Cancer-associated Fibroblasts Subpopulations Research Articles

Cancer associated fibroblasts serve as an ovarian cancer stem cell niche through noncanonical Wnt5a signaling

Frequent relapse and chemoresistance cause poor outcome in ovarian cancer (OC) and cancer stem cells (CSCs) are important contributors. While most studies focus exclusively on CSCs, the role of the microenvironment in providing optimal conditions to maintain their tumor-initiating potential remains poorly understood. Cancer associated fibroblasts (CAFs) are a major constituent of the OC tumor microenvironment and we show that CAFs and CSCs are enriched following chemotherapy in patient tumors. CAFs significantly increase OC cell resistance to carboplatin. Using heterotypic CAF-OC cocultures and in vivo limiting dilution assay, we confirm that the CAFs act by enriching the CSC population. CAFs increase the symmetric division of CSCs as well as the dedifferentiation of bulk OC cells into CSCs. The effect of CAFs is limited to OC cells in their immediate neighborhood, which can be prevented by inhibiting Wnt. Analysis of single cell RNA-seq data from OC patients reveal Wnt5a as the highest expressed Wnt in CAFs and that certain subpopulations of CAFs express higher levels of Wnt5a. Our findings demonstrate that Wnt5a from CAFs activate a noncanonical Wnt signaling pathway involving the ROR2/PKC/CREB1 axis in the neighboring CSCs. While canonical Wnt signaling is found to be predominant in interactions between cancer cells in patients, non-canonical Wnt pathway is activated by the CAF-OC crosstalk. Treatment with a Wnt5a inhibitor sensitizes tumors to carboplatin in vivo. Together, our results demonstrate a novel mechanism of CSC maintenance by signals from the microenvironmental CAFs, which can be targeted to treat OC chemoresistance and relapse.

Single-cell RNA sequencing reveals the heterogeneity of epithelial cell and fibroblast cells from non- to metastatic lymph node OTSCC.

Lymph node metastasis (LNM) is one of the common features of oral tongue squamous cell carcinoma (OTSCC). LNM is also taken as a sign of advanced OTSCC and poor survival rate. Recently, single-cell RNA sequencing has been applied in investigating the heterogeneity of tumor microenvironment and discovering the potential biomarkers for helping the diagnosis and prognosticating. Pathogenesis of LNM in OTSCC remains unknown. Specifically, cancer-associated fibroblasts (CAFs) and epithelial tumor cells could foster the progression of tumors. Thus, in this study, we aimed to comprehensively analyze the roles of subpopulations of CAFs and epithelial tumor cells in lymph node metastatic OTSCC using the integration of OTSCC single-cell RNA sequencing datasets. Four distinct subtypes of CAFs, namely vascular CAFs, myofibroblast CAFs, inflammatory CAFs, and growth arrest CAFs were successfully discovered in LNM tumor and confirmed the roles of GAS and PTN pathways in the progression of tumor metastasis. In addition, NKAIN2+ epithelial cells and FN1+ epithelial cells specifically exhibited an upregulation of PTN, NRG, MIF, and SPP1 signaling pathways in the metastatic OTSCC. In doing so, we put forth some potential biomarkers that could be utilized for the purpose of diagnosing and prognosticating OTSCC during its metastatic phase and tried to confirm by immunofluorescence assays.

Single-cell and spatial transcriptomics reveal POSTN+ cancer-associated fibroblasts correlated with immune suppression and tumour progression in non-small cell lung cancer.

Cancer-associated fibroblasts (CAFs) are potential targets for cancer therapy. Due to the heterogeneity of CAFs, the influence of CAF subpopulations on the progression of lung cancer is still unclear, which impedes the translational advances in targeting CAFs. We performed single-cell RNA sequencing (scRNA-seq) on tumour, paired tumour-adjacent, and normal samples from 16 non-small cell lung cancer (NSCLC) patients. CAF subpopulations were analyzed after integration with published NSCLC scRNA-seq data. SpaTial enhanced resolution omics-sequencing (Stereo-seq) was applied in tumour and tumour-adjacent samples from seven NSCLC patients to map the architecture of major cell populations in tumour microenvironment (TME). Immunohistochemistry (IHC) and multiplexed IHC (mIHC) were used to validate marker gene expression and the association of CAFs with immune infiltration in TME. A subcluster of myofibroblastic CAFs, POSTN+ CAFs, were significantly enriched in advanced tumours and presented gene expression signatures related to extracellular matrix remodeling, tumour invasion pathways and immune suppression. Stereo-seq and mIHC demonstrated that POSTN+ CAFs were in close localization with SPP1+ macrophages and were associated with the exhausted phenotype and lower infiltration of T cells. POSTN expression or the abundance of POSTN+ CAFs were associated with poor prognosis of NSCLC. Our study identified a myofibroblastic CAF subpopulation, POSTN+ CAFs, which might associate with SPP1+ macrophages to promote the formation of desmoplastic architecture and participate in immune suppression. Furthermore, we showed that POSTN+ CAFs associated with cancer progression and poor clinical outcomes and may provide new insights on the treatment of NSCLC.

