Cancer-associated Fibroblasts Subpopulations Research Articles

EBF1-COX4I2 signaling axis promotes a myofibroblast-like phenotype in cancer-associated fibroblasts (CAFs) and is associated with an immunosuppressive microenvironment

Immunotherapy has limited response rates in colorectal cancer (CRC) due to an immunosuppressive tumor microenvironment (TME). Combining transcriptome sequencing, clinical specimens, and functional experiments, we identified a unique group of CAF subpopulations (COX4I2 + ) with inhibited mitochondrial respiration and enhanced glycolysis. Through bioinformatics predictions and luciferase reporter assays, we determined that EBF1 can upstreamly regulate COX4I2 transcription. COX4I2 + CAFs functionally and phenotypically resemble myofibroblasts, are important for the formation of the fibrotic TME, and are capable of activating the M2 phenotype of macrophages. In vitro experiments demonstrated that COX4I2 + CAFs promote immunosuppressive TME by blocking CD8 + T cell infiltration and inducing CD8 + T cell dysfunction. Using multiple independent cohorts, we also found a strong correlation between the immunotherapy response rate of CRC patients and COX4I2 expression in their tumors. Our results identify a CAF subpopulation characterized by activation of the EBF1-COX4I2 axis, and this group of CAFs can be targeted to improve cancer immunotherapy outcomes.

Exploring the diversity of cancer-associated fibroblasts: insights into mechanisms of drug resistance.

Introduction: Among the various stromal cell types within the tumor microenvironment, cancer-associated fibroblasts (CAFs) emerge as the predominant constituent, exhibiting a diverse array of oncogenic functions not intrinsic to normal fibroblasts. Their involvement spans across all stages of tumorigenesis, encompassing initiation, progression, and metastasis. Current understanding posits the coexistence of distinct subpopulations of CAFs within the tumor microenvironment across a spectrum of solid tumors, showcasing both pro- and antitumor activities. Recent advancements in single-cell transcriptomics have revolutionized our ability to meticulously dissect the heterogeneity inherent to CAF populations. Furthermore, accumulating evidence underscores the pivotal role of CAFs in conferring therapeutic resistance to tumors against various drug modalities. Consequently, efforts are underway to develop pharmacological agents specifically targeting CAFs. Methods: This review embarks on a comprehensive analysis, consolidating data from 36 independent single-cell RNA sequencing investigations spanning 17 distinct human malignant tumor types. Results: Our exploration centers on elucidating CAF population markers, discerning their prognostic relevance, delineating their functional contributions, and elucidating the underlying mechanisms orchestrating chemoresistance. Discussion: Finally, we deliberate on the therapeutic potential of harnessing CAFs as promising targets for intervention strategies in clinical oncology.

Cancer-associated fibroblasts expressing fibroblast activation protein and podoplanin in non-small cell lung cancer predict poor clinical outcome.

Cancer-associated fibroblasts (CAFs) are a dominant cell type in the stroma of non-small cell lung cancer (NSCLC). Fibroblast heterogeneity reflects subpopulations of CAFs, which can influence prognosis and treatment efficacy. We describe the subtypes of CAFs in NSCLC. Primary human NSCLC resections were assessed by flow cytometry and multiplex immunofluorescence for markers of fibroblast activation which allowed identification of CAF subsets. Survival data were analysed for our NSCLC cohort consisting of 163 patients to understand prognostic significance of CAF subsets. We identified five CAF populations, termed CAF S1-S5. CAF-S5 represents a previously undescribed population, and express FAP and PDPN but lack the myofibroblast marker αSMA, whereas CAF-S1 populations express all three. CAF-S5 are spatially further from tumour regions then CAF-S1 and scRNA data demonstrate an inflammatory phenotype. The presence of CAF-S1 or CAF-S5 is correlated to worse survival outcome in NSCLC, despite curative resection, highlighting the prognostic importance of CAF subtypes in NSCLC. TCGA data suggest the predominance of CAF-S5 has a poor prognosis across several cancer types. This study describes the fibroblast heterogeneity in NSCLC and the prognostic importance of the novel CAF-S5 subset where its presence correlates to worse survival outcome.

