Recruitment Of Cancer-associated Fibroblasts Research Articles

Reprogramming of cancer-associated fibroblasts combined with immune checkpoint inhibitors: A potential therapeutic strategy for cancers.

Activated fibroblasts, namely cancer-associated fibroblasts (CAFs), are highly heterogeneous in phenotypes, functions, and origins. CAFs originated from varieties of cell types, including local resident fibroblasts, epithelial cells, mesenchymal stromal cells, or others. These cells participate in tumor angiogenesis, mechanics, drug access, and immune suppression, with the latter being particularly important. It was difficult to distinguish CAFs by subsets due to their complex origins until the use of scRNA-seq. Reprogramming CAFs with TGFβ-RI inhibitor, a CXCR4 blocker, or other methods increases T cells activation and infiltration, together with a decrease in CAFs recruitment, thus improving the prognosis. As depletion of CAFs can't bring clinical benefit, the combination of reprogramming CAFs and immune checkpoint inhibitors (ICIs) come into consideration. It has shown better outcomes compared with monotherapy respectively in basic/preclinical researches, and needs more data on clinical trials. Combination therapy may be a promising and expecting method for treatment of cancer.

Hepatocellular carcinoma cells remodel the pro-metastatic tumour microenvironment through recruitment and activation of fibroblasts via paracrine Egfl7 signaling

BackgroundThe tumour microenvironment consists of a complex and dynamic milieu of cancer cells, including tumour-associated stromal cells (leukocytes, fibroblasts, vascular cells, etc.) and their extracellular products. During invasion and metastasis, cancer cells actively remodel the tumour microenvironment and alterations of microenvironment, particularly cancer-associated fibroblasts (CAFs), can promote tumour progression. However, the underlying mechanisms of the CAF formation and their metastasis-promoting functions remain unclear.MethodsPrimary liver fibroblasts and CAFs were isolated and characterized. CAFs in clinical samples were identified by immunohistochemical staining and the clinical significance of CAFs was also analysed in two independent cohorts. A transwell coculture system was used to confirm the role of HCC cells in CAF recruitment and activation. qRT-PCR, western blotting and ELISA were used to screen paracrine cytokines. The role and mechanism of Egfl7 in CAFs were explored via an in vitro coculture system and an in vivo mouse orthotopic transplantation model.ResultsWe showed that CAFs in hepatocellular carcinoma (HCC) are characterized by the expression of α-SMA and that HCC cells can recruit liver fibroblasts (LFs) and activate them to promote their transformation into CAFs. High α-SMA expression, indicating high CAF infiltration, was correlated with malignant characteristics. It was also an independent risk factor for HCC survival and could predict a poor prognosis in HCC patients. Then, we demonstrated that EGF-like domain multiple 7 (Egfl7) was preferentially secreted by HCC cells, and exhibited high potential to recruit and activate LFs into the CAF phenotype. The ability of Egfl7 to modulate LFs relies upon increased phosphorylation of FAK and AKT via the receptor ανβ3 integrin. Strikingly, CAFs activated by paracrine Egfl7 could further remodel the tumour microenvironment by depositing fibrils and collagen and in turn facilitate HCC cell proliferation, invasion and metastasis.ConclusionOur data highlighted a novel role of Egfl7 in remodelling the tumour microenvironment: it recruits LFs and activates them to promote their transformation into CAFs via the ανβ3 integrin signaling pathway, which further promotes HCC progression and contributes to poor clinical outcomes in HCC patients.3o7rdHzTehVwN25qa8k-ATVideo

Modulation of EZH2 Activity Induces an Antitumoral Effect and Cell Redifferentiation in Anaplastic Thyroid Cancer.

Anaplastic thyroid cancer (ATC) is a rare and lethal form of thyroid cancer that requires urgent investigation of new molecular targets involved in its aggressive biology. In this context, the overactivation of Polycomb Repressive Complex 2/EZH2, which induces chromatin compaction, is frequently observed in aggressive solid tumors, making the EZH2 methyltransferase a potential target for treatment. However, the deregulation of chromatin accessibility is yet not fully investigated in thyroid cancer. In this study, EZH2 expression was modulated by CRISPR/Cas9-mediated gene editing and pharmacologically inhibited with EZH2 inhibitor EPZ6438 alone or in combination with the MAPK inhibitor U0126. The results showed that CRISPR/Cas9-induced EZH2 gene editing reduced cell growth, migration and invasion in vitro and resulted in a 90% reduction in tumor growth when EZH2-edited cells were injected into an immunocompromised mouse model. Immunohistochemistry analysis of the tumors revealed reduced tumor cell proliferation and less recruitment of cancer-associated fibroblasts in the EZH2-edited tumors compared to the control tumors. Moreover, EZH2 inhibition induced thyroid-differentiation genes' expression and mesenchymal-to-epithelial transition (MET) in ATC cells. Thus, this study shows that targeting EZH2 could be a promising neoadjuvant treatment for ATC, as it promotes antitumoral effects in vitro and in vivo and induces cell differentiation.