Abstract PR03: Hypoxia promotes inflammatory fibroblast formation in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy characterized by poor response to all existing therapies. Although immunotherapy has shown great promise against multiple deadly cancers, it has been largely ineffective in PDAC. This lack of response is in part attributed to its extensive, fibroinflammatory stroma and hypoxic microenvironment. Cancer-associated fibroblasts (CAFs) are a predominant and heterogeneous stromal cell type in PDAC, and single-cell transcriptomics of human and mouse PDAC has recently revealed a distinct CAF subpopulation, inflammatory CAFs (iCAFs). These inflammatory fibroblasts produce high levels of cytokines and chemokines in PDAC and have the potential to contribute to its immunosuppressive microenvironment and tumorigenesis. By injecting a hypoxia probe into PDAC mouse models, we recently found that iCAFs predominantly reside in hypoxic tumor regions. We also observed that the hypoxia-related gene signature is positively enriched in iCAFs in human PDAC samples. Importantly, by exposing three-dimensional (3D) co-cultures of pancreatic cancer cells and fibroblasts to either hypoxia or normoxia, we showed that hypoxia induces IL1α from cancer cells and that IL1α is required for hypoxia-mediated iCAF formation. Our data implicate hypoxia as a critical regulator of tumor stroma. Efforts are ongoing to understand the role of hypoxia in the crosstalk between cancer cells, fibroblasts, and immune cells in driving an immunosuppressive tumor microenvironment. Citation Format: Ashley M. Mello, Tenzin Ngodup, Yusoo Lee, Katelyn L. Donahue, Jinju Li, Arvind Rao, Eileen S. Carpenter, Howard C. Crawford, Marina Pasca Di Magliano, Kyoung Eun Lee. Hypoxia promotes inflammatory fibroblast formation in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr PR03.

Integrating single-cell and spatial transcriptomics reveals endoplasmic reticulum stress-related CAF subpopulations associated with chordoma progression.

With cancer-associated fibroblasts (CAFs) as the main cell type, the rich myxoid stromal components in chordoma tissues may likely contribute to its development and progression. Single-cell RNA sequencing (scRNA-seq), spatial transcriptomics, bulk RNA-seq, and multiplexed quantitative immunofluorescence (QIF) were used to dissect the heterogeneity, spatial distribution, and clinical implication of CAFs in chordoma. We sequenced here 72 097 single cells from 3 primary and 3 recurrent tumor samples, as well as 3 nucleus pulposus samples as controls using scRNA-seq. We identified a unique cluster of CAF in recurrent tumors that highly expressed hypoxic genes and was functionally enriched in endoplasmic reticulum stress (ERS). Pseudotime trajectory and cell communication analyses showed that this ERS-CAF subpopulation originated from normal fibroblasts and widely interacted with tumoral and immune cells. Analyzing the bulk RNA-seq data from 126 patients, we found that the ERS-CAF signature score was associated with the invasion and poor prognosis of chordoma. By integrating the results of scRNA-seq with spatial transcriptomics, we demonstrated the existence of ERS-CAF in chordoma tissues and revealed that this CAF subtype displayed the most proximity to its surrounding tumor cells. In subsequent QIF validation involving 105 additional patients, we confirmed that ERS-CAF was abundant in the chordoma microenvironment and located close to tumor cells. Furthermore, both ERS-CAF density and its distance to tumor cells were correlated with tumor malignant phenotype and adverse patient outcomes. These findings depict the CAF landscape for chordoma and may provide insights into the development of novel treatment approaches.

Inhibition of EphA3 Expression in Tumour Stromal Cells Suppresses Tumour Growth and Progression.

Tumour progression relies on interactions with untransformed cells in the tumour microenvironment (TME), including cancer-associated fibroblasts (CAFs), which promote blood supply, tumour progression, and immune evasion. Eph receptor tyrosine kinases are cell guidance receptors that are most active during development but re-emerge in cancer and are recognised drug targets. EphA3 is overexpressed in a wide range of tumour types, and we previously found expression particularly in stromal and vascular tissues of the TME. To investigate its role in the TME, we generated transgenic mice with inducible shRNA-mediated knockdown of EphA3 expression. EphA3 knockdown was confirmed in aortic mesenchymal stem cells (MSCs), which displayed reduced angiogenic capacity. In mice with syngeneic lung tumours, EphA3 knockdown reduced vasculature and CAF/MSC-like cells in tumours, and inhibited tumour growth, which was confirmed also in a melanoma model. Single cell RNA sequencing analysis of multiple human tumour types confirmed EphA3 expression in CAFs, including in breast cancer, where EphA3 was particularly prominent in perivascular- and myofibroblast-like CAFs. Our results thus indicate expression of the cell guidance receptor EphA3 in distinct CAF subpopulations is important in supporting tumour angiogenesis and tumour growth, highlighting its potential as a therapeutic target.

