Cancer-associated Fibroblasts Phenotype Research Articles

Abstract A030: Polycomb repressive complex 2 (PRC2) mediates the epigenetic regulation of cancer-associated fibroblasts in esophageal cancer

Abstract Cancer-associated fibroblasts (CAFs) are a heterogenous cell population that can promote esophageal cancer progression through multiple molecular mechanisms including increased cancer cell proliferation and migration, immunosuppression, and therapeutic resistance. Despite the divergence of CAFs from normal fibroblasts with regards to phenotype and functions, CAFs rarely exhibit widespread genetic alterations. Instead, it is proposed that CAFs are regulated at the epigenetic level. Our objective is to delineate this epigenetic regulation of CAFs in esophageal cancer. Ultimately, we aim to identify the epigenetic regulatory complexes that mediate CAF heterogeneity and pro-tumorigenic functions. To this end, we completed RNA-seq and ATAC-seq on fibroblasts isolated from esophageal squamous cell carcinoma (ESCC), esophageal adenocarcinoma (EAC) and non-cancerous esophageal tissue. Enrichment analysis identified the polycomb repressive complex 2 (PRC2) as a potential regulator of ESCC and EAC CAFs. PRC2 catalyzes the methylation (mono-, di- or tri-) of lysine 27 on histone 3 (H3K27me1/2/3) resulting in transcriptional repression. Inhibition of two key proteins of PRC2 (EZH2 and SUZ12) through either pharmacological or genetic modulation, altered expression of multiple CAF phenotype related proteins and inhibited CAF functions including CAF proliferation. Ongoing studies, using co-cultures of fibroblasts and tumor cells, seek to identify if inhibition of PRC2 can prevent the initial transition of normal esophageal fibroblasts into CAFs. Importantly, we are conducting in vivo studies to determine if selective PRC2 inhibition in CAFs can inhibit esophageal tumor progression with specific focus on primary tumor growth and metastatic spread. In summary, initial studies have identify that PRC2 complex mediates CAF phenotype and functions, and we aim to establish the larger impact of these CAF specific effects on esophageal cancer. Citation Format: Karen J. Dunbar, Raul Navaridas Fernandez de Bobadil, Katherine M. Cunningham, Emily Esquea, Gizem Efe, Anil K. Rustgi. Polycomb repressive complex 2 (PRC2) mediates the epigenetic regulation of cancer-associated fibroblasts in esophageal cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A030.

Abstract PR009: FMRP upregulation in cancer: Implicating FMRP-expressing cancer-associated fibroblasts in immune evasion

Abstract Our research has uncovered a novel and significant role for Fragile-X-mental-retardation-protein (FMRP) in cancer biology, extending its well-established functions beyond neuronal development. In a recent Science publication (Zeng et al., 2022), we demonstrated that elevated FMRP levels drive immune evasion in various murine and human tumors by modulating immune cell subsets, particularly through the suppression of effector T-cells. To further elucidate the mechanisms of FMRP-mediated immune evasion, we investigated its influence on the stromal component of the tumor microenvironment. Single-cell RNA sequencing of pancreatic ductal adenocarcinoma (PDAC) tumors revealed cancer-associated fibroblasts (CAFs) as the primary stromal cell subtype expressing FMRP. Given the crucial roles that CAFs play in tumor progression and therapeutic resistance, we explored whether FMRP expression contributes to the immunosuppressive characteristics of CAFs in PDAC and breast tumors. Our findings reveal that FMRP expression is markedly upregulated in CAFs from both breast and PDAC tumors, in contrast to its absence in naïve fibroblasts from healthy tissues. Knockdown of FMRP in CAFs resulted in substantial changes in their myofibroblastic and inflammatory properties, while overexpression of FMRP in naïve fibroblasts was sufficient to induce a CAF-like phenotype. Notably, FMRP overexpression led to significantly elevated inflammatory CAF (iCAF) and myofibroblastic CAF (myCAF) signatures, highlighting its role as a key pan-CAF regulator that influences both extracellular matrix (ECM) modulation and the secretory phenotype of CAFs. We utilized the full-body knockout (KO) mice model of the Fmr1 gene, which encodes FMRP, to further delineate the functional importance of FMRP expression in CAFs in stimulating an immunosuppressive program in vivo. Through bulk RNA sequencing, we developed an FMRP- associated gene signature for CAFs and applied it to human datasets, where we observed a pronounced increase in FMRP signatures in CAFs from human PDAC and breast tumors, highlighting its translational relevance. In conclusion, our research underscores the pivotal role of FMRP in cancer biology, impacting both the immune and stromal components of the tumor microenvironment. Targeting FMRP, particularly within CAFs, emerges as a promising therapeutic strategy to improve cancer treatment outcomes. Citation Format: Simge Yucel, Nadine Fournier, Mireia Blanco Gomez, Douglas Hanahan. FMRP upregulation in cancer: Implicating FMRP-expressing cancer-associated fibroblasts in immune evasion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR009.

