Cancer-associated Fibroblasts Population Research Articles

Senescence defines a distinct subset of myofibroblasts that orchestrates immunosuppression in pancreatic cancer.

PDAC therapeutic resistance is largely attributed to a unique tumor microenvironment embedded with an abundance of cancer associated fibroblasts (CAFs). Distinct CAF populations were recently identified, but the phenotypic drivers and specific impact of CAF heterogeneity remain unclear. In this study, we identify a subpopulation of senescent myofibroblastic CAFs (SenCAFs) in mouse and human PDAC. These SenCAFs are a phenotypically distinct subset of myofibroblastic CAFs that localize near tumor ducts and accumulate with PDAC progression. To assess the impact of endogenous SenCAFs in PDAC, we employed a LSL-KRASG12D;p53flox;p48-CRE;INK-ATTAC (KPPC-IA) mouse model of spontaneous PDAC with inducible senescent cell depletion. Depletion of senescent stromal cells in genetic and pharmacologic PDAC models relieved immune suppression by macrophages, delayed tumor progression and increased responsiveness to chemotherapy. Collectively, our findings demonstrate that SenCAFs promote PDAC progression and immune cell dysfunction.

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.

FTO elicits tumor neovascularization in cancer-associated fibroblasts through eliminating m6A modifications of multiple pro-angiogenic factors

Cancer-associated fibroblasts (CAFs) exhibit notable versatility, plasticity, and robustness, actively participating in cancer progression through intricate interactions within the tumor microenvironment (TME). N6-methyladenosine (m6A) modification is the most prevalent modification in eukaryotic mRNA, playing essential roles in mRNA metabolism and various biological processes. Howbeit, the precise involvement of m6A in CAF activation remains enigmatic. In this study, we revealed that the m6A demethylase FTO supports CAF-mediated angiogenesis through activation of EGR1 and VEGFA in conjunctival melanoma (CoM). First, single-cell transcriptome analysis revealed that FTO was specifically upregulated in the CAF population, thereby contributing to the hypo-m6A status in the TME of CoM. Moreover, CAFs of CoM displayed extensive proangiogenic potential, which was largely compromised by FTO inhibition, both in vitro and in vivo. By employing multi-omics analysis, we showed that FTO effectively eliminates the m6A modifications of VEGFA and EGR1. This process subsequently disrupts the YTHDF2-dependent mRNA decay pathway, resulting in increased mRNA stability and upregulated expression of these molecules. Collectively, our findings initially indicate that the upregulation of FTO plays a pivotal role in tumor development by promoting CAF-mediated angiogenesis. Therapeutically, targeting FTO may show promise as a potential antiangiogenic strategy to optimize cancer treatment.

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.

Abstract 2019: Primary cancer associated fibroblasts increase the chemoresistance of triple negative breast cancer cells by mitochondria metabolic upregulation

Abstract Breast cancers (BCs) are the most prevalent cancers occurring in women. Tumor reoccurrence attributed to chemotherapy resistance remains a main challenge in fighting disease relapse. Cancer associated fibroblasts (CAFs) are the most prominent component of the tumor microenvironment (TME) and have already been implicated in therapy resistances. Although the exact molecular mechanisms behind this resistance remain unclear. In this regard, we recently identified 4 CAF populations (S1-S4) in ovarian and BCs, and showed that only CAF-S1 promote immunotherapy resistance as well as metastasis. Based on these observations, we are now investigating the impact of CAF-S1 on the chemotherapy response of BCs. To study the interactions between CAF-S1 and BC cells, we developed a tumor on chip (ToC) device, mimicking the TME in 3D. Using ToC live imaging, we show that primary CAF-S1 mediate a chemoprotective effect over luminal and triple negative BC (TNBC) cells by significantly decreasing their apoptosis rate as early as 10h after Doxorubicin and 40h after Paclitaxel treatment. Interestingly, CAF-S1 display a highly heterogenous response to Doxorubicin, allowing the classification into chemo-sensitive and -resistant CAF-S1. Regardless of premature CAF-S1 cell death, the chemoprotective effect over BC cells is maintained for at least 3 days. This indicates a crucial CAF-S1 - cancer cell cross talk at early timepoints. Additionally, by applying machine learning algorithms on live imaging videos, we demonstrate that physical proximity of CAF-S1 to BC cells immediately after treatment initiation correlates with BC cell survival, even after several days of treatment. This again implies cancer cell priming by CAF-S1 as a possible mechanism of chemotherapy resistance. To investigate this molecular interplay further, we performed scRNA-seq of ToC extracted and treatment naïve CAF-S1 and TNBC cells as well as bulk RNA-seq of patient derived TNBC tumors before/after chemotherapy treatment. We identify mitochondrial metabolic pathways to be upregulated in TNBC cells after CAF-S1 co-culture inside ToC devices and in TNBC cells of chemo-resistant patients enriched in CAF-S1 at baseline. We are now validating the impact of target gene knock downs on the survival of TNBC cells in CAF-S1 co-cultures after chemotherapy treatment on chip. Overall, these results provide insights into molecular mechanisms by which CAF-S1 promote chemotherapy resistance of breast cancers. Citation Format: Isabella Hofer, Arianna Mencattini, Yann Kieffer, Eugenio Martinelli, Maria Carla Parrini, Fatima Mechta-Grigoriou. Primary cancer associated fibroblasts increase the chemoresistance of triple negative breast cancer cells by mitochondria metabolic upregulation [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 2019.

