cancer cells (MIA PaCa‐2 and Panc‐1) were cocultured with CAF or treated with CAF conditioned medium, after which their migration and invasion ability, epithelial‐mesenchymal transition (EMT)‐related marker expression and C‐X‐C motif chemokine 10 (CXCL10) expression and secretion were detected. Nab‐PTX treatment was carried out during the coculture system or during preparation of CAF conditioned medium. Then cancer cell migration and invasion ability, EMT‐related marker expression, CXCL10 expression and secretion, and interleukin‐6 (IL‐6) expression and secretion by CAF were checked After coculture with CAF, migration and invasion ability of cancer cells increased. CAF also downregulated E‐cadherin and upregulated N‐cadherin and vimentin expression in cancer cells. During coculture or stimulation with cancer cell‐cultured medium, CAF significantly increased IL‐6 expression and secretion. However, nab‐PTX in the coculture system canceled CAF‐induced migration and invasion promotion and EMT‐related gene changes. Moreover, nab‐PTX increased CXCL10 expression of cancer cells which blocked CAF IL‐6 expression and secretion. Nab‐PTX treatment could increase CXCL10 expression of cancer cells which blocks CAF cancer cell migration and invasion‐promoting effect by inhibiting IL‐6 expression.

Abstract 210: Uncovering a novel layer of complexity in the architecture of pancreatic cancer

Abstract Single cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single cell phenotypes has not been well defined. We integrated single cell RNA-seq (scRNA-Seq) and phospho mass cytometry (CyTOF) with high-content digital imaging to investigate tumor heterogeneity of human pancreatic cancer (PDAC) using a novel tumor-architecture approach. Patient-derived PDAC cells co-cultured at different ratios with cancer associated fibroblasts (CAFs) were found to heterogeneously acquire proliferative (PRO) and invasive (EMT) phenotypes. scRNA-Seq enabled the identification of a novel cell phenotype with upregulation of both PRO and EMT programs, which we named the double positive (DP) phenotype. Functional studies confirmed the heterogeneous activation of PRO and EMT programs with different patterns of tumor growth and metastasis in mice orthotopically xenografted with distinct PDAC:CAF ratios. In a time-course experiment of PDAC cells with CAF conditioned media (CM), mass spectrometry-based phosphoproteomics demonstrated selective enrichment of MAPK and STAT3 pathways. This was validated with mass cytometry (CyTOF) at the single cell level in our cell line model as well as from primary PDAC tumors, which identified the dual enrichment of MAPK and STAT3 signaling in the DP cell population. Functional validation of MAPK and STAT3 signaling was performed with small molecule inhibitors of MAPK (Trametinib) and STAT3 (pyrimethamine) in our cell line models. To evaluate the generalizability of these cell subpopulations, we performed RNA-ISH for EMT (FN1) and PRO (MKI67) across 195 human PDAC primary tumors and scored 319,626 cancer cells revealing significant heterogeneity of PRO, EMT, and DP cells among patients. Single-cell analysis within the context of tissue architecture revealed these cells were grouped together in discrete tumor glands. Using each of the 3 cell types (PRO, EMT, and DP), we classified 8 distinct types of tumor glands in these primary tumors. This provided the ability to define inter and intra-tumor heterogeneity at the tumor gland level, which provides an extra layer of tumor cell organization not appreciable with single cell analysis alone. We showed that tumor glands are independent functional units in human PDACs carrying distinct prognostic information. In fact, by comparing their prognostic power we noticed that some tumor glands are positively linked with worsened survival (Type I p=0.003, Type II p=0.04, Type II p=0.001 and Type VII p=0.02), while Type III glands are associated with a favorable patient prognosis (p=0.01, log-rank test). In conclusion, our study showed for the first time at this scale level that integrating single-cell technologies with tissue architectural information provides a more comprehensive landscape of intra-tumoral cancer cell heterogeneity that has implications on PDAC cell behavior and patient outcomes. Citation Format: Matteo Ligorio, Srinjoy Sil, Jose Malagon-Lopez, Sandra Misale, Murat Karabacak, Linda Nieman, Shyamala Maheswaran, Daniel A. Haber, Andrew L. Warshaw, Carlos Fernandez-Del Castillo, Cristina R. Ferrone, Wilhelm Haas, Martin Aryee, David T. Ting. Uncovering a novel layer of complexity in the architecture of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 210.