Pirfenidone antagonizes TGF-β1-mediated gabapentin resistance via reversal of desmoplasia and the ‘cold’ microenvironment in pancreatic cancer

Owing to the desmoplastic stroma constituted by cancer-associated fibroblasts (CAFs), few immune cells infiltrate the pancreatic ductal adenocarcinoma (PDAC). Gabapentin can impede the production of ketoacids by CAFs to support cancer cells. However, in our study, we discovered a dose-dependent increase in transforming growth factor β1 (TGF-β1) levels in cancer cells in response to gabapentin. This reverse increase of TGF-β1 contributes to 'Gabapentin-resistance', leading to the antitumor effects on PDAC cell lines are negatively negotiated in the presence of pancreatic stellate cells. Pirfenidone synergistically inhibited the growth and apoptosis resistance of PDAC when combined with Gabapentin. In a mouse orthotopic PDAC model, Fe3+-mediated coordination nanodrugs, which contain gabapentin, pirfenidone and the natural polyphenol (EGCG), efficiently promoted the infiltration of naïve CD8+ T cells (CD44lowCD62Lhigh) and the accumulation of inflammatory CAFs (α-SMAlowIL-6high). This led to a nearly two-fold increase in survival compared to the control. Furthermore, we identified a new subpopulation as Hmox1highiCAFs following treatment with our nanodrugs. Hmox1highiCAFs overexpressed the Cxcl10 receptor (Sdc4) and facilitated functional CD8+ T-cell infiltration through the Tnfsf9-Tnfrsf9 axis. Overall, our nanodrugs reshape the phenotype of CAFs and enhance functional CD8+ T-cell infiltration into tumors, holding the potential to be a safe and promising therapy for PDAC.

Abstract A039: Repeat RNA mediated disruption of cellular plasticity in pancreatic cancer

Abstract Aberrant expression of repeat RNAs in pancreatic ductal adenocarcinoma (PDAC) mimics viral-like responses with implications for tumor cell state and the surrounding microenvironment. To better understand the relationship of repeat RNAs in human PDAC, we employed the NanoString CosMx™ spatial molecular imaging (SMI) platform, which utilizes a 1,000-plex RNA panel with custom repeat RNA probes targeting long interspersed nuclear element 1 (LINE-1) retrotransposon ORF1 and ORF2, HSATII satellite (SAT) repeat, and two human endogenous retroviruses (HERV-K and HERV-H) in 46 primary tumors. This analysis revealed correlations of high repeat RNA expression with alterations in the epithelial state of PDAC cells and the myofibroblast phenotype in cancer-associated fibroblasts (CAFs). The loss of cellular identity observed with dosing of extracellular vesicles (EVs) and individual repeat RNAs in PDAC and CAF cell culture models points to cell-cell intercommunication involving these viral-like elements. Differences in the PDAC and CAF response are driven by distinct innate immune signaling pathways through interferon regulatory transcription factor 3 (IRF3). Altogether, our data indicate that cell context-specific viral-like responses driven by tumor cells have broad impacts on single-cell heterogeneity within cancer cells and the pancreatic cancer microenvironment. Citation Format: Eunae You, Patrick Danaher, Chenyue Lu, Siyu Sun, Luli Zou, Ildiko Phillips, Alexandra Rojas, Natalie Ho, Yuhui Song, Michael Raabe, Katherine Xu, Peter Richieri, Hao Li, Natalie Aston, Rebecca Porter, Bidish Patel, Linda Nieman, Nathan Schurman, Briana Hudson, Khrystyna North, Sarah Church, Vikram Deshpande, Andrew Liss, Tae Kim, Yi Cui, Youngmi Kim, Benjamin Greenbaum, Martin Aryee, David Ting. Repeat RNA mediated disruption of cellular plasticity in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A039.