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.

Cancer associated fibroblasts modulate the cytotoxicity of anti-cancer drugs in breast cancer: An in vitro study.

Tumour microenvironment (TME) contributes to resistance to anti-cancer drugs through multiple mechanisms including secretion of pro-survival factors by cancer associated fibroblasts (CAFs). In this study, we determined the chemotherapy resistance producing potential of CAFs in molecular subtypes of breast cancer. The CAFs were isolated from fresh lumpectomy/mastectomy specimens of different molecular subtypes of breast cancer. The CAFs were cultured and secretome was collected from each breast cancer subtype. Breast cancer cell lines MCF-7, SK-BR3, MDA-MB-231, and MDA-MB-468 were treated with different doses of tamoxifen, trastuzumab, cisplatin, and doxorubicin alone respectively and in combination with secretome of CAFs from respective subtypes. MTT assay was done to check cell death after drug treatment. Liquid chromatography-mass spectrometry (LCMS) analysis of CAF secretome was also done. MTT assay showed that anti-cancer drugs alone had growth inhibitory effect on the cancer cells however, presence of CAF secretome reduced the anti-cancer effect of the drugs. Resistant to drugs in the presence of secretome, was determined by increased cell viability i.e., MCF-7, 51.02% to 63.02%; SK-BR-3, 34.22% to 44.88%; MDA-MB-231, 52.59% to 78.63%; and MDA-MB-468, 48.92% to 55.08%. LCMS analysis of the secretome showed the differential abundance of CAFs secreted proteins across breast cancer subtypes. The treatment of breast cancer cell lines with anti-cancer drugs in combination with secretome isolated from molecular subtype specific CAFs, reduced the cytotoxic effect of the drugs. In addition, LCMS data also highlighted different composition of secreted proteins from different breast cancer associated fibroblasts. Thus, TME has heterogenous population of CAFs across the breast cancer subtypes and in vitro experiments highlight their contribution to chemotherapy resistance which needs further validation.

Abstract 4335: High-resolution characterization of age-specific cell type composition changes and signaling events in HPV-negative HNSCC tumors