Tumor cell-intrinsic epigenetic dysregulation shapes cancer-associated fibroblasts heterogeneity to metabolically support pancreatic cancer

The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) involves a significant accumulation of cancer-associated fibroblasts (CAFs) as part of the host response to tumor cells. The origins and functions of transcriptionally diverse CAF populations in PDAC remain poorly understood. Tumor cell-intrinsic genetic mutations and epigenetic dysregulation may reshape the TME; however, their impacts on CAF heterogeneity remain elusive. SETD2, a histone H3K36 trimethyl-transferase, functions as a tumor suppressor. Through single-cell RNA sequencing, we identify a lipid-laden CAF subpopulation marked by ABCA8a in Setd2-deficient pancreatic tumors. Our findings reveal that tumor-intrinsic SETD2 loss unleashes BMP2 signaling via ectopic gain of H3K27Ac, leading to CAFs differentiation toward lipid-rich phenotype. Lipid-laden CAFs then enhance tumor progression by providing lipids for mitochondrial oxidative phosphorylation via ABCA8a transporter. Together, our study links CAF heterogeneity to epigenetic dysregulation in tumor cells, highlighting a previously unappreciated metabolic interaction between CAFs and pancreatic tumor cells.

Deciphering the spatial landscape and plasticity of immunosuppressive fibroblasts in breast cancer

Although heterogeneity of FAP+ Cancer-Associated Fibroblasts (CAF) has been described in breast cancer, their plasticity and spatial distribution remain poorly understood. Here, we analyze trajectory inference, deconvolute spatial transcriptomics at single-cell level and perform functional assays to generate a high-resolution integrated map of breast cancer (BC), with a focus on inflammatory and myofibroblastic (iCAF/myCAF) FAP+ CAF clusters. We identify 10 spatially-organized FAP+ CAF-related cellular niches, called EcoCellTypes, which are differentially localized within tumors. Consistent with their spatial organization, cancer cells drive the transition of detoxification-associated iCAF (Detox-iCAF) towards immunosuppressive extracellular matrix (ECM)-producing myCAF (ECM-myCAF) via a DPP4- and YAP-dependent mechanism. In turn, ECM-myCAF polarize TREM2+ macrophages, regulatory NK and T cells to induce immunosuppressive EcoCellTypes, while Detox-iCAF are associated with FOLR2+ macrophages in an immuno-protective EcoCellType. FAP+ CAF subpopulations accumulate differently according to the invasive BC status and predict invasive recurrence of ductal carcinoma in situ (DCIS), which could help in identifying low-risk DCIS patients eligible for therapeutic de-escalation.

Abstract 5500: Single cell sequencing reveals cancer associated fibroblast heterogeneity at the core of the tumor microenvironment in brain metastasis

Abstract Introduction: Brain metastasis (BM) remains incurable with a dismal 2-year survival of less than 2%. BM 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), the most prominent cell type within the TME. We have previously shown that patient-derived BM CAFs can either support or inhibit tumor growth in vivo, suggesting that BM CAFs are heterogenous. Yet, there remains a dearth of data on the functional and mechanistic underpinnings of CAFs in the BM TME. Methods: To explore both the molecular profiles of CAFs and their heterogeneity in the BM TME, we performed a single cell Multiome (RNA and Assay for Transposase-Accessible Chromatin (ATAC)) sequencing (scMultiome-seq) analysis of 9 human BM tissue samples and 4 BM patient-derived CAF cell lines, from both lung and breast primary cancers. Next, we investigated the tumor supportive or inhibitory capabilities of BM CAF cell lines on the growth of BM tumor cells in vitro. Results: ScMultiome-seq analysis of BM tissue samples identified 4 distinct CAF subpopulations, that we termed desmoplastic, immune, contractile, and neural CAFs, in the BM TME. These CAF subpopulations are respectively involved in either regulating the ECM organization, orchestrating the neuroimmune response, triggering tissue contraction or regulating neural components upon BM growth. CellPhoneDB analysis revealed that CAFs had more cell-cell interactions with other cells than any other cell types in the BM TME. Pseudotime analysis demonstrated the potential cell of origin of each BM CAF subpopulation and the cell state transition they undergo. We observed that the end cell state of the desmoplastic CAFs expressed high levels of immunoglobulin superfamily containing leucine rich repeat (ISLR). ISLR was also observed to be highly expressed in a BM patient-derived CAF cell line which was previously reported by our lab to restrain tumor growth in vivo, suggesting that desmoplastic CAFs with high ISLR expression (ISLR-CAFs) might be tumor inhibitory. ScMultiome-seq analysis of BM CAF cell lines validated that tumor inhibitory CAF cell lines were primarily of the desmoplastic ISLR-CAF subpopulation. Conditioned media obtained from ISLR-CAFs containing high levels of ISLR inhibited tumor cell viability in vitro compared to controls. Conclusion: Our study is the first to describe the heterogeneity of CAFs and their potential central roles in the BM TME. We also identify that CAF subpopulations are dynamic and can transition to tumor inhibitory cell states, such as ISLR-CAFs. These data have potential to help design therapeutic strategies that selectively target tumor supportive CAFs whilst protecting tumor inhibitory ones. Citation Format: Thomas Simon, David N. Buckley, Ben Y. Tew, Matthew Salomon, Zeyi Yang, Gerald C. Gooden, Steven A. Toms, Gabriel Zada, Bodour Salhia. Single cell sequencing reveals cancer associated fibroblast heterogeneity at the core of the tumor microenvironment in brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5500.