Abstract 2115: An authenticated in vitro model for prostate microenvironment studies utilizing prostate epithelial cells and stromal-derived cells

Abstract Prostate cancer remains one of the most common cancers diagnosed in men and one of the leading causes of cancer death in men. Tumor development and progression have been shown to be highly influenced not simply by the genetic makeup of a cell, but by its surrounding stroma, particularly fibroblasts. It has been demonstrated that prostate cancer-associated fibroblasts (CAFs, which are located marginal to the prostate tumor), differ from prostate normal-associated fibroblast (NAFs, which are located distal to the prostate tumor), on their contribution to tumor progression. However, human prostate cancer in-vitro model systems have focused largely on prostate cancer epithelial cells exclusively. A need exists for a more physiologically relevant human cell model system to study prostate cancer progression within the context of its tumor microenvironment. In this study, we utilized prostate cancer-associated fibroblasts (CAFs), prostate normal-associated fibroblasts (NAFs) and normal prostate epithelial (PrE) cells; all three lines were immortalized by hTERT (human telomerase reverse transcriptase) alone and they were continuously passaged for at least 15 passages without any indications of a decrease in growth rate. All cell lines express appropriate specific cell lineage markers for either fibroblasts or epithelial cells. Fibroblasts expressed TE7 and alpha smooth muscle actin (a-SMA), while prostate epithelial cells expressed cytokeratin 5, low levels of prostate specific antigen (PSA) and high levels of p63 throughout their continuous passage; all characteristics in accord with their primary cell counterparts. Next, cell proliferation was measured for various prostate-derived epithelial cells under the influence of CAFs and NAFs cells. Normal prostate epithelial cell proliferation was inhibited and produced a visible morphological change in the cells in the presence of CAFs or CAF-conditioned medium. Meanwhile, the effects of stromal cells on prostate cancer cells was cell line dependent, demonstrating both promotion and inhibition of growth of selected cancer cells. Surprisingly, both CAFs and NAFs promoted cancer cell growth, but CAFs promoted a greater increase in cell proliferation than NAFs in some cancer cell lines. This study demonstrates that these three hTERT immortalized cells from human prostate are a valuable model system for the study of prostate cancer cell progression and tumor micro environment studies. Citation Format: Luis G. Rodriguez, Russell E. McDaniel, Xiangshan Zhao, Chaozhong Zou. An authenticated in vitro model for prostate microenvironment studies utilizing prostate epithelial cells and stromal-derived cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2115.

Cancer-associated fibroblasts promote gastric tumorigenesis through EphA2 activation in a ligand-independent manner.

Under physiologic conditions, the binding of erythropoietin-producing hepatocellular (Eph) A2 receptor and its ligand ephrinA1 results in decreased EphA2 level and tumor suppression. However, EphA2 and ephrinA1 are highly expressed in human cancers including gastric adenocarcinoma. In this study, we tested our hypothesis that cancer-associated fibroblasts (CAFs) promote gastric tumorigenesis through EphA2 signaling in a ligand-independent manner. Expression of EphA2 protein in primary tumor tissues of 91 patients who underwent curative surgery for gastric adenocarcinoma was evaluated by immunohistochemistry and western blotting. Conditioned medium of cancer-associated fibroblasts (CAF-CM) was used to evaluate the tumorigenic effect of CAFs on gastric cancer cell lines. Epithelial-mesenchymal transition (EMT), cell proliferation, migration, and invasion were assessed. EphrinA1-Fc ligand was used to determine the suppressor role of EphA2 receptor-ligand binding. CAF-CM-induced EMT and promoted cancer cell motility even without cell-cell interaction. Treatment with a selective EphA2 inhibitor (ALW-II-41-27) or EphA2-targeted siRNA markedly reduced CAF-CM-induced gastric tumorigenesis. EphrinA1-Fc ligand treatment showing ligand-dependent tumor suppression diminished the EphA2 expression and EMT progression. In contrast, ephrinA1-targeted siRNA did not significantly affect CAF-CM-mediated increases in EphA2 expression and EMT progression. Treatment with VEGF showed effects like CAF-CM in terms of EphA2 activation and EMT progression. CAFs may contribute to gastric tumorigenesis by activating EphA2 signaling pathway in a ligand-independent manner. Our results suggest that ligand-independent activation of EphA2 was triggered by VEGF released from CAF-CM. Our result may partially explain why ligand-dependent tumor suppressor roles of EphA2 are not evident in gastric cancer despite the prominent level of ephrinA1.