Abstract A047: Fibroblast-specific IL1 receptor inhibition reprograms the inflammatory stroma to overcome the immunosuppressive tumor microenvironment in pancreatic cancer

Abstract OBJECTIVE: KRAS-driven inflammatory signaling in pancreatic ductal adenocarcinoma (PDAC) promotes cancer-associated fibroblast (CAF) polarization and an immunosuppressive tumor microenvironment (iTME), driving therapeutic resistance. IL1α, a downstream product of KRAS signaling, activates the pro-inflammatory CAF (iCAF) phenotype and correlates with worse survival. Using patient and PDAC mouse model single-cell RNA sequencing (scRNAseq) data, we have shown that the IL1 Receptor (IL1R1) is highly enriched in the fibroblast compartment. However, the role of IL1R1 in modulating iCAFs and the iTME remains poorly understood. We hypothesize that CAF-specific disruption of IL1R1 signaling will reprogram cellular components of the PDAC TME to overcome the immunosuppressive milieu and therapeutic resistance. METHODS: The effects of IL1R1 inhibition on iCAF modulation in vivo were determined in Ptf1aCre/+, LSL-KrasG12D/+, Tgfbr2flox/flox (PKT) mice treated with IL1R1 antagonist. To determine if CAF-specific silencing of IL1R1 recapitulates the TME remodeling after IL1R1 inhibition, we generated a fibroblast-specific IL1R1KO mouse model Col1a2Cre/+;Il1r1flox/flox (CAF-Il1r1KO) and orthotopically injected KPC tumor cells for endpoint analysis. Flow cytometry was performed in primary tumors of CAF-Il1r1KO and littermate controls. Functional experiments evaluating myeloid-derived suppressor cell (MDSC) trafficking were performed using ex vivo bone marrow-derived cells (BMDCs) co-cultured with CAF/tumor-conditioned media (CM) with/without IL1R1 inhibition in a transwell system. MDSC contribution to iTME was assessed by co-culturing MDSCs from CAF-Il1r1KO tumors and splenic T cells from naïve C57BL/6 mice. To investigate the effects of IL1R1 deletion in overcoming therapeutic resistance, survival studies in CAF-Il1r1KO mice with chemotherapy were conducted. RESULTS: Treatment of PKT mice with IL1R1 antagonist diminished transcriptional levels of Il6 and Cxcl1, iCAF-associated cytokines, in the CAF compartment. Compared to littermate controls, orthotopic tumors in CAF-Il1r1KO showed significant reduction in tumor weights, increased CD8+ T cell infiltration, and overall reduction in polymorphonuclear (PMN) MDSC trafficking by flow cytometry. Moreover, migration of BMDCs was reduced when co-cultured with CAF/tumor CM and IL1R1 antagonist. In addition, co-culture of MDSCs from CAF-Il1r1KO mice with naïve T cells showed diminished inhibition of T cell functional activity. Chemotherapy treatment of orthotopic tumors in CAF-Il1r1KO mice showed significantly decreased tumor size and improved survival compared to tumors in littermate controls. CONCLUSION: Our findings illustrate a novel mechanism of CAF-specific disruption of IL1R1 signaling inhibiting trafficking of immunosuppressive MDSCs and increased infiltration of cytotoxic CD8+ T cells in the PDAC TME. These data pave the way for novel combinatorial treatment strategies targeting IL1R1 to overcome chemotherapeutic resistance through reprogramming of the stromal compartment in PDAC. Citation Format: Camille Acevedo, Samara Singh, Austin Dosch, Edmond W Box, Iago C Silva, Anna Bianchi, Siddharth Mehra, Haleh Amirian, Andrew Adams, Nagaraj Nagathihalli, Jashodeep Datta, Nipun B Merchant. Fibroblast-specific IL1 receptor inhibition reprograms the inflammatory stroma to overcome the immunosuppressive tumor microenvironment in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A047.