Abstract 716: Depletion of stromal cells remodels tumor microenvironment and potentiates immune checkpoint blockade in pancreatic cancer

Abstract During carcinoma progression, mesenchymal stromal cells (MSCs) become recruited to tumors and contribute to the pool of cancer-associated fibroblasts (CAFs). Sub-populations of CAFs, have diverse and incompletely understood effects on disease progression and resistance to therapy. Adipose stromal cells (ASCs), the MSCs from fat tissue increasingly recruited by carcinomas in the context of obesity, have been shown to support cancer progression. Here, the roles of perivascular CAFs expressing platelet-derived growth factor receptor beta (Pdgfrb) and of CAFs expressing non-glycanated decorin (ngDCN), a marker of ASCs, were analyzed. We used mice orthotopically grafted with pancreatic ductal adenocarcinoma (KPC) cells. Ablation of cells expressing thymidine kinase under the control of Pdgfrb promoter with ganciclovir resulted in suppression of primary pancreatic tumor growth. However, ablation Pdgfrb+ cells induced spontaneous metastases to the liver. For depletion of ngDCN+ cells, we used a hunter-killer peptide D-CAN, which has been previously reported to induce apoptosis of ASCs and CAFs in mouse models. Here, D-CAN also had a negative effect on pancreatic tumor growth, however, there was no significant effect on metastatic dissemination. Single cell RNA sequencing of tumors demonstrated that targeting of Pdgfrb+ and ngDCN+ stromal cells had distinct effects on sub-populations of CAFs. Endothelial cell and cancer cell survival was decreased by depletion of either Pdgfrb+ or ngDCN+ stroma. However, pathway analysis revealed that depletion of Pdgfrb+ and ngDCN+ stromal cells has different effects on the markers of cancer cell aggressiveness. D-CAN treatment, and to a less extent Pdgfrb+ cell ablation, suppressed macrophage M2 polarization and increased infiltration of cytotoxic T-lymphocytes. This observation, confirmed in the MyCap graft model of prostate cancer, suggested that ablation of ngDCN+ stroma should synergize with immune checkpoint inhibitors. We tested the effect of D-CAN on the efficacy of anti-PDL1 antibody in the orthotopic KPC model. Compared to D-CAN and anti-PDL1 antibody alone, combination treatment had a synergistic effect on tumor growth. Importantly, liver metastases were suppressed by the D-CAN/anti-PDL1 antibody combination, but not by individual agents. We conclude that improved approaches to target mesenchymal stroma in tumors may be effective in combination with immunotherapy. Citation Format: Joseph Rupert, Alexes Daquinag, Lingyi Cai, Yulin Dai, Zhongming Zhao, Dimitris Anastassiou, Mikhail Kolonin. Depletion of stromal cells remodels tumor microenvironment and potentiates immune checkpoint blockade in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 716.