Abstract This study is aimed at testing the hypothesis that increased patient age fosters conditions that are favorable for head and neck squamous cell carcinomas (HNSCC) of the oral cavity development. There is a well-documented increase in cancer incidence with age observed across diverse cancer types, where elderly patients exhibit diminished survival outcomes compared to their younger counterparts. While the convergence of aging and cancer hallmarks offers valuable insights, further work is needed to elucidate the age-specific mechanisms influencing HNSCC, beyond the shared characteristics of these biological processes. To this end, we have assembled a high-quality human single-cell RNA-sequencing HNSCC atlas profiling more than 230,000 cells across more than 50 patients with ages ranging between 18 and 89, which provides a resource to investigate age-associated changes in the disease heterogeneity. For this library, we integrated six publicly available single-cell RNAseq datasets from 54 HPV-negative patients to create the atlas. Cells were clustered, classified, characterized by gene set enrichment analysis, both in the epithelial cell compartment and in the tumor microenvironment (TME). Differential cell type proportion analysis was performed to identify cell types enriched in young (<49 years) or old (>70 years) patients. Cell-cell communication analysis was performed to identify signaling events occurring between different populations, and how these and specific ligand-receptor pairs differed in patients of different ages. CNV analyses were performed to identify cancer subclones and assess level of variation across tumors. Interestingly, we identified distinct cell populations and signaling events that associate with age, and we were able to compare, validate and recapitulate these observations in the 4MOSC1 OSCC isograft tumor model in old and young mice. Additional notable findings include a differential enrichment for cytotoxic CD8 T cells over dysfunctional CD8 T cells in young patients when compared to old, suggesting younger patients have a more effective immune TME. We also found an enrichment for cancer-associated fibroblast populations in old patients (myCAFs) that send collagen signals to malignant epithelial cells, a result we have recapitulated using the 4MOSC1 model, and hypothesize may play a role in ECM stiffening, EMT, and metastasis. Further analyses are ongoing, and we plan to validate the hypotheses generated, specifically the presence of differential abundance of specific cell populations, and age-specific ligand-receptor signaling events that lead to tumor growth. Citation Format: Lina Kroehling, Anthony Spinella, Maria Kukuruzinska, Xaralabos Varelas, Stefano Monti. High-resolution characterization of age-specific cell type composition changes and signaling events in HPV-negative HNSCC tumors [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 4335.

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 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.

Cancer-Associated Fibroblasts in Pancreatic Ductal Adenocarcinoma or a Metaphor for Heterogeneity: From Single-Cell Analysis to Whole-Body Imaging.

Pancreatic ductal adenocarcinoma (PDAC) represents a formidable challenge due to its aggressive nature and poor prognosis. The tumor microenvironment (TME) in PDAC, characterized by intense stromal desmoplastic reactions and a dominant presence of cancer-associated fibroblasts (CAFs), significantly contributes to therapeutic resistance. However, within the heterogeneous CAF population, fibroblast activation protein (FAP) emerges as a promising target for Gallium-68 FAP inhibitor positron emission tomography (Ga68FAPI-PET) imaging. Notably, 68Ga-FAPI-PET demonstrates promising diagnostic sensitivity and specificity, especially in conjunction with low tracer uptake in non-tumoral tissues. Moreover, it provides valuable insights into tumor-stroma interactions, a critical aspect of PDAC tumorigenesis not adequately visualized through conventional methods. The clinical implications of this innovative imaging modality extend to its potential to reshape treatment strategies by offering a deeper understanding of the dynamic TME. However, while the potential of 68Ga-FAPI-PET is evident, ongoing correlative studies are essential to elucidate the full spectrum of CAF heterogeneity and to validate its impact on PDAC management. This article provides a comprehensive review of CAF heterogeneity in PDAC and explores the potential impact of 68Ga-FAPI-PET on disease management.

INHBA(+) cancer-associated fibroblasts generate an immunosuppressive tumor microenvironment in ovarian cancer

Effective targeting of cancer-associated fibroblasts (CAFs) is hindered by the lack of specific biomarkers and a poor understanding of the mechanisms by which different populations of CAFs contribute to cancer progression. While the role of TGFβ in CAFs is well-studied, less attention has been focused on a structurally and functionally similar protein, Activin A (encoded by INHBA). Here, we identified INHBA(+) CAFs as key players in tumor promotion and immunosuppression. Spatiotemporal analyses of patient-matched primary, metastatic, and recurrent ovarian carcinomas revealed that aggressive metastatic tumors enriched in INHBA(+) CAFs were also enriched in regulatory T cells (Tregs). In ovarian cancer mouse models, intraperitoneal injection of the Activin A neutralizing antibody attenuated tumor progression and infiltration with pro-tumorigenic subsets of myofibroblasts and macrophages. Downregulation of INHBA in human ovarian CAFs inhibited pro-tumorigenic CAF functions. Co-culture of human ovarian CAFs and T cells revealed the dependence of Treg differentiation on direct contact with INHBA(+) CAFs. Mechanistically, INHBA/recombinant Activin A in CAFs induced the autocrine expression of PD-L1 through SMAD2-dependent signaling, which promoted Treg differentiation. Collectively, our study identified an INHBA(+) subset of immunomodulatory pro-tumoral CAFs as a potential therapeutic target in advanced ovarian cancers which typically show a poor response to immunotherapy.