TAK1 Promotes an Immunosuppressive Tumor Microenvironment through Cancer-Associated Fibroblast Phenotypic Conversion in Pancreatic Ductal Adenocarcinoma.

We aim to clarify the precise function of Transformed growth factor-beta 1 activated kinase-1 (TAK1) in cancer-associated fibroblasts (CAFs) within human pancreatic ductal adenocarcinoma (PDAC) by investigating its role in cytokine-mediated signaling pathways. The expression of TAK1 in pancreatic cancer was confirmed by TCGA data and human pancreatic cancer specimens. CAFs from freshly resected PDAC specimens were cultured and used in a three-dimensional model for direct and indirect co-culture with PDAC tumors to investigate TAK1 function. Additionally, organoids from KPC (LSL-K-RasLSLG12D/+; LSL-p53R172H/+; Pdx1-Cre) mice were mixed with CAFs and injected subcutaneously into C57BL/6 mice to explore in vivo functional interactions of TAK1. TCGA data revealed significant upregulation of TAK1 in PDAC, associating with a positive correlation with the T-cell exhaustion signature. Knockdown of TAK1 in CAFs decreased the iCAF signature and increased the myCAF signature both in vitro and in vivo. The absence of TAK1 hindered CAF proliferation, blocked several inflammatory factors via multiple pathways associated with immunosuppression, and hindered EMT, outgrowth in vitro in spheroid co-cultures with PDAC cells. Additionally, TAK1 inhibitor restrained tumor growth, increased CD4+ and CD8+ T cell abundance, and reduced immunosuppressive cells present in vivo. Blocking the TAK1+CAF phenotype leads to the conversion of protumorigenic CAFs to antitumorigenic CAFs. This highlights TAK1 as a potential therapeutic target, particularly in CAFs, and represents a novel avenue for combined immunotherapy in PDAC.

Senescence-associated secretory phenotype regulation by dual drug delivery biomimetic nanoplatform for enhanced tumor chemotherapy

Many chemotherapies, which are still the main clinical treatment for primary tumors, will induce persistent DNA damage in non-tumor stromal cells, especially cancer-associated fibroblasts (CAFs), and activate them to secrete senescence associated secretory phenotype (SASP). The transition could further result in the formation of tumor immunosuppressive microenvironment and cause drug resistance of neighboring tumor cells. To solve this dilemma, a multi-functional biomimetic drug delivery system (named mPtP@Lipo) was rationally developed by combining CAFs reshaper ginsenoside 20(S)-protopanaxadiol (PPD) and cisplatin prodrug (PtLA) to inhibit tumor progression and the formation of SASP. To achieve effective delivery of these molecules deep into the desmoplastic tumor, fibroblast membrane was fused with liposomes as a targeting carrier. In vitro and in vivo results showed that mPtP@Lipo could penetrate deep into the tumor, reverse CAFs phenotype and inhibit SASP formation, which then blocked the immunosuppressive progresses and thus reinforced anti-tumor immune response. The combination of chemotherapeutics and CAFs regulator could achieve both tumor inhibition and tumor immune microenvironment remodeling. In conclusion, mPtP@Lipo provides a promising strategy for the comprehensive stromal-desmoplastic tumor treatment.