Abstract 5185: A multimodal, spatially defined, nanomechanical signature as predictive biomarker for breast cancer outcome: A 20 year survival study

Abstract Background: With the recent advancement in perioperative treatment protocols, the search for clinical biomarkers that can predict overall response and long-term cancer outcome independent from treatment acquires even more importance. Recently, it has become clear that mechanical properties of the tumor microenvironment in solid tumors play a key role in driving outcome, mediating immune infiltration and activation, and response to therapy. Moreover, a growing body of evidence supports the understanding that mechanical changes induced by a plethora of therapeutic regimens should be leveraged to elicit favorable outcome. Methods: In this work we propose a dual approach to reveal mechanical biomarkers predictive of long-term outcome in solid cancers. Our first approach is based on a newly developed, 60 channel, biomechanical imaging mass cytometry panel to elucidate biomechanical cues that drive tumor aggressiveness and immune infiltration. Our second approach utilizes the AFM-based Automated and Reliable Tissue Diagnostics (ARTIDIS) investigational device to measure mechanical properties of fresh clinical biopsy samples at a nanoscale resolution. Our analysis was performed on over 300 clinical samples, from routine baseline clinical biopsies collected from both the Breast Clinic, University Hospital Basel, and the University of Dundee, in the UK. Patients in these two cohorts were treated with either adjuvant or neoadjuvant approaches, including radiation, chemotherapy, hormonal, and surgical treatment regimens, with individual patient follow up as long as 20 years. Results: We demonstrated with two complimentary technologies that biomechanical spatial patterns drive outcome in breast cancer. Our results include a multi-platform elucidation of the biomechanical phenotypes of epithelial to mesenchymal transitions driven by hypoxic environments. Furthermore, we identified several biomechanical subpopulations of cancer associated fibroblasts, and their role in mediating cancer outcome, in relationship with survival time and several clinical variables (e.g. metastasis, nodes, grade). We mechanistically corroborated these results by evaluating the immune modulatory effect of the identified biomechanical microenvironments in breast cancer. Lastly, we correlated the identified biomechanical microenvironments with the ARTIDIS nanomechanical signature as a clinically integrated, rapid, single biomechanics signature at the nanoscale. Conclusion: Our results further validate the importance of biomechanics in driving tumor response and immune infiltration across a variety of clinical treatment setting. These result support the integration of ARTIDIS nanomechanical signature in clinical settings as a single predictive biomarker able to rapidly and accurately capture biomechanical alterations that drive cancer outcome. Citation Format: Sara Nizzero, Maria Pelaez Soni, Gregory Zaugg, Yitian Xu, Licheng Zhang, Junjun Zheng, Lee B. Jordan, Colin A. Purdie, Philip R. Quinlan, Chandandeep Nagi, Karla A. Sepulveda, Philipp Oertle, Tobias A. Appenzeller, Marko Loparic, Shu-Hsia Chen, Vittorio Cristini, Marija Plodinec, Alastair M. Thompson. A multimodal, spatially defined, nanomechanical signature as predictive biomarker for breast cancer outcome: A 20 year survival study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5185.

Abstract 5485: Spatial characterization of the immunosuppressive tumor microenvironment in estrogen receptor positive breast cancer at single cell level

Abstract We characterized immune cell exclusion in estrogen receptor positive (ER+) breast cancer through the study of spatial relationships among various subsets of cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), and other immune cells using a novel in-situ spatial transcriptomic technology. ER+ breast cancer is the most common breast cancer subtype. While endocrine therapy and immune checkpoint inhibitors have considerably reduced relapse and mortality for breast cancer patients, tumor resistance to treatment remains a major challenge. The tumor microenvironment (TME) plays an important role in this mechanism of resistance. Our previous spatial proteomic analysis of ER+ breast FFPE samples demonstrated the intricate relationship between tumor and immunosuppressive cells such as CAFs and TAMs via the CXCR4-CXCL12 axis associated with the exclusion of CD8 T cells from the TME. However, the limited number of antibodies in the panel made it challenging to investigate the diversity of those CAF and TAM populations and better understand their contribution to T cell exclusion. For this purpose, we further characterized the TME of 4 human ER+ breast cancer FFPE cores using the Xenium in-situ transcriptomic platform with a breast cancer-focused panel of 280 genes from 10x Genomics plus 100 additional genes selected from literature and publicly available single cell datasets to delineate CAF and TAM sub-populations. Image analysis enabled the quantification and spatial mapping of the target genes within each cell boundary. Cells were manually annotated through unsupervised clustering of over 1 million cells. The cell phenotyping analysis allowed us to annotate major cell types including tumor cells, macrophages, T cells and fibroblasts. With our curated panel, we further characterized CAF and TAM sub-populations by identifying myofibroblastic CAFs (myCAFs), inflammatory CAFs (iCAFs), and antigen-presenting CAFs (apCAFs) in addition to M1 and M2-like macrophages as reported in the literature. We then performed spatial neighborhood analysis based on the similarity of the cell type composition in the vicinity of each cell. The results showed that myCAFs and M2-like macrophages are found in proximity to tumor cells and may play a role in T-cell exclusion. In addition, iCAFs mainly express CXCL12, a chemokine known to attract immunosuppressive cells such as regulatory T cells and M2-like macrophages as well as repel cytotoxic T cells from infiltrating the tumor via the CXCR4-CXCL12 axis. Overall, our work shows the potential of spatial transcriptomics to elucidate the spatial distribution of cells and their contribution to the immunosuppressive TME, which may enable researchers to improve understanding of tumor biology and drug target discovery efforts. Citation Format: Julien Tessier, Sandra Curras-Alonso, Joon Sang Lee, Fabien Delahaye, Hong Ma, Katie Malley, Dinesh Bangari, Donald Jackson, Angela Hadjipanayis. Spatial characterization of the immunosuppressive tumor microenvironment in estrogen receptor positive breast cancer at single cell level [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5485.