Abstract B013: Tumor-derived CCL5 recruits’ cancer-associated fibroblasts and promotes tumor cell proliferation in esophageal carcinomas

Abstract The tumor microenvironment (TME), at both the primary tumor and sites of metastases, is a key determinant in the success of metastatic colonization. Cancer-associated fibroblasts (CAFs) are a predominant cell type in the TME that, through deposition and remodeling of extracellular matrix (ECM) components, can mediate the TME and promote cancer progression. In addition to cancer progression and metastasis, CAFs have also been shown to mediate resistance to chemotherapy and radiotherapy. Therefore, there is a need to identify the factors which activate and recruit CAFs to solid tumors. Though cytokine arrays, we identified CC motif chemokine ligand 5 (CCL5) as a secreted factor which is increased upon co-culture of esophageal cancer cell lines and CAFs. Using CRISPR/Cas9 technology, we determined that CCL5 is predominantly tumor-cell derived and loss of tumor-cell derived CCL5 reduced esophageal carcinoma cell proliferation in vitro and in vivo. We propose that this reduction in cell proliferation is mediated by ERK1/2 signaling as loss of tumor-cell derived CCL5 reduces expression of p-ERK1/2 and cyclin D1, an ERK1/2 target gene which is essential for cell cycle progression. Interestingly, the loss of tumor-derived CCL5 also reduced the percentage of CAFs, identified by αSMA positivity, recruited to xenograft tumors in vivo. CCL5 is a ligand for the CC motif receptor 5 (CCR5), for which a clinically approved inhibitor exists called Maraviroc. We treated xenografts generated from esophageal carcinoma cell lines or patient derived tumor fragments with Maraviroc. Maraviroc treated xenografts had reduced tumor volume, CAF recruitment and ERK1/2 signaling. Thus, mimicking the effects of the genetic loss of CCL5. Analysis of the TCGA database, identified that high CCL5 or CCR5 expression is associated with poorer prognosis in low grade esophageal carcinomas. Together, we propose that this data highlights the importance of CCL5 in esophageal carcinomas with regards to CAF recruitment and tumor cell proliferation, and that the maraviroc in vivo experiments suggest that targeting the CCL5-CCR5 axis may have therapeutic potential in esophageal carcinomas. Citation Format: Karen J. Dunbar, Qiaosi Tang, Tatiana A. Karakasheva, Anil K. Rustgi. Tumor-derived CCL5 recruits’ cancer-associated fibroblasts and promotes tumor cell proliferation in esophageal carcinomas [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B013.

Cancer-Associated Fibroblasts in Cholangiocarcinoma: Current Knowledge and Possible Implications for Therapy.

Cholangiocarcinoma (CCA) is an aggressive neoplasia with an increasing incidence and mortality. It is characterized by a strong desmoplastic stroma surrounding cancer cells. Cancer-associated fibroblasts (CAFs) are the main cell type of CCA stroma and they have an important role in modulating cancer microenvironments. CAFs originate from multiple lines of cells and mainly consist of fibroblasts and alpha-smooth muscle actin (α-SMA) positive myofibroblast-like cells. The continuous cross-talking between CCA cells and desmoplastic stroma is permitted by CAF biochemical signals, which modulate a number of pathways. Stromal cell-derived factor-1 expression increases CAF recruitment to the tumor reactive stroma and influences apoptotic pathways. The Bcl-2 family protein enhances susceptibility to CAF apoptosis and PDGFRβ induces fibroblast migration and stimulates tumor lymphangiogenesis. Many factors related to CAFs may influence CCA prognosis. For instance, a better prognosis is associated with IL-33 expression and low stromal IL-6 (whose secretion is stimulated by microRNA). In contrast, a worst prognosis is given by the expression of PDGF-D, podoplanin, SDF-1, α-SMA high expression, and periostin. The maturity phenotype has a prognostic relevance too. New therapeutic strategies involving CAFs are currently under study. Promising results are obtained with anti-PlGF therapy, nintedanib (BIBF1120), navitoclax, IPI-926, resveratrol, and controlled hyperthermia.

Discoidin Domain Receptor 2 Expression as Worse Prognostic Marker in Invasive Breast Cancer.

Discoidin domain receptor 2 (DDR2) is arising as a promising therapeutic target in breast carcinoma (BC). The ability of DDR2 to bind to collagen promotes protumoral responses in cancer cells that influence the tumor microenvironment (TME). Nonetheless, the interrelation between DDR2 expression and TME modulation during BC progression remains poorly known. For this reason, we aim to evaluate the correlation between intratumoral expression of DDR2 and the infiltration of the main TME cell populations, cancer-associated fibroblasts (CAFs), and tumor-associated macrophages (TAMs). First, collagen and DDR2 expression levels were analyzed in human invasive BC samples. Then, DDR2 status correlation with tumor aggressiveness and patient survival were retrieved from different databases. Subsequently, the main pathways, cell types, and tissues correlated with DDR2 expression in BC were obtained through bioinformatics approach. Finally, we studied the association of DDR2 expression with the recruitment of CAFs and TAMs. Our findings showed that, together with the expected overexpression of TME markers, DDR2 was upregulated in tumor samples. Besides, we uncovered that altered TME markers were linked to DDR2 expression in invasive BC patients. Consequently, DDR2 modulates the stromal reaction through CAFs and TAMs infiltration and could be used as a potential worse prognostic factor in the treatment response of invasive BC.