NNMT switches the proangiogenic phenotype of cancer-associated fibroblasts via epigenetically regulating ETS2/VEGFA axis.

Cancer-associated fibroblasts (CAFs) are known to promote angiogenesis in oral squamous cell carcinoma (OSCC). However, the epigenetic mechanisms through which CAFs facilitate angiogenesis within the tumor microenvironment are still poorly characterized. Nicotinamide N'-methyltransferase (NNMT), a member of the N-methyltransferase family, was found to be a key molecule in the activation of CAFs. This study shows that NNMT in fibroblasts contributes to angiogenesis and tumor growth through an epigenetic reprogramming-ETS2-VEGFA signaling axis in OSCC. Single-cell RNA Sequencing (scRNA-seq) analysis suggests that NNMT is mainly highly expressed in fibroblasts of head and neck squamous cell carcinoma (HNSCC). Moreover, analysis of the TCGA database and multiple immunohistochemical staining of clinical samples also identified a positive correlation between NNMT and tumor angiogenesis. This research further employed an assembled organoid model and a fibroblast-endothelial cell co-culture model to authenticate the proangiogenic ability of NNMT. At the molecular level, high expression of NNMT in CAFs was found to promote ETS2 expression by regulating H3K27 methylation level through mediating methylation deposition. Furthermore, ETS2 was verified to be an activating transcription factor of VEGFA in this study. Collectively, our findings delineate an epigenetic molecular regulatory network of angiogenesis and provide a theoretical basis for exploring new targets and clinical strategy in OSCC.

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.

BAG6 restricts pancreatic cancer progression by suppressing the release of IL33-presenting extracellular vesicles and the activation of mast cells

Recent studies reveal a critical role of tumor cell-released extracellular vesicles (EVs) in pancreatic cancer (PC) progression. However, driver genes that direct EV function, the EV-recipient cells, and their cellular response to EV uptake remain to be identified. Therefore, we studied the role of Bcl-2-associated-anthanogene 6 (BAG6), a regulator of EV biogenesis for cancer progression. We used a Cre recombinase/LoxP-based reporter system in combination with single-cell RNA sequencing to monitor in vivo EV uptake and tumor microenvironment (TME) changes in mouse models for pancreatic ductal adenocarcinoma (PDAC) in a Bag6 pro- or deficient background. In vivo data were validated using mouse and human organoids and patient samples. Our data demonstrated that Bag6-deficient subcutaneous and orthotopic PDAC tumors accelerated tumor growth dependent on EV release. Mechanistically, this was attributed to mast cell (MC) activation via EV-associated IL33. Activated MCs promoted tumor cell proliferation and altered the composition of the TME affecting fibroblast polarization and immune cell infiltration. Tumor cell proliferation and fibroblast polarization were mediated via the MC secretome containing high levels of PDGF and CD73. Patients with high BAG6 gene expression and high protein plasma level have a longer overall survival indicating clinical relevance. The current study revealed a so far unknown tumor-suppressing activity of BAG6 in PDAC. Bag6-deficiency allowed the release of EV-associated IL33 which modulate the TME via MC activation promoting aggressive tumor growth. MC depletion using imatinib diminished tumor growth providing a scientific rationale to consider imatinib for patients stratified with low BAG6 expression and high MC infiltration.EVs derived from BAG6-deficient pancreatic cancer cells induce MC activation via IL33/Il1rl1. The secretome of activated MCs induces tumor proliferation and changes in the TME, particularly shifting fibroblasts into an inflammatory cancer-associated fibroblast (iCAF) phenotype. Blocking EVs or depleting MCs restricts tumor growth.