Cancer-associated fibroblasts: Just on the opposite side of antitumour immunity?

The tumour microenvironment (TME) is critical for the initiation, progression, and metastasis of tumours, and cancer-associated fibroblasts (CAFs) are the most dominant cells and have attracted interest as targets for cancer therapy among the stromal components within the TME. Currently, most of the identified CAF subpopulations are believed to exhibit suppressive effects on antitumour immunity. However, accumulating evidence indicates the presence of immunostimulatory CAF subpopulations, which play an important role in the maintenance and amplification of antitumour immunity, in the TME. Undoubtedly, these findings provide novel insights into CAF heterogeneity. Herein, we focus on summarizing CAF subpopulations that promote antitumour immunity, the surface markers of these populations, and possible immunostimulatory mechanisms in the context of recent advances in research on CAF subpopulations. In addition, we discuss the possibility of new therapies targeting CAF subpopulations and conclude with a brief description of some prospective avenues for CAF research.

267. IDENTIFYING CANCER-ASSOCIATED FIBROBLAST POPULATIONS DRIVING THERAPY RESISTANCE IN GASTROESOPHAGEAL ADENOCARCINOMA

Abstract Background Peri-operative docetaxel-based triplet chemotherapy is the standard-of-care treatment for advanced gastroesophageal adenocarcinoma (GEA). However, most patients recur due to innate or acquired resistance. Although distinct populations of cancer-associated fibroblasts (CAFs) within the tumor microenvironment play important roles in conferring chemoresistance in other cancer types, this paradigm has not been extensively studied in GEA. We aim to characterize CAF heterogeneity in a well-annotated cohort of GEA patients, identifying potential biomarkers and targets to overcome chemoresistance. Methods Immunofluorescence and flow cytometry assays were performed to validate previously reported CAF markers from the literature in primary patient fibroblasts. To identify specific CAF markers for GEA, an atlas was developed using single-cell RNA sequencing (scRNA-seq) data obtained from 46 GEA patient samples, including 28 patients with longitudinal samples over the docetaxel-based triplet chemotherapy treatment trajectory. Differential and gene ontology analyses were performed to characterize distinct CAF subpopulations and identify dynamic CAF markers across treatment timepoints, while correlating with in-patient treatment response to chemotherapy. In parallel, tumor organoid-CAF co-cultures were established for subsequent in vitro drug testing with standard-of-care chemotherapy. Results CAF markers (VIM, FAP, PDPN, and S100A4) were found to be differentially expressed in fibroblasts isolated from good and poor pathological-response tumours. Analysis of scRNA-seq data reveals two main subpopulations of CAFs: myofibroblast CAFs (myCAFs) and inflammatory CAFs (iCAFs). Two markers, CCL20 and CHRDL1, were identified as differentially expressed between good and poor pathological responders in iCAFs, while nine markers, including ISG20 and C20orf27, were differentially expressed between clinical partial responders and poor responders. Dynamic CAF markers, including STAG2 and HAT1, were differentially expressed across treatment timepoints and were associated with pathological or clinical response. Conclusions GEA CAFs demonstrate extensive heterogeneity and comprise two major subpopulations. Several CAF markers were found to be associated with patient chemotherapy pathological or clinical response to chemotherapy, either in the chemonaïve setting or in the context of changes over the course of therapy. These putative biomarkers will be further validated and investigated, both in additional samples and in co-culture settings, to gain a better understanding of their clinical relevance and targeting potential.

Novel CAF-identifiers via transcriptomic and protein level analysis in HNSC patients

Cancer-associated fibroblasts (CAFs), a prominent component of the tumor microenvironment, play an important role in tumor development, invasion, and drug resistance. The expression of distinct “CAF-markers” which separates CAFs from normal fibroblasts and epithelial cells, have traditionally been used to identify them. These commonly used CAF-markers have been reported to differ greatly across different CAF subpopulations, even within a cancer type. Using an unbiased -omic approach from public data and in-house RNAseq data from patient derived novel CAF cells, TIMP-1, SPARC, COL1A2, COL3A1 and COL1A1 were identified as potential CAF-markers by differential gene expression analysis using publicly available single cell sequencing data and in-house RNAseq data to distinguish CAF populations from tumor epithelia and normal oral fibroblasts. Experimental validation using qPCR and immunofluorescence revealed CAF-specific higher expression of TIMP-1 and COL1A2 as compared to other markers in 5 novel CAF cells, derived from patients of diverse gender, habits and different locations of head and neck squamous cell carcinoma (HNSC). Upon immunohistochemical (IHC) analysis of FFPE blocks however, COL1A2 showed better differential staining between tumor epithelia and tumor stroma. Similar data science driven approach utilizing single cell sequencing and RNAseq data from stabilized CAFs can be employed to identify CAF-markers in various cancers.