Abstract 5565: Multiomic characterization of colorectal cancer using MICS technology reveals interaction of antigen presenting cancer associated fibroblasts and T cells

Abstract In solid tumors, the tumor microenvironment (TME) is composed of diverse cell types including cancer cells, immune cells, stromal cells, and other tissue specific cell types. The complex intercellular interactions that occur between these various cell populations determine cancer development and progression. Cancer-associated fibroblasts (CAFs) have been identified as key players in the TME, capable of promoting tumor cell growth and invasion, as well as manipulating immune responses. To better resolve potential subpopulations, spatial relationships, and signaling occurring between cell types within the TME, we performed same-section multiomic profiling of colorectal cancer (CRC) using the MACSima™ Imaging Cyclic Staining (MICS) technology. We combined a custom 40plex RNA panel with a panel of antibodies to characterize the drivers of tumorigenesis and the activation state of immune cells. FFPE CRC specimens were reviewed by a pathologist for clinical assessment and region of interest selection. Then, gene expression profiles were generated using RNAsky™ technology with each gene being detected via cyclic rounds of detection probe hybridization, image acquisition, and signal erasure. Subsequently, fluorescently labeled antibodies were applied to the same section following an equivalent acquisition process. Finally, multiomic clustering and cell population analyses were performed using MACS® iQ View software. The analysis revealed spatially separated subpopulations of CAFs. Functional characterization of cellular neighborhoods and the cell-to-cell interactions occurring within the TME showed that one of the CAF populations potentially promoted cancer cell growth while another subpopulation, resembling antigen-presenting CAFs (apCAFs), closely interacted with T cells in the TME. These findings will deepen our understanding of tumor progression in colorectal cancer and potentially other solid tumors. Citation Format: Emily Neil, Rebecca C. Hennessey, David Agorku, Dongju Park, Julia Femel, Michael DiBuono, Hanna Lafayette, Erica Lloyd, Hsinyi Lo, Alex Makrigiorgos, Shaina Lu, John Lee, Sameh Soliman, Dominic Mangiardi, Paurush Praveen, Philip Ströbel, Silvia Rüberg, Fabian Staubach, Ryan Hindman, Thomas Rothmann, Olaf Hardt, Hansueli Meyer, Tanya Wantenaar, Jinling Wang, Werner Müller, Robert Pinard, Andreas Bosio. Multiomic characterization of colorectal cancer using MICS technology reveals interaction of antigen presenting cancer associated fibroblasts and T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5565.

Abstract 291: Unveiling heterogeneity of cancer-associated fibroblasts (CAFs) across multiple solid cancer types