Synthetic retinoid-mediated preconditioning of cancer-associated fibroblasts and macrophages improves cancer response to immune checkpoint blockade

BackgroundThe proliferation of cancer-associated fibroblasts (CAFs) hampers drug delivery and anti-tumor immunity, inducing tumor resistance to immune checkpoint blockade (ICB) therapy. However, it has remained a challenge to develop therapeutics that specifically target or modulate CAFs.MethodsWe investigated the involvement of Meflin+ cancer-restraining CAFs (rCAFs) in ICB efficacy in patients with clear cell renal cell carcinoma (ccRCC) and urothelial carcinoma (UC). We examined the effects of Am80 (a synthetic retinoid) administration on CAF phenotype, the tumor immune microenvironment, and ICB efficacy in cancer mouse models.ResultsHigh infiltration of Meflin+ CAFs correlated with ICB efficacy in patients with ccRCC and UC. Meflin+ CAF induction by Am80 administration improved ICB efficacy in the mouse models of cancer. Am80 exerted this effect when administered prior to, but not concomitant with, ICB therapy in wild-type but not Meflin-deficient mice. Am80-mediated induction of Meflin+ CAFs was associated with increases in antibody delivery and M1-like tumor-associated macrophage (TAM) infiltration. Finally, we showed the role of Chemerin produced from CAFs after Am80 administration in the induction of M1-like TAMs.ConclusionOur data suggested that Am80 administration prior to ICB therapy increases the number of Meflin+ rCAFs and ICB efficacy by inducing changes in TAM phenotype.

Identification of Small-Molecule Inhibitors Targeting Different Signaling Pathways in Cancer-Associated Fibroblast Reprogramming under Tumor–Stroma Interaction

Cancer-associated fibroblasts (CAFs) interact reciprocally with tumor cells through various signaling pathways in many cancer types, including cutaneous squamous cell carcinoma. Among normal fibroblast subtypes, papillary fibroblasts (PFs) and reticular fibroblasts (RFs) respond distinctly to tumor cell signaling, eventuating the differentiation of RFs rather than PFs into CAFs. The regulation of subtype differentiation in fibroblasts remains poorly explored. In this study, we assessed the differences between PFs, RFs, and CAFs and examined the effects of small-molecule inhibitors targeting the TGFβ, phosphoinositide 3-kinase/protein kinase B/mTOR, and NOTCH pathways on the tumor-promoting property of CAFs and CAF reprogramming in 2-dimensional and 3-dimensional cultures. Blocking TGFβ and phosphoinositide 3-kinase strongly deactivated and concurrently induced a PF phenotype in RFs and CAFs. Three-dimensional coculturing of a cutaneous squamous cell carcinoma cell line MET2 with RFs or CAFs led to enhanced tumor invasion, RF-CAF transition, and cytokine production, which were further repressed by blocking TGFβ and phosphoinositide 3-kinase/mTOR pathways but not NOTCH pathway. In conclusion, the study identified biomarkers for PFs, RFs, and CAFs and displayed different effects of blocking key signaling pathways in CAFs and tumor cell-CAF interplay. These findings prompted a CAF-to-PF therapeutic strategy and provided perspectives of using included inhibitors in CAF-based cancer therapy.

HIF2A mediates lineage transition to aggressive phenotype of cancer-associated fibroblasts in lung cancer brain metastasis

ABSTRACT Brain metastasis is the most devasting form of lung cancer. Recent studies highlight significant differences in the tumor microenvironment (TME) between lung cancer brain metastasis (LCBM) and primary lung cancer, which contribute significantly to tumor progression and drug resistance. Cancer-associated fibroblasts (CAFs) are the major component of pro-tumor TME with high plasticity. However, the lineage composition and function of CAFs in LCBM remain elusive. By reanalyzing single-cell RNA sequencing (scRNA-seq) data (GSE131907) from lung cancer patients with different stages of metastasis comprising primary lesions and brain metastasis, we found that CAFs undergo distinctive lineage transition during LCBM under a hypoxic situation, which is directly driven by hypoxia-induced HIF-2α activation. Transited CAFs enhance angiogenesis through VEGF pathways, trigger metabolic reprogramming, and promote the growth of tumor cells. Bulk RNA sequencing data was utilized as validation cohorts. Multiplex immunohistochemistry (mIHC) assay was performed on four paired samples of brain metastasis and their primary lung cancer counterparts to validate the findings. Our study revealed a novel mechanism of lung cancer brain metastasis featuring HIF-2α-induced lineage transition and functional alteration of CAFs, which offers potential therapeutic targets.