Abstract 2414: HMGA1 induces FGF19 to drive tumor progression and recruit cancer associated fibroblasts in pancreatic adenocarcinoma

Abstract Pancreatic ductal adenocarcinomas (PDACs) are highly lethal tumors characterized by a dense desmoplastic stroma comprised of cancer associated fibroblasts (CAFs) and fibrotic scar tissue. While stromal CAFs act as a barrier to therapy and release signals promoting tumor growth and invasion, the stroma restricts tumor growth in mouse models. The High Mobility Group A1 (HMGA1) protein is an oncofetal protein and epigenetic regulator that amplifies signals from the microenvironment to foster stem cell properties within intestinal epithelium. Although HMGA1 is highly expressed in embryonic and adult stem cells, it is silenced postnatally in most differentiated cells. HMGA1 becomes aberrantly re-expressed in diverse tumors where high levels predict adverse clinical outcomes. In PDAC, HMGA1 is detected only in late-stage precursor lesions or invasive tumors, but not in normal pancreas nor in early precursor lesions. Moreover, high HMGA1 nuclear staining associates with poor differentiation status and decreased patient survival. Here, we discovered a novel epigenetic program whereby HMGA1 recruits stromal CAFs and drives tumor progression by inducing FGF19. Silencing HMGA1 in PDAC cell lines slows proliferation, disrupts oncogenic properties in vitro (migration, invasion, clonogenicity, 3D sphere formation), and depletes tumor initiator cells in xenograft assays. RNA sequencing revealed transcriptional networks up-regulated by HMGA1 that function in cell signaling and proliferation; we focused on FGF19 as a potential mediator of tumor-stromal crosstalk. HMGA1 binds directly to the FGF19 promoter and recruits active histone marks (H3K4me2, H3K27Aac) to induce gene expression and protein secretion from PDAC cells. Silencing FGF19 recapitulates effects of HMGA1 silencing, disrupting oncogenic and cancer stem cell properties in vitro while depleting tumor initiator cells in vivo. In co-culture experiments, FGF19 is required for CAF migration across a membrane towards PDAC cells. Silencing HMGA1, FGF19, or treatment with FGF19 inhibitors disrupts CAF recruitment. Silencing HMGA1 or FGF19 in PDAC cells also decreases both the desmoplastic stroma and tumor growth in mouse xenografts. In primary tumors, co-expression of HMGA1 and FGF19 predict decreased survival. Together, our results reveal a novel paradigm whereby tumor cells collaborate with CAFs via HMGA1 and FGF19 to drive progression, thus illuminating FGF19 as a rational therapeutic target for PDACs overexpressing HMGA1 and FGF19. Citation Format: Lionel Chia, Shuai Shuai, Jung-Hyun Kim, Woo Jung Sung, Ruitao Zhang, Tait Huso, Leslie Cope, Karen Reddy, Linda M. Resar. HMGA1 induces FGF19 to drive tumor progression and recruit cancer associated fibroblasts in pancreatic adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2414.