Single-cell and bulk RNA sequencing reveal cancer-associated fibroblast heterogeneity and a prognostic signature in prostate cancer.

Cancer-associated fibroblasts (CAFs), the central players in the tumor microenvironment (TME), can promote tumor progression and metastasis via various functions. However, the properties of CAFs in prostate cancer (PCa) have not been fully assessed. Therefore, we aimed to examine the CAF characteristics in PCa and construct a CAF-derived signature to predict PCa prognosis. CAFs were identified using single-cell RNA sequencing (scRNA-seq) data from 3 studies. We performed the FindAllMarkers function to extract CAF marker genes and constructed a signature to predict the biochemical relapse-free survival (bRFS) of PCa in the Cancer Genome Atlas (TCGA) cohort. Subsequently, different algorithms were applied to reveal the differences of the TME, immune infiltration, treatment responses in the high- and low-risk groups. Additionally, the CAF heterogeneity was assessed in PCa, which were confirmed by the functional enrichment analysis, gene set enrichment analysis (GSEA), and AUCell method. The scRNA-seq analysis identified a CAF cluster with 783 cells and determined 183 CAF marker genes. Cell-cell communication revealed extensive interactions between fibroblasts and immune cells. A CAF-related prognostic model, containing 7 genes (ASPN, AEBP1, ALDH1A1, BGN, COL1A1, PAGE4 and RASD1), was developed to predict bRFS and validated by 4 independent bulk RNA-seq cohorts. Moreover, the high-risk group of the signature score connected with an immunosuppressive TME, such as a higher level of M2 macrophages and lower levels of plasma cells and CD8+ T cells, and a reduced reaction rate for immunotherapy compared with low-risk group. After re-clustering CAFs via unsupervised clustering, we revealed 3 biologically distinct CAF subsets, namely myofibroblast-like CAFs (myCAFs), immune and inflammatory CAFs (iCAFs) and antigen-presenting CAFs (apCAFs). In conclusion, the CAF-derived signature, the first of its kind, can effectively predict PCa prognosis and serve as an indicator for immunotherapy. Furthermore, our study identified 3 CAF subpopulations with distinct functions in PCa.

Friend or foe? The elusive role of hepatic stellate cells in liver cancer.

Liver fibrosis is a substantial risk factor for the development and progression of liver cancer, which includes hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA). Studies utilizing cell fate mapping and single-cell transcriptomics techniques have identified quiescent perisinusoidal hepatic stellate cells (HSCs) as the primary source of activated collagen-producing HSCs and liver cancer-associated fibroblasts (CAFs) in HCCand liver metastasis, complemented in iCCA by contributions from portal fibroblasts. At the same time, integrative computational analysis of single-cell, single-nucleus and spatial RNA sequencing data have revealed marked heterogeneity among HSCs and CAFs, with distinct subpopulations displaying unique gene expression signatures and functions. Some of these subpopulations have divergent roles in promoting or inhibiting liver fibrogenesis and carcinogenesis. In this Review, we discuss the dual roles of HSC subpopulations in liver fibrogenesis and their contribution to liver cancer promotion, progression and metastasis. We review the transcriptomic and functional similarities between HSC and CAF subpopulations, highlighting the pathways that either promote or prevent fibrosis and cancer, and the immunological landscape from which these pathways emerge. Insights from ongoing studies will yield novel strategies for developing biomarkers, assessing prognosis and generating new therapies for both HCC and iCCA prevention and treatment.

Medical Biology of Cancer-Associated Fibroblasts in Pancreatic Cancer.

Pancreatic cancer is one of the deadliest forms of cancer with one of the lowest 5-year survival rates of all cancer types. A defining characteristic of pancreatic cancer is the existence of dense desmoplastic stroma that, when exposed to stimuli such as cytokines, growth factors, and chemokines, generate a tumor-promoting environment. Cancer-associated fibroblasts (CAFs) are activated during the progression of pancreatic cancer and are a crucial component of the tumor microenvironment (TME). CAFs are primarily pro-tumorigenic in their activated state and function as promoters of cancer invasion, proliferation, metastasis, and immune modulation. Aided by many signaling pathways, cytokines, and chemokines in the tumor microenvironment, CAFs can originate from many cell types including resident fibroblasts, mesenchymal stem cells, pancreatic stellate cells, adipocytes, epithelial cells, endothelial cells, and other cell types. CAFs are a highly heterogeneous cell type expressing a variety of surface markers and performing a wide range of tumor promoting and inhibiting functions. Single-cell transcriptomic analyses have revealed a high degree of specialization among CAFs. Some examples of CAF subpopulations include myofibrotic CAFs (myCAFs), which exhibit a matrix-producing contractile phenotype; inflammatory CAFs (iCAF) that are classified by their immunomodulating, secretory phenotype; and antigen-presenting CAFs (apCAFs), which have antigen-presenting capabilities and express Major Histocompatibility Complex II (MHC II). Over the last several years, various attempts have been undertaken to describe the mechanisms of CAF-tumor cell interaction, as well as CAF-immune cell interaction, that contribute to tumor proliferation, invasion, and metastasis. Although our understanding of CAF biology in cancer has steadily increased, the extent of CAFs heterogeneity and their role in the pathobiology of pancreatic cancer remains elusive. In this regard, it becomes increasingly evident that further research on CAFs in pancreatic cancer is necessary.