Hydrogen Therapy Reverses Cancer-Associated Fibroblasts Phenotypes and Remodels Stromal Microenvironment to Stimulate Systematic Anti-Tumor Immunity.

Tumor microenvironment (TME) plays an important role in the tumor progression. Among TME components, cancer-associated fibroblasts (CAFs) show multiple tumor-promoting effects and can induce tumor immune evasion and drug-resistance. Regulating CAFs can be a potential strategy to augment systemic anti-tumor immunity. Here, the study observes that hydrogen treatment can alleviate intracellular reactive oxygen species of CAFs and reshape CAFs' tumor-promoting and immune-suppressive phenotypes. Accordingly, a controllable and TME-responsive hydrogen therapy based on a CaCO3 nanoparticles-coated magnesium system (Mg-CaCO3) is developed. The hydrogen therapy by Mg-CaCO3 can not only directly kill tumor cells, but also inhibit pro-tumor and immune suppressive factors in CAFs, and thus augment immune activities of CD4+ T cells. As implanted in situ, Mg-CaCO3 can significantly suppress tumor growth, turn the "cold" primary tumor into "hot", and stimulate systematic anti-tumor immunity, which is confirmed by the bilateral tumor transplantation models of "cold tumor" (4T1 cells) and "hot tumor" (MC38 cells). This hydrogen therapy system reverses immune suppressive phenotypes of CAFs, thus providing a systematic anti-tumor immune stimulating strategy by remodeling tumor stromal microenvironment.

Integration of proteomic and metabolomic analysis reveal distinct metabolic alterations of prostate cancer-associated fibroblasts compared to normal fibroblasts from patient's stroma samples

The prostate gland is a complex and heterogeneous organ composed of epithelium and stroma. Whilst many studies into prostate cancer focus on epithelium, the stroma is known to play a key role in disease with the emergence of a cancer-associated fibroblasts (CAF) phenotype associated upon disease progression. In this work, we studied the metabolic rewiring of stromal fibroblasts following differentiation to a cancer-associated, myofibroblast-like, phenotype. We determined that CAFs were metabolically more active compared to normal fibroblasts. This corresponded with a heightened lipogenic metabolism, as both reservoir species and building block compounds. Interestingly, lipid metabolism affects mitochondria functioning yet the mechanisms of lipid-mediated functions are unclear. Data showing oxidised fatty acids and glutathione system are elevated in CAFs, compared to normal fibroblasts, strengthens the hypothesis that increased metabolic activity is related to mitochondrial activity. This manuscript describes mechanisms responsible for the altered metabolic flux and shows that prostate cancer-derived extracellular vesicles can increase basal respiration in normal fibroblasts, mirroring that of the disease-like phenotype. This indicates that extracellular vesicles derived from prostate cancer cells may drive an altered oxygen-dependent metabolism associated to mitochondria in CAFs.

Development of 42 marker panel for in-depth study of cancer associated fibroblast niches in breast cancer using imaging mass cytometry.

Imaging Mass Cytometry (IMC) is a novel, and formidable high multiplexing imaging method emerging as a promising tool for in-depth studying of tissue architecture and intercellular communications. Several studies have reported various IMC antibody panels mainly focused on studying the immunological landscape of the tumor microenvironment (TME). With this paper, we wanted to address cancer associated fibroblasts (CAFs), a component of the TME very often underrepresented and not emphasized enough in present IMC studies. Therefore, we focused on the development of a comprehensive IMC panel that can be used for a thorough description of the CAF composition of breast cancer TME and for an in-depth study of different CAF niches in relation to both immune and breast cancer cell communication. We established and validated a 42 marker panel using a variety of control tissues and rigorous quantification methods. The final panel contained 6 CAF-associated markers (aSMA, FAP, PDGFRa, PDGFRb, YAP1, pSMAD2). Breast cancer tissues (4 cases of luminal, 5 cases of triple negative breast cancer) and a modified CELESTA pipeline were used to demonstrate the utility of our IMC panel for detailed profiling of different CAF, immune and cancer cell phenotypes.