Residual ANTXR1+ myofibroblasts after chemotherapy inhibit anti-tumor immunity via YAP1 signaling pathway

Although cancer-associated fibroblast (CAF) heterogeneity is well-established, the impact of chemotherapy on CAF populations remains poorly understood. Here we address this question in high-grade serous ovarian cancer (HGSOC), in which we previously identified 4 CAF populations. While the global content in stroma increases in HGSOC after chemotherapy, the proportion of FAP+ CAF (also called CAF-S1) decreases. Still, maintenance of high residual CAF-S1 content after chemotherapy is associated with reduced CD8+ T lymphocyte density and poor patient prognosis, emphasizing the importance of CAF-S1 reduction upon treatment. Single cell analysis, spatial transcriptomics and immunohistochemistry reveal that the content in the ECM-producing ANTXR1+ CAF-S1 cluster (ECM-myCAF) is the most affected by chemotherapy. Moreover, functional assays demonstrate that ECM-myCAF isolated from HGSOC reduce CD8+ T-cell cytotoxicity through a Yes Associated Protein 1 (YAP1)-dependent mechanism. Thus, efficient inhibition after treatment of YAP1-signaling pathway in the ECM-myCAF cluster could enhance CD8+ T-cell cytotoxicity. Altogether, these data pave the way for therapy targeting YAP1 in ECM-myCAF in HGSOC.

Abstract PR07: Unravelling the epigenetic landscape of pancreatic cancer: The role of cancer-associated fibroblasts

Abstract Pancreatic cancer is set to become the 3rd deadliest cancer by 2025. Pancreatic ductal adenocarcinoma (PDAC) represents the majority of cases and is the most aggressive subtype, with a 5-year survival rate of around 10% and patients often diagnosed at a metastatic stage. Current treatments offer dismal survival benefit for the patients, highlighting the need for innovative therapeutic strategies. PDAC is characterized by genetic and epigenetic modifications and an abundant tumor microenvironment (TME) largely composed of cancer-associated fibroblasts (CAFs). Multiple populations of CAFs have been described and implicated in PDAC progression, however, little is known regarding the molecular crosstalk between them and the cancer cells. This project aims to investigate how the epigenetic landscape of PDAC is affected by recently described CAF populations. We have optimized a co-culture model consisting of cancer cells and genetically-locked inflammatory CAFs (iCAFs) and myofibroblastic CAFs (myCAFs) to study the link between the TME and the gene regulatory machinery in PDAC through proteomic and genomic approaches. Firstly, a full proteome analysis revealed the two CAF populations drive expression of different subsets of proteins in PDAC cells, including transcription factors. We then interrogated chromatin accessibility via ATAC-seq, which showed that CAF subsets differentially affect chromatin dynamics in epithelial cells. Transcription factor interactomes were investigated using rapid immunoprecipitation of endogenous proteins (RIME), a method developed in our lab that allows the discovery of protein complexes in a robust manner. We leverage this technique to discover druggable transcription factor interactors. RIME data shows that upon co-culture with each CAF population, distinct interactors are recruited towards FOXA1, a pioneer factor that drives a transcriptional program associated with PDAC metastasis to the liver, and towards FOXA2, which has been shown to play distinct roles in different grades of PDAC. We are now assessing the potential of these interactors as drug candidates in each context. Chromatin immunoprecipitation assays have shown that the CAF populations also affect the binding profile of FOXA1 and FOXA2 to the chromatin. We are currently investigating how CAFs impact tumor progression in vivo using an orthotopic implantation model. Preliminary data suggests that CAF subsets affect the metastatic profile in PDAC, as tumors co-injected with iCAFs metastasize to different organs than those co-injected with myCAFs. Ongoing experiments aim to explore the differences between tumors and metastases at the epigenetic level. Subsequent studies will aim to employ distinct approaches that target tumor progression in each context. PDAC is a highly heterogeneous cancer with several layers of complexity; therefore, a more holistic approach is critical to tackle the disease. Understanding tumor-stroma interactions from an epigenetics standpoint may enable identification of druggable molecules much needed for PDAC patients. Citation Format: Catarina Pelicano, Igor Chernukhin, Lisa Young, Naomi Vranas, Yi Cheng, Joshua Kent, Kamal Kishore, Clive D'Santos, Alasdair Russell, Giulia Biffi, Shalivi V. Rao, Jason S. Carroll. Unravelling the epigenetic landscape of pancreatic cancer: The role of cancer-associated fibroblasts [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 PR07.