Abstract 297: HMGA1 induces FGF-19 to foster tumor-stromal cell crosstalk and drive tumor progression in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinomas (PDACs) are highly lethal tumors characterized by a dense desmoplastic stroma comprised of cancer associated fibroblasts (CAFs) and fibrotic scar tissue. While stromal CAFs provide a barrier to therapy and release signals that foster tumor growth and invasion, the stroma also restrains tumor growth in murine models. The High Mobility Group A1 (HMGA1) chromatin remodeling gene encodes an oncofetal protein and epigenetic regulator that amplifies signals from the microenvironment to foster stem cell properties within intestinal epithelium. HMGA1 is also highly expressed during embryogenesis and in adult stem cells, but silenced postnatally in most differentiated cells. In diverse, aggressive cancers, HMGA1 becomes aberrantly re-expressed where high levels portend adverse clinical outcomes. In PDAC, HMGA1 protein is detected in late stage precursor lesions and invasive tumors, but not in normal pancreas nor in early precursor lesions. Furthermore, high HMGA1 levels correlate with poor differentiation status and decreased patient survival. Here, we discovered a novel epigenetic program mediated by HMGA1 that recruits CAFs to drive PDAC progression. We discovered that silencing HMGA1 in multiple PDAC cell lines halts proliferation and disrupts oncogenic properties, including migration, invasion, clonogenicity, and xenograft tumorigenesis. HMGA1 silencing also impairs three-dimensional sphere formation and depletes tumor initiator cells in limiting dilution assays. Through RNA sequencing analysis comparing PDAC cells overexpressing HMGA1 to those with HMGA1 silencing, we identified FGF-19 as a potential transcriptional target of HMGA1. HMGA1 binds specifically to the FGF-19 promoter and recruits the active histone mark, H3K4me3, to activate its expression. HMGA1 is also required for FGF-19 secretion from PDAC cells. Similar to HMGA1, silencing FGF-19 blocks oncogenic and cancer stem cell properties in vitro while disrupting tumorigenesis and depleting tumor initiator cells in vivo. In co-culture experiments, HMGA1 expressed in PDAC cells amplifies FGF-19 secretion, thereby stimulating CAF migration to tumor cells across a membrane. Silencing HMGA1 or FGF-19 prevents CAF recruitment to PDAC tumor cells. Furthermore, CAF recruitment is blocked by either FGF-19 blocking antibodies or an inhibitor to the FGF-19 receptor (FGR4). In murine models, silencing HMGA1 also decreases formation of a fibroblastic stroma. Moreover, overexpression of HMGA1 together with that of FGF-19 predict decreased survival in primary human PDAC. Our results reveal a novel paradigm whereby PDAC cells collaborate with stromal CAFs via HMGA1 and FGF-19 to drive tumor progression. These data also provide insight into mechanisms for tumor progression and illuminate FGF-19 as a rational therapeutic target in PDACs with up-regulation of HMGA1 and FGF-19. Citation Format: Lionel Chia, Shuai Shuai, Lingling Xian, Jung-Hyun Kim, Ruitao Zhang, Tait Huso, David Huso, Leslie Cope, Karen Reddy, Linda Resar. HMGA1 induces FGF-19 to foster tumor-stromal cell crosstalk and drive tumor progression in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 297.

TMIC-22. IDENTIFICATION OF CANCER-ASSOCIATED FIBROBLASTS IN GLIOBLASTOMA and Defining Their Protumoral Effects

Abstract While cancer-associated fibroblasts (CAFs) and their pro-tumoral effects have been demonstrated in systemic cancers, CAFs had been presumed absent in glioblastoma given the lack of normal fibroblasts in the healthy brain. Here, we show that 5–26% (mean=12%) of cells in human glioblastomas express CAF markers α-SMA or PDGFR-β, morphologically resemble fibroblasts, and transcriptomically resemble by RNA-seq CAFs from other cancers. Glioblastoma CAFs were chemotactically attracted to glioblastoma-initiating stem cells (P=0.02). While glioblastoma CAFs did not affect differentiated glioblastoma cell proliferation (P=0.4), CAFs increased glioblastoma stem cell proliferation (P=0.002) and expression of glioblastoma stem cell-associated genes (P< 0.001). To identify mediators of CAF/glioblastoma stem cell interactions, we created a resource of inferred crosstalk by mapping the expression of receptors to that of their cognate ligands/agonists, using our RNA-seq results from glioblastoma CAFs and stem cells, revealing PDGF-β/PDGFR and osteopontin/CD44 to mediate stem cell recruitment of CAFs and CAF enrichment of stem cells, as confirmed by blocking antibodies (P=0.02–0.03). CAFs also render the glioblastoma microenvironment more pro-tumoral by promoting M2 polarization of tumor-associated macrophages (P=0.01), an effect we found to arise from unique CAF production of the EDA splice variant of fibronectin binding toll-like receptor 4 (TLR4), a known EDA receptor expressed by macrophages (P=0.02). In patient glioblastomas, CAFs were enriched 3-fold in the subventricular zone (SVZ) (P=0.04) which houses the neural stem cells that generate glioblastoma stem cells. SVZs from epilepsy cases or autopsies of glioblastoma-containing brains without ventricular involvement lacked CAFs. Depleting CAFs in xenografts derived from neurosphere-containing glioblastoma stem cells slowed their growth in vivo (P< 0.001). These findings are among the first to identify and profile glioblastoma CAFs. CAF recruitment by glioblastoma stem cells and creation of a pro-tumoral microenvironment in the perivascular niche housing glioblastoma stem cells, particularly in the SVZ, makes them an intriguing therapeutic target.

Pancreatic cancer-derived exosomes promoted pancreatic stellate cells recruitment by pancreatic cancer.