Abstract Background: Stromal cells, including CAFs, are a heterogeneous group of cells in the tumor microenvironment. Many models describe their heterogeneity like the classification into 3 main subtypes in breast cancer: myCAF (myofibroblasts), iCAF (inflammatory CAF), and PVL (perivascular-like) cells. However, a pan-cancer model has not been established to exhaustively characterize stromal cell heterogeneity, undermining efforts in targeting CAFs with anti-tumor therapies. We investigated the heterogeneity of stromal cells in diverse diagnoses with single-cell RNA sequencing (scRNA-seq) data and bulk RNA-seq of sorted stromal cells to define consistent subtypes. Design: First, a meta-cohort of 11 scRNA-seq open-source datasets (>500,000 cells, >100 patients, 6 diagnoses) was analyzed. We also generated a scRNA-seq dataset of CD90+ cells derived from tumor tissue of an ovarian cancer patient. Additionally, bulk RNA-seq of 6 PVL, 2 iCAF, and 4 myCAF sorted cell samples across four solid cancers was performed. scRNA-seq data was analyzed with the SCANPY Python package. Results: Stromal cell clusters were defined as positive for PDGFRB, THY1 (CD90), and COL1A1 expression, and negative for the expression of known non-stromal and endothelial cell markers, by separately analyzing the open-source scRNA-seq datasets. Analysis of stromal cell heterogeneity revealed that the CAFs were consistently classified into 3 subtypes across all datasets: myCAF (high expression: FAP and ACTA2), PVL (high expression: MCAM (CD146) and ACTA2), and iCAF (high expression: APOD and cytokines IL6, CXCL12, CXCL2). Differential gene expression analysis of the scRNA-seq meta-cohort allowed us to develop functional gene expression signatures to define each subtype. All scRNA-sequenced CD90+ cells from ovarian cancer tissue were classified into the CAF subtypes. The myCAF and PVL signatures were specifically upregulated in the corresponding cell types (p<0.01, Mann-Whitney U test). While the iCAF signature was enriched in both iCAFs and myCAFs, myCAF and PVL signatures were downregulated in iCAFs. Finally, we developed a fluorescence-activated cell sorting (FACS) panel to separate CAF subtypes, using negative markers (CD45, CD31, EPCAM), a positive marker for stromal cells (CD90) and cell-type surface markers (CD146, FAP, CD91, CD44) for myCAF, iCAF and PVL cells. The high expression of CAF-specific genes in the sorted CAF populations denoted high sample purity. Importantly, the 3 CAF subpopulations separated by gating based on different levels of cell-type surface markers showed expression patterns congruent with the developed signatures across diagnoses. Summary: Altogether, the consistent classification of stromal cells with our cell-specific expression profiles demonstrated that the 3 CAF subtypes are conserved in many cancer types, offering groundwork for a pan-cancer model of stromal cell heterogeneity. Citation Format: Andrey Kravets, Boris Shpak, Anastasia Zotova, Maria Savchenko, Sofya Kust, Olga Golubeva, Mary Abdou, Fedor Grigoryev, Irina Bulusheva, Beniamin Sargsyan, Polina Pavlovich, Vlad Maximov, Vladimir Kushnarev, Michael Goldberg, Nikita Kotlov, Alexander Bagaev. Unveiling heterogeneity of cancer-associated fibroblasts (CAFs) across multiple solid cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 291.

Abstract 1408: Intrahepatic IgA complex induces polarization of cancer-associated fibroblasts to matrix phenotypes in the tumor microenvironment of HCC

Abstract Background and Aims: Cancer-associated fibroblasts (CAFs) are activated fibroblasts that play key roles in the tumor microenvironment (TME). Immunoglobulin A (IgA) contributes to inflammation and anti-tumor immunity dismantling in the human liver. We investigated the effects of the IgA complex on CAFs in the TME of hepatocellular carcinoma (HCC). Approach and Results: CAF dynamics in HCC TME were analyzed via single-cell RNA sequencing of HCC samples. CAFs were isolated from 40 HCC samples. The isolated CAFs were treated with mock or serum-derived IgA dimers. Following treatment with the mock or serum IgA dimers in vitro, co-culture experiments were performed using CAF and CD8+ T cells. Single-cell analysis showed that sub-cluster proportions in the CAF-fibroblast activation protein-α (FAP) matrix were significantly increased in patients with high serum IgA levels. We were performed flow cytometry on fresh surgical tissues and observed a significant increase in the mean fluorescence intensity (MFI) of FAP in the CD68+ cells from patients with high serum IgA levels compared to those with low serum IgA levels (p<0.001). We validated that the transferrin receptor (CD71) is expressed in CAFs (p<0.01). IgA-treated CAFs exhibited higher programmed death-ligand 1 (PD-L1) expression levels than mock-treated CAFs (p<0.05). Co-culture with CAFs induced the attenuation of the cytotoxic function of activated CD8+ T cell, and these cells co-cultured with IgA-treated CAFs exhibited increased expression levels of programmed death-1 (PD-1) compared to those co-cultured with mock-treated CAFs (p<0.05). Conclusions: Intrahepatic IgA induces polarization of HCC CAFs into more malignant matrix phenotype and attenuates cytotoxic T cell function. Our study uncovers five dynamic CAF subpopulations within HCC tissues and highlights their potential roles in tumor progression and immune suppression. Citation Format: Pil Soo Sung. Intrahepatic IgA complex induces polarization of cancer-associated fibroblasts to matrix phenotypes in the tumor microenvironment of HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1408.