Abstract B034: Antigen-presenting cancer-associated fibroblasts are found in immunotherapy-sensitive murine models of pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related deaths. Despite the advent of immune checkpoint inhibition (ICI) that has markedly changed the treatment landscape for select tumors with respect to both outcomes and tolerability, PDAC remains unresponsive to such an approach. One feature of the PDAC tumor microenvironment (TME) that remains poorly understood is the interaction between cancer-associated fibroblasts (CAFs) and immune and tumor cells. Unique subsets of CAFs have been identified, including antigen-presenting CAFs (apCAFs) which are defined by expression of MHC-II and can activate CD4 T cells in genetically engineered mouse models of PDAC. apCAFs are rarely found in human PDAC; in a human PDAC atlas consisting of six published human single cell RNA-sequencing (scRNA-seq) datasets, we identified 370 apCAFs out of 8736 total CAFs, the majority of which were found in one dataset. Here, we have orthotopically implanted cell lines derived from the autochthonous KPC murine model of PDAC which mimics the stromal components of human disease to further study the influence of apCAFs on the anti-tumor response. We have found that although the overall CAF population is equivalent between tumors, apCAFs are present primarily in tumors derived from KPC cell lines that display ICI sensitivity. apCAFs sorted from these tumors and pulsed with OVA peptides upregulated CD25 and CD69 expression on naïve OT-II CD4 T cells, confirming that apCAFs in our system can present via MHC-II. Furthermore, these apCAFs increased CD4 activation markers upon ex-vivo coculture with dissociated KPC tumors. We therefore hypothesize that apCAFs are required for the ICI-mediated anti-tumor effect and do so via modulation of CD4 T cell responses. Although a small proportion of the total compartment, the apCAF subset may be enhanced in number and function by CAF-targeted modulators to synergize with ICI to affect the antitumor response, which could provide a solution to the currently unmet need for new and more effective treatments for PDAC patients. Citation Format: Saumya Maru, Kathryn Howe, Lalitya Andaloori, James Leatherman, Joseph Tandurella, Edward J. Pearce, Elizabeth M. Jaffee. Antigen-presenting cancer-associated fibroblasts are found in immunotherapy-sensitive murine models of pancreatic ductal adenocarcinoma [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 B034.

Abstract C025: Fibroblast-specific IL1R1-p38 MAPK signaling sustains stromal inflammation and contributes to therapeutic resistance in pancreatic cancer