Cancer-associated fibroblasts (CAFs), which are an important component of the tumor microenvironment, have been identified in the blood circulation of patients with cancer metastasis, and metastatic cancer cells can recruit circulating CAFs. However, primary carcinoma sites usually regulate the behavior of metastatic cancer cells through exosomes. Here, we hypothesized that cancer-derived exosomes could enhance CAF recruitment. Exosomes secreted by pancreatic cancer cells (PANC-1 and MIA PaCa-2) were isolated and characterized. The ability of pancreatic cancer to recruit pancreatic stellate cells (PSCs) was assessed with Transwell assays in vitro and bioluminescent imaging in a mouse model in vivo, and the underlying molecular mechanism was also investigated. The results showed that pancreatic cancer cell-derived exosomes (Exo-Pan and Exo-Mia) promoted the pancreatic cancer recruitment of PSCs. This effect was mediated partially by the transfer of the exosomal protein Lin28B to the recipient cells to activate the Lin28B/let-7/HMGA2/PDGFB signaling pathway. These results suggested that exosomes derived from local cancer could promote the formation of distant metastases through transferring the exosomal protein Lin28B to the metastatic cancer cells.

Expressional analysis of disease-relevant signalling-pathways in primary tumours and metastasis of head and neck cancers

Head and neck squamous cell carcinoma (HNSCC) often metastasize to lymph nodes resulting in poor prognosis for patients. Unfortunately, the underlying molecular mechanisms contributing to tumour aggressiveness, recurrences, and metastasis are still not fully understood. However, such knowledge is key to identify biomarkers and drug targets to improve prognosis and treatments. Consequently, we performed genome-wide expression profiling of 15 primary HNSSCs compared to corresponding lymph node metastases and non-malignant tissue of the same patient. Differentially expressed genes were bioinformatically exploited applying stringent filter criteria, allowing the discrimination between normal mucosa, primary tumours, and metastases. Signalling networks involved in invasion contain remodelling of the extracellular matrix, hypoxia-induced transcriptional modulation, and the recruitment of cancer associated fibroblasts, ultimately converging into a broad activation of PI3K/AKT-signalling pathway in lymph node metastasis. Notably, when we compared the diagnostic and prognostic value of sequencing data with our expression analysis significant differences were uncovered concerning the expression of the receptor tyrosine kinases EGFR and ERBB2, as well as other oncogenic regulators. Particularly, upregulated receptor tyrosine kinase combinations for individual patients varied, implying potential compensatory and resistance mechanisms against specific targeted therapies. Collectively, we here provide unique transcriptional profiles for disease predictions and comprehensively analyse involved signalling pathways in advanced HNSCC.

Abstract A21: Chemokine directed migration of activated stellate cells in pancreatic ductal adenocarcinoma

Abstract Multiple studies have shown that the extreme desmoplasia observed in pancreatic tumors modulates the effectiveness of chemotherapeutic agents. Despite this, the specific mechanisms by which stroma develops in pancreatic tumors are unknown and the role that it plays in pathogenesis of the disease is unsettled. Chemokines are a family of nearly 50 different secreted proteins playing a key role in the recruitment of a wide variety of cell types in health and disease. While we and others have extensively explored the role of the chemokine CXCL12 in pancreatic cancer, the expression and potential role of other chemokines, or their cognate receptors, in pancreatic cancer remains an area of active exploration. To this end, we profiled the expression of the chemokine family in pancreatic cancer. RT-PCR of a battery of established human pancreatic cancer cell lines revealed elevated transcript levels of the chemokine ligands CCL28 and CXCL16, as well as their cognate receptors CCR10 and CXCR6, respectively. These results were mirrored in cell lines derived from transgenic murine models of pancreatic cancer. Immunohistochemical analysis of both diseased and normal human pancreatic tissue showed that the expression of CCL28, CXCL16, CCR10, and CXCR6 proteins were upregulated in pancreatic adenocarcinoma compared with normal exocrine ductal epithelium. Interestingly, CCR10 and CXCR6 were also expressed in pancreatic tumor fibroblasts, while their cognate ligands, CCL28 and CXCL16 were absent in the stromal compartment. Based on those data we hypothesized that expression of chemokines in cancer cells with the concomitant expression of their cognate receptors by activated fibroblasts facilitates recruitment of those cancer-associated fibroblasts into the tumor microenvironment. Analysis of tissue from patients with pancreatitis revealed that CCL28/CCR10, but not CXCL16/CXCR6, proteins were upregulated. Consistent with its known regulation by inflammatory mediators, we found that interferon-gamma stimulated significant increases in CCL28 secretion by human pancreatic cancer cell lines. In contrast, interferon-gamma had little if any effect on CCL28 secretion by an immortalized activated human pancreatic stellate cell (HPSC) line. Stimulation with exogenous CCL28 elicited the prototypical dose dependent calcium signaling expected of a ligand activated chemokine receptor. CCL28 stimulated the dose dependent directional migration of HPSC without altering cell viability. These data are consistent with HPSC being a functional target for the chemokine. Finally, in a 2-D co-culture model, CCL28 secreted from human pancreatic cancer cells induced HPSC chemotactic migration. Pretreatment with neutralizing antibody to the chemokine blocked HPSC chemotaxis toward the cancer cells. Together, these data suggest that the CCL28-CCR10 chemokine axis plays a role in the inflammation-mediated recruitment of cancer-associated fibroblasts into the stromal compartment of pancreatic tumors. More broadly, we have demonstrated a new role for chemokines in the pancreatic tumor microenvironment, suggestive of their potential role in stromal remodeling in pancreatic cancer. This is the first study to establish the differential expression and functional role for the CCL28 chemokine in pancreatic disease. Citation Format: Ishan Roy, Kathleen A. Boyle, A Craig Mackinnon, Rosa F. Hwang, Susan Tsai, Douglas B. Evans, Michael B. Dwinell.{Authors}. Chemokine directed migration of activated stellate cells in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A21.