Cancer-associated fibroblasts: from basic science to anticancer therapy.

Cancer-associated fibroblasts (CAFs), as a central component of the tumor microenvironment in primary and metastatic tumors, profoundly influence the behavior of cancer cells and are involved in cancer progression through extensive interactions with cancer cells and other stromal cells. Furthermore, the innate versatility and plasticity of CAFs allow their education by cancer cells, resulting in dynamic alterations in stromal fibroblast populations in a context-dependent manner, which highlights the importance of precise assessment of CAF phenotypical and functional heterogeneity. In this review, we summarize the proposed origins and heterogeneity of CAFs as well as the molecular mechanisms regulating the diversity of CAF subpopulations. We also discuss current strategies to selectively target tumor-promoting CAFs, providing insights and perspectives for future research and clinical studies involving stromal targeting.

Fibroblasts in pancreatic cancer: molecular and clinical perspectives.

Pancreatic stellate cells (PSCs) and cancer-associated fibroblasts (CAFs) are highly abundant cells in the pancreatic tumor microenvironment (TME) that modulate desmoplasia. The formation of a dense stroma leads to immunosuppression and therapy resistance that are major causes of treatment failure in pancreatic ductal adenocarcinoma (PDAC). Recent evidence suggests that several subpopulations of CAFs in the TME can interconvert, explaining the dual roles (antitumorigenic and protumorigenic) of CAFs in PDAC and the contradictory results of CAF-targeted therapies in clinical trials. This highlights the need to clarify CAF heterogeneity and their interactions with PDAC cells. This review focuses on the communication between activated PSCs/CAFs and PDAC cells, as well as on the mechanisms underlying this crosstalk. CAF-focused therapies and emerging biomarkers are also outlined.

Hypoxia promotes inflammatory fibroblast formation in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy characterized by poor response to all existing therapies. Although immunotherapy has shown great promise against multiple deadly cancers, it has been largely ineffective in PDAC. This lack of response is in part attributed to its extensive, fibroinflammatory stroma and hypoxic microenvironment. Cancer-associated fibroblasts (CAFs) are the predominant stromal cell type in PDAC, and single-cell transcriptomics of human and mouse PDAC has recently revealed a distinct CAF subpopulation, inflammatory CAFs (iCAFs). These inflammatory fibroblasts produce high levels of cytokines and chemokines in PDAC, and have the potential to contribute to its immunosuppressive microenvironment and tumorigenesis. By injecting a hypoxia probe into PDAC mouse models, we recently found that iCAFs predominantly reside in hypoxic tumor regions. We also observed that the hypoxia-related gene signature is positively enriched in iCAFs in human PDAC samples. Importantly, by exposing three-dimensional (3D) co-cultures of pancreatic cancer cells and fibroblasts to either hypoxia or normoxia, we showed that hypoxia induces IL1α from cancer cells and that IL1α is required for hypoxia-mediated iCAF formation. Our data implicate hypoxia as a critical regulator of tumor stroma. Efforts are ongoing to understand the role of hypoxia in the crosstalk between cancer cells, fibroblasts, and immune cells in driving an immunosuppressive tumor microenvironment. Supported by a grant from the NIH (T32 AI007413) and the University of Michigan Rackham Merit Fellowship.

Abstract 1338: Subpopulations of cancer associated fibroblasts serve as cancer stem cell niche in ovarian cancer via activation of non-canonical Wnt signaling