Abstract Introduction: The desmoplastic stroma of pancreatic ductal adenocarcinoma (PDAC) perpetuates therapeutic resistance by impairing drug delivery and effector immune cell infiltration and activation. We have identified tumor cell-derived interleukin-1α (IL1α) as a critical activator of the stroma, comprised of cancer-associated fibroblasts (CAF), towards a pro-inflammatory phenotype via the IL1 receptor, IL1R1, and subsequent activation of p38 MAPK. Our hypothesis is that disruption of the CAF-specific IL1α/p38 MAPK signaling axis can improve chemotherapeutic resistance by remodeling the fibrotic stromal landscape and the overall immune microenvironment in PDAC. Methods: p38 MAPK was pharmacologically inhibited with pexmetinib and genetically with an shRNA lentiviral system. Bulk RNA and ATAC sequencing (RNAseq and ATACseq) were performed on human pancreatic stellate cells (hPSC). Gene set enrichment analyses and motif enrichment analyses were performed on the RNAseq and ATACseq datasets, respectively. Tumor cells (KPC-6694), derived from LSL-KrasG12D/+; LSL-Tp53R172H/+; Pdx1Cre/+ (KPC) murine tumors, were injected orthotopically into Col1a2Cre/+; Mapk14f/f and Col1a2Cre/+; Il1r1f/f mice, and tumors were harvested for downstream analysis. Ptf1aCre/+; LSL-KrasG12D/+; Tgfbr2flox/flox (PKT) mice were treated with pexmetinib (PEX, 30mg/kg, daily PO), anakinra (AKA, 50mg/kg IP, BID) or vehicle control for 2.5 weeks prior to sacrifice for downstream analyses and single-cell RNA sequencing (scRNA) on tumors. Results: In vitro inhibition of p38 MAPK prevented CAF activation into an inflammatory fibroblast, when stimulated with IL1α, demonstrated through bulk RNAseq and ATACseq. Bulk ATACseq revealed epigenetic regulation of the inflammatory CAF phenotype by IL1-mediated activation of p38 MAPK. Stromal-specific deletion of Il1r1 or Mapk14 resulted in signification reduction of tumor weight and intratumoral myeloid-derived suppressor cells. Additionally, pharmacologic blockade of both IL1R1 (AKA) and p38 MAPK inhibition (PEX) altered intratumoral immune cell and CAF populations, by flow cytometry and scRNA. Lastly, p38 inhibition or IL1R1 blockade combined with chemotherapy significantly reduced tumor burden and favorably altered the immune landscape in a PDAC genetically engineered mouse model. Conclusions: These findings provide important mechanistic data in preclinical PDAC models to explore p38 MAPK inhibition in combination therapy to target the fibrotic stroma and immunosuppressive tumor microenvironment. Citation Format: Samara P. Singh, Austin R. Dosch, Siddharth Mehra, Iago de Castro Silva, Vanessa Tonin Garrido, Anna Bianchi, Haleh Amirian, Edmond W. Box, Jashodeep Datta, Nagaraj S. Nagathihalli, Nipun B. Merchant. Fibroblast-specific IL1R1-p38 MAPK signaling sustains stromal inflammation and contributes to therapeutic resistance 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 C025.

Abstract B067: Patient-specific differences in cancer-associated fibroblasts alter tumor organoid phenotype and chemosensitivity in pancreatic ductal adenocarcinoma

Abstract Pancreatic adenocarcinoma (PDAC) is the 3rd leading cause of cancer-related deaths in Canada with a 5-year survival rate ~10%. Unfortunately, only 20% of patients are eligible for surgical resection while the other 80% of patients are treated with chemotherapy (i.e. nab-paclitaxel, Gemcitabine, or FOLFIRINOX) that shows limited efficiency. Also, the tumor microenvironment (TME), and specifically cancer-associated fibroblasts (CAFs), contribute to PDAC chemoresistance. Recent studies suggest heterogeneity of CAF subtypes in patients, including myofibroblast (my), inflammatory (i), and antigen-presenting (ap) CAFs, is linked to more aggressive PDAC. While there is clear evidence CAFs effect tumor growth, chemoresistance and metastasis, the mechanisms underlying these effects are unknown. Given the involvement of environmental cues from the TME and the recent literature involving HDACs and epigenetic regulators in chemoresistance, we propose epigenetic reprogramming occurs in pancreatic cancer cells in response to CAFs, thereby affecting their response to chemotherapy. Using patient-derived tumor cells we examine the cross talk that occurs between cancer cells and CAFs in a patient-specific manner. We hypothesized that patient-specific CAF populations alter cancer cell phenotypes and chemosensitivity through epigenetic reprograming. Supported by the Baker Centre for Pancreatic Cancer, we established a living biobank of patient-derived organoids (PDO) grown in 3D cultures and CAFs grown in 2D cultures. These samples are linked to DERIVE (Determination of Response to Therapy in Individual Patients), a clinical database that includes patient response to therapy. Characterization of low passage, patient CAFs by flow cytometry shows significant heterogeneity between samples based on the proportions of myCAFs, iCAFs and apCAFs, and also identified CAFs that do not belong to any of these subtypes. Incubation of PDOs with CAF-conditioned media showed an increase in growth regardless of the CAF samples but, interestingly, myCAF-high (myCAFHI) conditioned media promoted a cystic-like phenotype in PDOs while iCAF-high conditioned media promoted a more complex PDO phenotype. myCAFHI-conditioned media also sensitized resistant PDOs to gemcitabine. Characterization of epigenetic mediators showed variable expression of histone deacetylases (HDACs), and combinatorial treatment with gemcitabine and the HDAC5 inhibitor, LMK-235 sensitized resistant PDOs to gemcitabine. We are currently performing single-cell RNA-seq, ATAC-seq and DNA methylation profiling on PDOs before and after treatment with CAF-conditioned media to identify unique CAF populations and examine the epigenetic targets underlying these outcomes. This study (1) shows CAFs present with significant heterogeneity between patients and secreted factors from CAFs induce responses in a patient-specific fashion that may change chemosensitivity, and (2) suggest targeting epigenetic mediators in PDOs and CAFs may help sensitize tumors to chemotherapy. Citation Format: Emilie Jaune-Pons, Zachary Klassen, Rachel Lu, Ye Shen, Nadeem Hussain, Michael Sey, Ken Leslie, Ephraim Tang, Anton Skaro, Matthew Cecchini, Crystal Engelage, Danielle Porplycia, Stephen Welch, Brian Yan, Christopher Pin. Patient-specific differences in cancer-associated fibroblasts alter tumor organoid phenotype and chemosensitivity in pancreatic ductal adenocarcinoma [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 B067.