Resveratrol suppresses migration, invasion and stemness of human breast cancer cells by interfering with tumor-stromal cross-talk

Cancer-associated fibroblasts (CAFs) constitute a major compartment of the tumor microenvironment. CAFs produce a variety of cytokines, growth factors and extracellular matrix proteins, thereby stimulating tumor progression. CAFs are distinct from normal fibroblasts for their overexpression of α-smooth muscle actin. Recent studies suggest that CAFs play an important role in proliferation and migration of cancer cells through cross-talk with them. Resveratrol (trans-3,4′5,-trihydroxystilbene), a phytoalexin present in grapes, has been reported to possess chemopreventive and chemotherapeutic activities. In the present study, we examined the effects of resveratrol on CAF-induced migration, invasion and self-renewal activity of breast cancer cells. Resveratrol inhibited proliferation, migration and invasion of human breast cancer cells treated with CAF-conditioned media (CAF-CM). Resveratrol treatment suppressed the CAF-CM-induced expression of Cyclin D1, c-Myc, MMP-2 and MMP-9. In addition, resveratrol inhibited Sox2 expression as well as activation of Akt and STAT3 induced by CAF-CM in breast cancer cells. Further, resveratrol abrogated stemness properties and reduced the expression of self-renewal signaling molecules in stem-like breast cancer cells. Taken together, the present study provides insights into the role of resveratrol in tumor microenvironment with focus on interaction between cancer cells and the hosting niche.

GPR68, a proton-sensing GPCR, mediates interaction of cancer-associated fibroblasts and cancer cells.

The microenvironment of pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense fibrotic stroma (desmoplasia) generated by pancreatic cancer-associated fibroblasts (CAFs) derived from pancreatic stellate cells (PSCs) and pancreatic fibroblasts (PFs). Using an unbiased GPCRomic array approach, we identified 82 G-protein-coupled receptors (GPCRs) commonly expressed by CAFs derived from 5 primary PDAC tumors. Compared with PSCs and PFs, CAFs have increased expression of GPR68 (a proton-sensing GPCR), with the results confirmed by immunoblotting, The Cancer Genome Atlas data, and immunohistochemistry of PDAC tumors. Co-culture of PSCs with PDAC cells, or incubation with TNF-α, induced GPR68 expression. GPR68 activation (by decreasing the extracellular pH) enhanced IL-6 expression via a cAMP/PKA/cAMP response element binding protein signaling pathway. Knockdown of GPR68 by short interfering RNA diminished low pH-induced production of IL-6 and enhancement of PDAC cell proliferation by CAF conditioned media. CAFs from other gastrointestinal cancers also express GPR68. PDAC cells thus induce expression by CAFs of GPR68, which senses the acidic microenvironment, thereby increasing production of fibrotic markers and IL-6 and promoting PDAC cell proliferation. CAF-expressed GPR68 is a mediator of low-pH-promoted regulation of the tumor microenvironments, in particular to PDAC cell-CAF interaction and may be a novel therapeutic target for pancreatic and perhaps other types of cancers.-Wiley, S. Z., Sriram, K., Liang, W., Chang, S. E., French, R., McCann, T., Sicklick, J., Nishihara, H., Lowy, A. M., Insel, P. A. GPR68, a proton-sensing GPCR, mediates interaction of cancer-associated fibroblasts and cancer cells.

Abstract A146: Stromal neuregulin-1 modulates the response to MEK inhibitors in WT BRAF/WT NRAS (WT/WT) melanomas

Abstract MEK-ERK1/2 signaling is elevated in the majority of melanomas. While MEK inhibitors (MEKi) are FDA-approved for mutant BRAF melanoma, and have some activity against NRAS mutant tumors, they have shown little activity in patients with melanomas that are wild type for BRAF and NRAS (WT/WT). Since the tumor microenvironment (TME) often regulates drug resistance, we tested the effects of growth factors on WT/WT melanoma growth in the presence of MEKi. Neuregulin-1 (NRG1) protected patient-derived WT/WT human melanoma cell lines from growth inhibition mediated by trametinib (MEKi). Phospho-proteomic analysis and clinical grade neutralizing antibodies implicated adaptive activation of ErbB3/ErbB2 complexes in the protective effects of NRG1. NRG1 was detected in fibroblasts and cancer-associated fibroblasts (CAF), and CAF-conditioned medium activated ErbB3/ErbB2 signaling in MEK inhibited WT/WT melanoma cells. ErbB3 and ErbB2 neutralizing antibodies blocked the protective effects of fibroblast- and CAF-derived NRG1 in vitro and cooperated with MEKi to delay xenograft growth in vivo. Together our results provide a rationale for the treatment of WT/WT melanomas with the combination of MEKi and anti-ErbB3/ErbB2 antibodies. Citation Format: Claudia Capparelli, Timothy J. Purwin, Shea Heilman, Inna Chervoneva, Michael A. Davies, Jeffrey E. Gershenwald, Andrew E. Aplin. Stromal neuregulin-1 modulates the response to MEK inhibitors in WT BRAF/WT NRAS (WT/WT) melanomas [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A146.