Abstract Ovarian cancer (OC) is the most lethal gynecologic cancer and the 5th leading cause of cancer-related deaths among women in the US. One of the main reasons for the poor outcome is a high recurrence rate and development of chemoresistance. The standard care involves debulking surgery and carbo-taxol chemotherapy. While most patients respond well initially, a small fraction of OC cells survive - these persister cells are enriched in cancer stem cells (CSCs), and are capable of tumor initiation and cause relapse. Most studies on OC stem cells (OCSCs) have focused on their cell-intrinsic properties and not much is known about the role of the tumor microenvironment (TME) in regulating their growth, fate, and sustenance. Cancer-associated fibroblasts (CAFs) are a key constituent of the OC TME and have been reported to play important roles in regulating tumorigenesis, metastasis, and immune evasion. Moreover, the residual tumors following neo-adjuvant chemotherapy are highly fibrotic. Our studies using matched pre- and post-chemotherapy OC patient tumors revealed an enrichment in OCSCs and CAFs following chemotherapy. Using heterotypic 3D cocultures of patient-derived CAFs and OC cells and in vivo limiting dilution assay, we found that CAFs could induce OCSCs. CAFs stimulated symmetric division of OCSCs as well as dedifferentiation of some non-OCSCs. Using a novel interface interaction assay that we developed, we found that CAFs could influence only those OC cells that are in their immediate neighborhood. We have identified the underlying mechanism involving a non-canonical Wnt signaling pathway in the OCSCs induced by Wnt5a secreted by CAFs. Using functional rescue experiments, we determined that Wnt5a acted via its coreceptor ROR2 on the OCSCs that activated PKC and CREB1 to induce stemness. Inhibiting the CAF-OCSC crosstalk using a Wnt5a-specific inhibitor, Box5 abrogated the effects of CAFs on OCSCs both in vitro and in vivo. While Box5 treatment alone was sufficient to inhibit tumor growth in vivo, its combination with carboplatin sensitized tumors to carboplatin by eradicating residual OCSCs. CAFs are heterogenous and not all CAFs are capable of inducing OCSCs, similarly, only a subpopulation of OC cells can respond to CAFs and dedifferentiate into OCSCs. We performed single-cell RNA-seq using heterotypic 3D cocultures of patient-derived CAFs and OC cells, and characterized the pathways activated in Wnt5ahigh CAFs. Using trajectory analysis, we identified OC subpopulations that respond to CAF signals and become OCSCs. In summary, we have identified a novel mechanism by which a subpopulation of CAFs in the OC TME act as OCSC niche. Our research provides compelling evidence in support of targeting Wnt5a, in combination with the standard of care carbo-taxol chemotherapy, to prevent OC relapse and chemoresistance. Citation Format: Xue Xiao, Yiming Fang, David Pepin, Dmitriy Zamarin, Anirban K. Mitra. Subpopulations of cancer associated fibroblasts serve as cancer stem cell niche in ovarian cancer via activation of non-canonical Wnt signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1338.

Abstract 5959: Dissecting the stromal drivers of gastroesophageal adenocarcinoma chemoresistance

Abstract Gastroesophageal adenocarcinoma (GEA) is the fastest rising cancer in North America. Over the course of five years, the survival rate is <20%, creating an urgent need for appropriate treatments against GEA. Currently, providing patients with peri-operative systemic docetaxel triplet-based chemotherapy (DCF or FLOT) is the most effective approach to treat GEA. Despite this, for 50% of patients that do present an initial response to therapy, the tumor returns due to pre-existing or newly acquired resistance (i.e., chemoresistance) by the cancer. Researchers have shifted their focus to the tumor microenvironment (TME) as one of the factors influencing chemoresistance in patients. The TME is composed of tumor cells, immune cells and their secreted products, as well as fibroblasts. The components of the TME have been shown to interact with one another to influence tumor growth and progression. Fibroblasts are wound-healing cells that can be transformed into cancer-associated fibroblasts (CAFs) in response to stress and the release of inflammatory products. CAFs are the most abundant cells in the TME, yet their role in the chemoresponse of GEA is still unclear. Previous studies on other cancer types demonstrated that CAF expression is distinct between chemoresistant and chemo-sensitive tumors and certain CAF subpopulations may confer this resistance. This project will investigate the role of CAFs in the chemo-response of GEA using patient-derived organoids (PDOs) and CAFs from >200 GEA patients. A single-cell RNA sequencing atlas developed from >30 GEA DCF- or FLOT-treated patient samples will be used to identify CAF markers and targetable processes. CAF sub-populations will then be elucidated and correlated to tumor response. IF, FACS, and ELISA will be performed for subsequent CAF marker validation and characterization. Ex vivo drug testing with DCF or FLOT will be conducted on PDO-CAF co-cultures to recapitulate their drug response. Citation Format: Kulsum Tai, Sanjima Pal, Julie Bérubé, Iris Kong, Adam Hoffman, Swneke Bailey, Aki Kirbizakis, Sui Huang, Michael Strasser, David Gibbs, Nicholas Bertos, Veena Sangwan, Lorenzo Ferri. Dissecting the stromal drivers of gastroesophageal adenocarcinoma chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5959.

Abstract 5842: Determining the tumor supportive and inhibitory capabilities of cancer associated fibroblast subpopulations in central nervous system metastasis