Cancer-associated fibroblast spatial heterogeneity and EMILIN1 expression in the tumor microenvironment modulate TGF-β activity and CD8+ T-cell infiltration in breast cancer.

Rationale: The tumor microenvironment (TME) and its multifaceted interactions with cancer cells are major targets for cancer treatment. Single-cell technologies have brought major insights into the TME, but the resulting complexity often precludes conclusions on function. Methods: We combined single-cell RNA sequencing and spatial transcriptomic data to explore the relationship between different cancer-associated fibroblast (CAF) populations and immune cell exclusion in breast tumors. The significance of the findings was then evaluated in a cohort of tumors (N=75) from breast cancer patients using immunohistochemistry analysis. Results: Our data show for the first time the degree of spatial organization of different CAF populations in breast cancer. We found that IL-iCAFs, Detox-iCAFs, and IFNγ-iCAFs tended to cluster together, while Wound-myCAFs, TGFβ-myCAFs, and ECM-myCAFs formed another group that overlapped with elevated TGF-β signaling. Differential gene expression analysis of areas with CD8+ T-cell infiltration/exclusion within the TGF-β signaling-rich zones identified elastin microfibrillar interface protein 1 (EMILIN1) as a top modulated gene. EMILIN1, a TGF-β inhibitor, was upregulated in IFNγ-iCAFs directly modulating TGFβ immunosuppressive function. Histological analysis of 75 breast cancer samples confirmed that high EMILIN1 expression in the tumor margins was related to high CD8+ T-cell infiltration, consistent with our spatial gene expression analysis. High EMILIN1 expression was also associated with better prognosis of patients with breast cancer, underscoring its functional significance for the recruitment of cytotoxic T cells into the tumor area. Conclusion: Our data show that correlating TGF-β signaling to a CAF subpopulation is not enough because proteins with TGF-β-modulating activity originating from other CAF subpopulations can alter its activity. Therefore, therapeutic targeting should remain focused on biological processes rather than on specific CAF subtypes.

Abstract B047: Phase I trial of αvβ3 integrin cytotoxin ProAgio in patients with previously treated advanced pancreatic cancer and other solid tumor malignancies