Deregulated matriptase activity in oral squamous cell carcinoma promotes the infiltration of cancer-associated fibroblasts by paracrine activation of protease-activated receptor 2.

Cancer-associated fibroblasts (CAFs) are known to contribute to cancer progression. We have reported that cell surface expression of hepatocyte growth factor activator inhibitor 1 (HAI-1) is decreased in invasive oral squamous cell carcinoma (OSCC) cells. This study examined if HAI-1-insufficiency contributes to CAF recruitment in OSCC. Serum-free conditioned medium (SFCM) from a human OSCC line (SAS) stimulated the migration of 3 human fibroblast cell lines, NB1RGB, MRC5 and KD. SFCM from HAI-1-knockdown SAS showed an additive effect on the migration of NB1RGB and MRC5, but not KD. SAS SFCM induced protease-activated receptor-2 (PAR-2) expression in NB1RGB and MRC5, but not in KD, and a PAR-2 antagonist blocked the stimulatory effect of HAI-1 knockdown on migration of the PAR-2 expressing cell lines. Moreover, HAI-1-deficient SFCM showed additive stimulatory effects on the migration of wild-type but not PAR-2-deficient mouse fibroblasts. Therefore, the enhanced migration induced by HAI-1-insufficiency was mediated by PAR-2 activation in fibroblasts. This activation resulted from the deregulation of the activity of matriptase, a PAR-2 agonist protease. HAI-1 may thus prevent CAF recruitment to OSCC by controlling matriptase activity. When HAI-1 expression is reduced on OSCC, matriptase may contribute to CAF accumulation by paracrine activation of fibroblast PAR-2. Immunohistochemical analysis of resected OSCC revealed increased PAR2-positive CAFs in 35% (33/95) of the cases studied. The increased PAR-2 positive CAFs tended to correlate with infiltrative histology of the invasion front and shorter disease-free survival of the patients.

MP92-09 COMBINATION THERAPY USING MOLECULAR-TARGETED DRUGS INHIBITING PLATELET-DERIVED GROWTH FACTOR RECEPTORS IN THE TUMOR MICROENVIRONMENT OF RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology III1 Apr 2016MP92-09 COMBINATION THERAPY USING MOLECULAR-TARGETED DRUGS INHIBITING PLATELET-DERIVED GROWTH FACTOR RECEPTORS IN THE TUMOR MICROENVIRONMENT OF RENAL CELL CARCINOMA Hiroyuki Kitano, Jun Teishima, Ryo Yuge, Syunsuke Shinmei, Hirotaka Nagamatsu, Keisuke Goto, Kouichi Shoji, Syogo Inoue, Tetsutaro Hayashi, Kazuhiro Sentani, Yasuhiko Kitadai, Wataru Yasui, and Akio Matsubara Hiroyuki KitanoHiroyuki Kitano More articles by this author , Jun TeishimaJun Teishima More articles by this author , Ryo YugeRyo Yuge More articles by this author , Syunsuke ShinmeiSyunsuke Shinmei More articles by this author , Hirotaka NagamatsuHirotaka Nagamatsu More articles by this author , Keisuke GotoKeisuke Goto More articles by this author , Kouichi ShojiKouichi Shoji More articles by this author , Syogo InoueSyogo Inoue More articles by this author , Tetsutaro HayashiTetsutaro Hayashi More articles by this author , Kazuhiro SentaniKazuhiro Sentani More articles by this author , Yasuhiko KitadaiYasuhiko Kitadai More articles by this author , Wataru YasuiWataru Yasui More articles by this author , and Akio MatsubaraAkio Matsubara More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2642AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Recent studies have shown that the tumor microenvironment, which is comprised of cancer cells and stroma, is involved in cancer progression and platelet-derived growth factor (PDGF) plays a major role in stromal activation. However, the impacts of molecular-targeted drugs on the stroma of renal cell carcinoma (RCC) remain unclear. Therefore, in this study, we used a tyrosine kinase inhibitor (TKI) and a mammalian target of rapamycin inhibitor (mTORI) targeting PDGF receptors with the aim of elucidating their effects on the RCC stroma and analyzing the treatment outcomes obtained with TKI/mTORI combination therapy. METHODS Orthotopic mouse models, created using Caki-1, a luciferase-expressing human RCC cell line, were divided into four groups: control (C), everolimus (E), sunitinib (S) and everolimus/sunitinib (ES). After four-week everolimus at 1 mg/kg/day and sunitinib at 10 mg/kg/day, tumors were removed. Cell viability was measured every 2 weeks by in vivo imaging. Cell proliferation was analyzed in extracted tissues by the Ki67 labeling index (Ki67-LI). Recruitment of cancer-associated fibroblasts (CAFs) and extracellular matrix growth were analyzed by immunostaining for a-smooth muscle actin and type 1 collagen, respectively, angiogenesis and lymphangiogenesis by immunofluorescence staining for CD31 and LYVE-1, respectively, and pericyte recruitment by double-immunofluorescence staining with antibodies against endothelial cell (CD31) and pericyte (desmin) markers. RESULTS Cell viability and Ki67-LI were significantly decreased in the E and ES groups as compared to the S group. We first confirmed expressions of PDGF receptors on CAFs and pericytes by double-immunofluorescence staining. Neither recruitment of CAFs nor growth of type 1 collagen was inhibited in the E group but both were significantly inhibited in the S and ES groups. Compared to the E group, angiogenesis and lymphangiogenesis were significantly inhibited in the S and ES groups. The C and E groups showed pericyte recruitment around blood vessels, but these cells were relatively sparse in the S and ES groups. CONCLUSIONS The comparison between TKI and mTORI revealed mTORI to have significantly greater inhibitory effects on cancer cells, while TKI exerted inhibitory effects on the stroma. Our findings suggest that combining these two molecular-targeted drugs, with the aim of targeting the tumor microenvironment, would be an effective treatment for RCC. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1165 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Hiroyuki Kitano More articles by this author Jun Teishima More articles by this author Ryo Yuge More articles by this author Syunsuke Shinmei More articles by this author Hirotaka Nagamatsu More articles by this author Keisuke Goto More articles by this author Kouichi Shoji More articles by this author Syogo Inoue More articles by this author Tetsutaro Hayashi More articles by this author Kazuhiro Sentani More articles by this author Yasuhiko Kitadai More articles by this author Wataru Yasui More articles by this author Akio Matsubara More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