Abstract Central nervous metastases (CM) are surrounded by a complex tumor microenvironment (TME) comprised of a meshwork of extracellular matrix (ECM) proteins and an assemblage of cell types including cancer-associated fibroblasts (CAFs). We have previously shown that patient-derived CM CAFs can either support or inhibit tumor growth in vivo, suggesting that CM CAFs comprise a heterogenous subgroup of cells. Accordingly, we recently reported, based on literature in other cancer types, that CAFs can be categorized into four main subpopulations: ‘desmoplastic’, ‘immune’, ‘contractile’ and ‘aggressive’. The purpose of our study is to identify different CAF subpopulations in the TME of CM and determine their impact on CM growth. To do this, we performed single cell RNA sequencing (scRNA-seq) on 10 CM patient tissues and 8 patient-derived CM CAF cell lines established in our lab. ScRNA-seq analysis of tumor tissues revealed that desmoplastic, immune, contractile, and aggressive CAF subpopulations are also present in the TME of CM. In addition, scRNA-seq showed that CM CAF cell lines were mainly comprised of the desmoplastic and aggressive CAF subpopulations. These two subpopulations showed differential expression of the newly described tumor inhibitory CAF marker Immunoglobulin Superfamily Containing Leucine Rich Repeat (ISLR) and classic tumor supportive CAF marker Actin Alpha 2, Smooth Muscle (ACTA2, α-SMA), with desmoplastic CAFs showing ISLRhigh/α-SMAlow expression while aggressive CAFs were associated with ISLRlow/α-SMAhigh expression. Next, we used fluorescence activated cell sorting (FACS) based on the expression of ISLR on the cell surface to enrich and expand the ISLRhigh/α-SMAlow desmoplastic CAF and the ISLRlow/α-SMAhigh aggressive CM CAF subpopulations. Reciprocal expression of α-SMA and ISLR was confirmed by western blot and immunofluorescence after FACS. Additionally, co-culture experiments showed that ISLRhigh/α-SMAlow CM CAFs inhibit CM cell proliferation. In summary, our data show that there are several molecularly defined subpopulations of CAFs in the TME of CM that can act to either inhibit or promote CM tumor growth in vitro and in vivo. Our results support that the desmoplastic CM CAFs characterized by ISLRhigh/α-SMAlow can inhibit tumor growth. Overall, the present study improves our understanding of the CM TME, which ultimately will help establish new strategies to harness the cancer restraining capabilities of tumor inhibitory CAFs in order to improve outcomes for patients with CM. Citation Format: Thomas Simon, David N. Buckley, Gerald C. Gooden, Ben Y. Tew, Gabriel Zada, Bodour Salhia. Determining the tumor supportive and inhibitory capabilities of cancer associated fibroblast subpopulations in central nervous system metastasis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5842.

Abstract 4530: Single-cell analysis of oral squamous cell carcinoma (OSCC) from smoker vs. non-smoker patients highlights two groups of tumors with distinct immune microenvironments

Abstract Background and Rationale: OSCC is the most frequent head and neck cancer. While it is mainly affecting patients with a smoking and/or alcohol history (SD), 10% to 15% of OSCC are diagnosed in patients with not known risk factors (NSND). We and others have shown that OSCC from NSND patients are characterized by an enrichment in interferon gamma (IFNγ) response and PD-1 signaling pathways together with a higher intratumor T-cell infiltrate. Herein, we seek to gain more insights into the immune and non-immune tumor microenvironment (TME) of OSCC in SD vs. NSND patients. Material and Methods: We made use of single-cell RNA seq (10x Genomics) from non-sorted and CD45 negative enriched cell suspensions of OSCC biopsies from 11 treatment naïve patients, human papillomavirus negative, including 6 SD and 5 NSND patients. Results: All samples were characterized by a strong immune cell infiltrate. Consistent with our previous results, the analysis of 51,629 immune cells confirmed that NSND patients’ samples displayed a significantly higher proportion of T cells (p = 0.005) compared to SD patients. NK cells were also increased, with 2% in SD patients versus 6% in NSND patients (p = 0.008). T cells strongly expressed IFNγ and remarkably, both myeloid cells and tumor cells from NSND patients had an increased enrichment in IFNγ response pathway in NSND as compared to SD patients. The TME of SD patients was characterized by the presence of 15% of plasma cells vs. 3% in NSND patients (p = 0.003). Thus, tumors from NSND versus SD patients represent two distinct groups of tumors characterized by a different immune TME. Moreover, tumors from SD patients displayed 2% vs. 4% Cancer Associated Fibroblasts (CAFs) (p = 0.01). In addition, among CAFs subpopulations, myofibroblast CAFs, defined as the subpopulation producing extracellular matrix proteins were found only in tumors from SD patients, suggesting that distinct CAFs subpopulations may shape the immune TME by affecting the recruitment and function of adaptive immune cells in SD versus NSND patients. Finally, we analyzed tumor cells using non-negative matrix factorization to find Meta Programs (MP), which are set of genes coordinately upregulated. We identified 5 MP associated with different biological functions: epithelial differentiation, stress, cell cycle (G2/M), partial epithelial-to-mesenchymal transition, and epithelial senescence. While the epithelial differentiation MP was found in all patients, the remaining ones were more frequently found in SD patients. Conclusion: Our results show that the TME of OSCC from NSND vs. SD differs in terms of T, NK, and plasma cells composition, CAFs, and tumor cells MP. Integration with spatial transcriptomics is ongoing. Those biological differences may represent an opportunity to refine the therapeutic approaches in the two populations of patients. Citation Format: Yannick Le Meitour, Béatrice Vanbervliet, Sonia Canjura-Rodriguez, Cyril Degletagne, Laurie Tonon, Lucas Michon, Jebrane Bouaoud, Philippe Zrounba, Aude Excoffier, Karène Mahtouk, Pierre Saintigny. Single-cell analysis of oral squamous cell carcinoma (OSCC) from smoker vs. non-smoker patients highlights two groups of tumors with distinct immune microenvironments. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4530.