Abstract Background: ProAgio is a pegylated peptide drug that targets and kills cells that express integrin αvβ3 including cancer associated fibroblasts (CAFs) and endothelial cells within the tumor microenvironment. ProAgio utilizes a novel binding site to directly trigger apoptosis of target cells in pre-clinical models. Here we describe the interim results of a first-in-human phase 1 trial of ProAgio in patients with advanced solid malignancies, including pancreatic cancer. Methods:  The trial is enrolling adults with previously treated and histologically confirmed advanced solid malignancy for which no curative therapy exists. The dose escalation cohort uses a modified 3+3 design to determine the recommended phase 2 dose (RP2D) of ProAgio. This will be followed by a dose expansion cohort which will enroll only participants with non-neuroendocrine advanced pancreatic cancer. The primary objective of the trial is to determine the RP2D of ProAgio. Secondary objectives assess the safety (CTCAE v. 4.03), pharmacokinetics and objective response rate (ORR) of Proagio. Ascending doses of ProAgio were administered as an intravenous infusion every 1-2 weeks depending on dose level. Response is determined by serial imaging (CT or MRI) and tumor marker assessment (CA 19-9 or appropriate tumor-specific marker). Exploratory objectives include assessing biologic effect of ProAgio on pre- and on-therapy tumor biopsy specimens (apoptosis of tumor cells, integrin αvβ3 expression, CAF population, tumor vasculature and architecture), as well as identifying putative surrogate circulating biomarkers (circulating αvβ3 expressing cells among others). Results: Sixteen participants (14 with pancreatic adenocarcinoma and 2 with other gastrointestinal malignancies) have been treated on the dose escalation as of June 30, 2023. Preliminary pharmacokinetic measurements demonstrate a plasma half-life of 17.35 hours, distribution volume of 17.62 L, and clearance of 0.522 L/hr. ProAgio has been exceptionally well tolerated with only transient grade 1 or 2 treatment-related adverse events observed in most participants. Two participants had grade 2 infusion related reaction but were able to continue treatment with increased dose of diphenhydramine pre-medication. A serious adverse event of grade 3 hyperglycemia was observed in 1 participant with insulin-dependent diabetes within 24 hours of ProAgio infusion. No other treatment-associated hyperglycemia has been observed. Two participants with pancreatic cancer receiving weekly dosing of ProAgio had stable disease for >4 months and interval stabilization in CA 19-9 tumor marker. Conclusions: ProAgio is safe and well-tolerated. Upon completion of the dose escalation, a dose expansion phase of 15 participants treated at the RP2D will begin enrolling. NCT05085548 Citation Format: Nebojsa Skorupan, Cody J. Peer, Eve Koehler, Seth Steinberg, Jane B Trepel, William D Figg, Zhi-Ren Liu, Christine Alewine. Phase I trial of αvβ3 integrin cytotoxin ProAgio in patients with previously treated advanced pancreatic cancer and other solid tumor malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B047.

Unravelling heterogeneous effects of cancer‑associated fibroblasts on poor prognosis markers in breast cancer EM‑G3 cell line: Invitro‑targeted treatment (anti‑IL-6, anti‑VEGF-A, anti‑MFGE8) based on transcriptomic profiling.

Breast cancer is the most frequently diagnosed cancer in women worldwide. Although dramatically increased survival rates of early diagnosed cases have been observed, late diagnosed patients and metastatic cancer may still be considered fatal. The present study's main focus was on cancer‑associated fibroblasts (CAFs) which is an active component of the tumor microenvironment (TME) regulating the breast cancer ecosystem. Transcriptomic profiling and analysis of CAFs isolated from breast cancer skin metastasis, cutaneous basal cell carcinoma, and squamous cell carcinoma unravelled major gene candidates such as IL6, VEGFA and MFGE8 that induced co‑expression of keratins‑8/‑14 in the EM‑G3 cell line derived from infiltrating ductal breast carcinoma. Western blot analysis of selected keratins (keratin‑8, ‑14, ‑18, ‑19) and epithelial‑mesenchymal transition‑associated markers (SLUG, SNAIL, ZEB1, E‑/N‑cadherin, vimentin) revealed specific responses pointing to certain heterogeneity of the studied CAF populations. Experimental invitro treatment using neutralizing antibodies against IL-6, VEGF‑A and MFGE8 attenuated the modulatory effect of CAFs on EM‑G3 cells. The present study provided novel data in characterizing and understanding the interactions between CAFs and EM‑G3 cells invitro. CAFs of different origins support the pro‑inflammatory microenvironment and influence the biology of breast cancer cells. This observation potentially holds significant interest for the development of novel, clinically relevant approaches targeting the TME in breast cancer. Furthermore, its implications extend beyond breast cancer and have the potential to impact a wide range of other cancer types.