GZD856, a novel potent PDGFRα/β inhibitor, suppresses the growth and migration of lung cancer cells in vitro and in vivo

Platelet-derived growth factor receptors (PDGFRα/β) play critical roles in the autocrine-stimulated growth and recruitment of cancer-associated fibroblasts (CAFs) of human lung cancer cells. We have identified GZD856 as a new PDGFR inhibitor that potently inhibits PDGFRα/β kinase activity and blocks this signaling pathway in lung cancer cells both in vitro and in vivo. GZD856 strongly suppresses the proliferation of PDGFRα-amplified H1703 (PDGFRβ-) human lung cancer cells and demonstrates significant in vivo antitumor efficacy in a xenograft mouse model. Although GZD856 displays only limited in vitro antiproliferative efficiency against PDGFRα-/PDGFRβ+ A549 lung cancer cells, it efficiently inhibits the in vivo growth and metastasis of A549 cancer cells in xenograft and orthotopic models, respectively. The promising in vivo antitumor activity of GZD856 in A549 models may result from its suppression of PDGFR-related microenvironment factors, such as recruitment of CAFs and collagen content in stromal cells. GZD856 may be considered as a promising new candidate for anti-lung cancer drug development.

Tumor-α9β1 integrin-mediated signaling induces breast cancer growth and lymphatic metastasis via the recruitment of cancer-associated fibroblasts.

Tumor-derived matricellular proteins such as osteopontin (OPN) and tenascin-C (TN-C) have been implicated in tumor growth and metastasis. However, the molecular basis of how these proteins contribute to tumor progression remains to be elucidated. Importantly, these matricellular proteins are known to interact with α9β1 integrin. Therefore, we hypothesized that tumor-derived α9β1 integrin may contribute to tumor progression. To clarify the roles of α9β1 integrin in tumor growth and lymphatic metastasis, we used an inhibitory anti-human α9β1 integrin antibody (anti-hα9β1 antibody) and a α9β1 integrin-positive human breast cancer cell line, MDA-MB-231 luc-D3H2LN (D3H2LN), in vitro functional assays, and an in vivo orthotopic xenotransplantation model. In this study, we demonstrated that tumor, but not host α9β1 integrin, contributes to tumor growth, lymphatic metastasis, recruitment of cancer-associated fibroblasts (CAFs), and host-derived OPN production. We also found that CAFs contributed to tumor growth, lymphatic metastasis, and host-derived OPN levels. Consistent with those findings, tumor volume was well-correlated with numbers of CAFs and levels of host-derived OPN. Furthermore, it was shown that the inoculation of D3H2LN cells into mammary fat pads with mouse embryonic fibroblasts (MEFs), obtained from wild type, but not OPN knock-out mice, resulted in enhancement of tumor growth, thus indicating that CAF-derived OPN enhanced tumor growth. These results suggested that tumor α9β1-mediated signaling plays a pivotal role in generating unique primary tumor tissue microenvironments, which favor lymphatic metastasis and tumor growth. Tumor α9β1 integrin promotes lymphatic metastasis through enhancing invasion. Tumor α9β1 integrin promotes tumor growth through CAFs. Tumor α9β1 integrin enhances the recruitment of CAFs into the primary tumor. Tumor cells induce the production of OPN by CAFs in the primary tumor. CAF-derived OPN promotes tumor growth.