Abstract P2-21-07: Exploring spatial correlations in Breast invasive Lobular Carcinoma subtypes using a novel CAF multiplex immunofluorescence panel

Abstract Background: Recent advances in immunotherapy led to development of new technologies to identify topographical distributions and correlations in the tumor microenvironment, enabling a better understanding of the tumor immune landscape. Based on the 2019 WHO classification Invasive lobular carcinoma (ILC) has various histological presentations. The pleomorphic variant has been reported to be biologically different to the classical counterpart. Cancer associated fibroblasts (CAFs) secrete cytokines and growth factors aiding in tumor growth. CAFs have been shown to express alpha-smooth muscle actin (α-SMA), Thy1, fibroblast activation protein (FAP), S-100. IHC based studies have shown differences in CAFs subpopulation between ILC and IDC. Here, we explored different CAFs subpopulations between Pleomorphic and Classical ILC using multiplex immunofluorescence (mIF). Study design: Multiplex immunofluorescence (mIF) was performed on formalin-fixed paraffin-embedded (FFPE) tissue sections [(n=6, classic ILC; n=6 pleomorphic ILC)] stained with Opal 7-color Kit using an automated system. Sections were stained consecutively with Pancytokeratin, CD45, A-SMA, FAP, S100, Thy-1. Slides were scanned using the Vectra 3.0 spectral imaging system (PerkinElmer). Five tumor ROIs were examined with Phenochart (Akoya/PerkinElmer) viewer and subsequent images were analyzed to explore spatial distances of CK+ cells to the CAFs. Data was generated using InForm and Phenoptr softwares (Akoya Labs) as a median value. Statistical analysis data correlation was performed using SPSS version 24.0 (IBM Corp) Results: Twelve invasive lobular carcinomas cases were evaluated. All cases with classical features were nuclear grade 2 and all pleomorphic cases (n=6) were nuclear grade 3. Six were Luminal B, five were Luminal A and one case was triple negative invasive lobular carcinoma. We studied nineteen different phenotypes of CAFs in the tumor, stroma, and overall tissue compartments. We found statistically significant differences (p-value< 0.05) between the distances of CAFs from the tumor cells in classic and pleomorphic ILC in three phenotypes. The A-SMA+, A-SMA+/S100+ phenotypes were closer to the tumor cells in classic subtype and the S100 only phenotype was closer to the tumor cells in pleomorphic carcinomas. In addition, there were two other phenotypes namely A-SMA+/Thy-1+(closer to tumor cells in classic ILC) and FAP+/S-100+ (closer to tumor cells in pleomorphic ILC), which showed a trend of significance (Table 1) Discussion: TME studies in invasive breast lobular carcinoma have been primarily based on chromogenic IHC. Multiplex immunofluorescence quantifies cell phenotype’s densities and positions in different tissue compartments (tumor vs stroma vs total tissue). Knowing the proximity of an individual TME cells to the tumor cells, aids in pinpointing possible therapeutic targets. Fibroblast dysregulation in cancers, enhances their pro-tumorigenic and anti-tumorigenic potential. Due to their heterogeneity, they can express a wide array of markers. Identifying the full possibility of CAF subsets is one of the keys to discovering actionable targets. Our results showed Alpha- SMA positive, Alpha-SMA/S-100 positive, Alpha SMA/Thy-1 positive CAFs in closer proximity to the classic ILC tumor cells as compared to pleomorphic ILC. Additionally, S-100 positive and FAP/S-100 positive CAFs showed a closer proximity to tumor cells in the pleomorphic subtype. Based on these results we hypothesize that pleomorphic ILCs are closely associated with pro-tumorigenic CAFs as compared to classic ILCs. Larger datasets are needed to confirm this. In conclusion we utilized an objective approach to quantify, phenotype and spatially correlate each cell in the tumor microenvironment. This has helped identifying CAFs subsets that differs in the spatial correlation to tumor cells in ILC subtypes, which can be potential actionable targets. Table 1 Phenotypes of Cancer associated fibroblasts and their statistical correlation between classic vs pleomorphic invasive Lobular carcinomas. Citation Format: Harsh Batra, Renganayaki K. Pandurengan, Heladio P. Ibarguen, Salome A McAllen, Qingqing Ding, Aysegul Sahin, Ignacio Wistuba, Edwin Roger Parra, Maria Gabriela Raso. Exploring spatial correlations in Breast invasive Lobular Carcinoma subtypes using a novel CAF multiplex immunofluorescence panel [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-21-07.