Abstract B039: Hypoxia promotes an inflammatory phenotype of fibroblasts in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is notable for its dense desmoplastic stroma, consisting of extracellular matrix, cancer-associated fibroblasts (CAFs), and immune cells. Another notable feature of the PDAC microenvironment is hypoxia, a condition of insufficient oxygen availability. Although hypoxia leads to adaptive responses in both cancer cells and stromal cells, the effects of hypoxia on the PDAC stroma and tumor-stroma interactions are not fully understood. 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. By exposing 3D cocultures of pancreatic tumor cells and fibroblasts to either hypoxia or normoxia, we demonstrated that hypoxia promotes iCAF formation via IL1ɑ secreted from tumor cells. Importantly, the presence of hypoxic fibroblasts further elevated IL1ɑ levels in tumor cells, implicating hypoxia as a modulator of bidirectional interactions between tumor cells and fibroblasts. Efforts are ongoing to understand the mechanism by which hypoxia promotes iCAF formation by modulating bidirectional interactions between tumor cells and fibroblasts in PDAC. Together, our study identifies hypoxia as a key environmental cue for inducing an iCAF phenotype, thus highlighting an instructive role of hypoxia in shaping the stromal microenvironment. Citation Format: Tenzin Ngodup, Ashley Mello. Hypoxia promotes an inflammatory phenotype of fibroblasts in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B039.

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 Analysis Reveals the Role of Cancer-associated Fibroblasts in Skin Melanoma.

Mechanism of fibroblasts in skin melanoma (SKME) revealed by single-cell RNA sequencing data. SKME is responsible for more than 80% of skin-related cancer deaths. Cancer-associated fibroblasts (CAFs) generate inflammatory factors, growth factors and extracellular matrix proteins to facilitate cancer cell growth, metastasis, drug resistance and immune exclusion. However, molecular mechanisms of CAFs in SKME are still lacking. Our goal was to reveal the role of CAFs in SKME. We downloaded the single-cell RNA sequencing (scRNA-seq) dataset from the Gene Expression Omnibus (GSE215120) database. Then, the Seurat package was applied to analyze the single-cell atlas of SKME data, and cell subsets were annotated with the CellMarker database. The molecular mechanisms of CAFs in SKME were disclosed via differential gene expression and enrichment analysis, Cellchat and SCENIC methods. Using scRNA-seq data, three SKME cases were used and downscaled and clustered to identify 11 cell subgroups and 5 CAF subsets. The enrichment of highly expressed genes among the 5 CAF subsets suggests that cell migration-inducing hyaluronan-binding protein (CEMIP) + fibroblasts and naked cuticle homolog 1 (NKD1) + fibroblasts were closely associated with epithelial to mesenchymal transition. Cellchat analysis revealed that CAF subpopulations promoted melanocyte proliferation through Jagged1 (JAG1)-Notch homolog 1 (NOTCH1), JAG1-NOTCH3 and migration through pleiotrophin (PTN)-syndecan-3 (SDC3) receptor-ligand pairs. The SCENIC analysis identified that most of the transcription factors in each CAF subpopulation played a certain role in the metastasis of melanoma and were highly expressed in metastatic SKME samples. Specifically, we observed that CEMIP+ fibroblasts and NKD1+ fibroblasts had potential roles in participating in immune therapy resistance. Collectively, we uncovered a single-- cell atlas of SKME and revealed the molecular mechanisms of CAFs in SKME development, providing a base for immune therapy and prognosis assessment. Our study reveals that 5 CAFs in SKME have a promoting effect on melanocyte proliferation and metastasis. More importantly, CEMIP+ fibroblasts and NKD1+ fibroblasts displayed close connections with immune therapy resistance. These findings help provide a good basis for future immune therapy and prognosis assessment targeting CAFs in SKME.

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.