Platelet-derived growth factor D: A new player in the complex cross-talk between cholangiocarcinoma cells and cancer-associated fibroblasts

Platelet-derived growth factor D: A new player in the complex cross-talk between cholangiocarcinoma cells and cancer-associated fibroblasts

Platelet-derived growth factor-D and Rho GTPases regulate recruitment of cancer-associated fibroblasts in cholangiocarcinoma

Cholangiocarcinoma (CCA) is characterized by an abundant stromal reaction. Cancer-associated fibroblasts (CAFs) are pivotal in tumor growth and invasiveness and represent a potential therapeutic target. To understand the mechanisms leading to CAF recruitment in CCA, we studied (1) expression of epithelial-mesenchymal transition (EMT) in surgical CCA specimens and CCA cells, (2) lineage tracking of an enhanced green fluorescent protein (EGFP)-expressing human male CCA cell line (EGI-1) after xenotransplantation into severe-combined-immunodeficient mice, (3) expression of platelet-derived growth factors (PDGFs) and their receptors in vivo and in vitro, (4) secretion of PDGFs by CCA cells, (5) the role of PDGF-D in fibroblast recruitment in vitro, and (6) downstream effectors of PDGF-D signaling. CCA cells expressed several EMT biomarkers, but not alpha smooth muscle actin (α-SMA). Xenotransplanted CCA masses were surrounded and infiltrated by α-SMA-expressing CAFs, which were negative for EGFP and the human Y-probe, but positive for the murine Y-probe. CCA cells were strongly immunoreactive for PDGF-A and -D, whereas CAFs expressed PDGF receptor (PDGFR)β. PDGF-D, a PDGFRβ agonist, was exclusively secreted by cultured CCA cells. Fibroblast migration was potently induced by PDGF-D and CCA conditioned medium and was significantly inhibited by PDGFRβ blockade with Imatinib and by silencing PDGF-D expression in CCA cells. In fibroblasts, PDGF-D activated the Rac1 and Cdc42 Rho GTPases and c-Jun N-terminal kinase (JNK). Selective inhibition of Rho GTPases (particularly Rac1) and of JNK strongly reduced PDGF-D-induced fibroblast migration. CCA cells express several mesenchymal markers, but do not transdifferentiate into CAFs. Instead, CCA cells recruit CAFs by secreting PDGF-D, which stimulates fibroblast migration through PDGFRβ and Rho GTPase and JNK activation. Targeting tumor or stroma interactions with inhibitors of the PDGF-D pathway may offer a novel therapeutic approach.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Macrophage Migration Inhibitory Factor Is Secreted by Rhabdomyosarcoma Cells, Modulates Tumor Metastasis by Binding to CXCR4 and CXCR7 Receptors and Inhibits Recruitment of Cancer-Associated Fibroblasts

Abstract The overexpression of macrophage migration inhibitory factor (MIF) has been observed in many tumors and is implicated in oncogenic transformation and tumor progression. MIF activates CXCR2 and CD74 receptors and, as recently reported, may also bind to the stromal-derived factor-1 (SDF-1)–binding receptor CXCR4. Here, we report that human rhabdomyosarcoma (RMS) cell lines secrete MIF and that this chemokine (a) induces phosphorylation of mitogen-activated protein kinase (MAPK) p42/44 and AKT, (b) stimulates RMS cell adhesion, (c) enhances tumor vascularization, but surprisingly (d) decreases recruitment of cancer-associated fibroblasts (CAF). Because RMS cells used in our studies do not express CXCR2 and CD74 receptors, the biological effects of MIF on RMS cells depend on its interaction with CXCR4, and as we report here for the first time, MIF may also engage another SDF-1–binding receptor (CXCR7) as well. Interestingly, downregulation of MIF in RMS cells inoculated into immunodeficient mice led to formation of larger tumors that displayed higher stromal cell support. Based on these observations, we postulate that MIF is an important autocrine/paracrine factor that stimulates both CXCR4 and CXCR7 receptors to enhance the adhesiveness of RMS cells. We also envision that when locally secreted by a growing tumor, MIF prevents responsiveness of RMS to chemoattractants secreted outside the growing tumor (e.g., SDF-1) and thereby prevents release of cells into the circulation. On the other hand, despite its obvious proangiopoietic effects, MIF inhibits in CXCR2/CD74-dependent manner recruitment of CAFs to the growing tumor. Our data indicate that therapeutic inhibition of MIF in RMS may accelerate metastasis and tumor growth. Mol Cancer Res; 8(10); 1328–43. ©2010 AACR.