Abstract 5903: Interaction with extracellular matrix enhance the chemoresistance via CXCL12/CXCR4 and integrin signal activation by cancer-associated fibroblasts in gastric cancers

Abstract Purpose: Gastric cancers (GCs) is an aggressive cancer metastasizing with a high propensity for peritoneal dissemination and liver metastasis. Some studies have been shown that not only cancer cells but also cancer environment, especially cancer-associated fibroblasts (CAFs) play an important role in tumor development. Previously, we reported that CXCL12-CXCR4 and integrin beta 1 signal activation by CAFs co-operates for promoting FAK phosphorylation and enhances the invasiveness of GC cells in extracellular matrix (ECM). On the other hand, a previous study presented that CXCR4 and integrin signaling co-operates in mediating adhesion and chemoresistance in small cell lung cancer cells. Therefore, we hypothesized that CXCL12-CXCR4 and integrin signal activation by CAFs enhances the chemoresistance in GCs as well. The aim of current study is to elucidate the significance of cancer microenvironment with CAFs mediating signal activation for chemoresistance in GCs. Experimental Design: We first performed reactive oxygen spices (ROS)-sensitive assay and chemo-sensitive assays using GC cells cultured with fresh medium or with CAF-conditioned-medium (CM), and these experiments were conducted on non-coated and Matrigel-coated plates. Next, we examined the CXCL12-CXCR4 and integrin related signaling by western blotting analysis. Furthermore, we investigated the critical molecule for chemoresistance using silencing CXCR4 or integrin beta 1 in GC cells. Results: ROS-sensitive assay and chemo-sensitive assays revealed that GC cells cultured with CAF-CM showed more resistant to ROS and cisplatin than those cultured with fresh medium. Moreover, GC cells with CAF-CM on Matrigel-coated plates exhibited remarkable resistance in these assays. On the other hand, western blotting analysis showed that Akt activity in GC cells cultured with CAF-CM was up-regulated in concurrence with FAK phosphorylation. Finally, the chemoresitance induced by CAFs was significantly suppressed by CXCR4 or integrin beta 1 silencing. Conclusions: These results suggest that interaction with ECM is important for enhance chemoresitance mediated CXCL12/CXCR4 and integrin beta 1 signal activation by CAFs in GCs. This mechanism underlying the chemoresistance may provide a novel therapeutic target in advanced GCs. Citation Format: Keisuke Miyake, Takatsugu Ishimoto, Daisuke Izumi, Kota Arima, Tsugio Eto, Daisuke Kuroda, Masaaki Iwatsuki, Yoshifumi Baba, Yasuo Sakamoto, Naoya Yoshida, Hideo Baba. Interaction with extracellular matrix enhance the chemoresistance via CXCL12/CXCR4 and integrin signal activation by cancer-associated fibroblasts in gastric cancers [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 5903. doi:10.1158/1538-7445.AM2017-5903

Human cancer-associated fibroblasts enhance glutathione levels and antagonize drug-induced prostate cancer cell death

Drug resistance is a major problem in cancer therapy. A growing body of evidence demonstrates that the tumor microenvironment, including cancer-associated fibroblasts (CAFs), can modulate drug sensitivity in tumor cells. We examined the effect of primary human CAFs on p53 induction and cell viability in prostate cancer cells on treatment with chemotherapeutic drugs. Co-culture with prostate CAFs or CAF-conditioned medium attenuated DNA damage and the p53 response to chemotherapeutic drugs and enhanced prostate cancer cell survival. CAF-conditioned medium inhibited the accumulation of doxorubicin, but not taxol, in prostate cancer cells in a manner that was associated with increased cancer cell glutathione levels. A low molecular weight fraction (<3 kDa) of CAF-conditioned medium had the same effect. CAF-conditioned medium also inhibited induction of reactive oxygen species (ROS) in both doxorubicin- and taxol-treated cancer cells. Our findings suggest that CAFs can enhance drug resistance in cancer cells by inhibiting drug accumulation and counteracting drug-induced oxidative stress. This protective mechanism may represent a novel therapeutic target in 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.

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

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