Abstract 233: Cabozantinib changes the landscape of the pancreatic tumor microenvironment and improves the efficacy of pembrolizumab

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with an approximate 10% 5-year survival rate. PDAC patients rarely benefit from treatment with immune checkpoint inhibitors (ICIs) due to a tumor microenvironment (TME) associated with constrained effector T cell infiltration, and the presence of a suppressive myeloid cell population. Objective: To identify a combinatorial therapeutic strategy targeting the myeloid derived suppressor cells (MDSCs) and cancer associated fibroblasts (CAFs) within the PDAC TME to increase the efficacy of ICIs. Methods: C57BL/6 mice were orthotopically transplanted with syngeneic 7940b PDAC cells derived from transgenic KPC mouse. 7 days post-transplantation mice were treated with InVivoPlus anti-mouse PD-1 (CD279), cabozantinib (cabo, tyrosine kinase inhibitor) or a combination of drugs for a further 7 days. NanoString CosMx™ spatial molecular imaging (CosMx™ SMI) was performed using FFPE sections collected from core biopsies and surgically resected tissues. PDAC patient derived organoids (PDOs) were generated from the core biopsies or surgical biospecimens. Cancer-associated fibroblasts (CAFs) were isolated from the organoid cultures. Autologous immune cells (IMM) were cultured from patient peripheral blood mononuclear cells. PDO/CAF/IMM co-cultures were used to predict the patient response to cabo plus pembrolizumab combinatorial therapy. Results: Mice treated with a combination of anti-PD-1 and cabo exhibited significant EpCAM/PD-L1-expressing cell death that correlated with increased CTL proliferation and decreased PMN-MDSC death. Mice that were depleted of CD8+ CTLs using anti-CD8 antibody in vivo did not exhibit tumor cell death in response to combinatorial therapy and thus supporting the role of CTLs in driving effective immunotherapy in combination with cabozantinib treatment. Unexpectedly, a significant decrease in extracellular matrix protein fibronectin was observed. Human PDOs orthotopically transplanted into NSG mice developed pathology consistent with human PDAC with strong expression of CD44v9, IL6, vimentin and αSMA. Using PDAC PDO/CAFs/IMM co-cultures, pembro significantly induced tumor cell death in the presence CTLs, MDSCs significantly inhibited this response. Depletion of MDSCs using cabo in the PDO/CAFs/IMM co-cultures resulted in a significant increase in tumor cell death with a concomitant increase in CTL proliferation. Compared to core biopsies, surgical biospecimens collected post-standard of-care demonstrated a shift in cell populations within defined NICHES (local cellular TME) whereby there was 1) increases MDSCs, 2) change in CAFs phenotype and 3) increased expression of CD44v9. Conclusions: Cabozantinib depletes the PDAC TME of MDCSs leading to sensitization to ICI and tumor cell death and thus offers a potential combinatorial therapeutic approach. Citation Format: Pritha Adhikary, Jayati Chakrabarti, Meenu Priya Resmi, Jiang Wang, Davendra Sohal, Rachna T. Shroff, Syed A. Ahmad, Yana Zavros. Cabozantinib changes the landscape of the pancreatic tumor microenvironment and improves the efficacy of pembrolizumab [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 233.

Phenotypic Heterogeneity of Cancer Associated Fibroblasts in Cervical Cancer Progression: FAP as a Central Activation Marker.

Cervical cancer (CC) is the fourth leading cancer among women and is one of the principal gynecological malignancies. In the tumor microenvironment, cancer-associated fibroblasts (CAFs) play a crucial role during malignant progression, exhibiting a variety of heterogeneous phenotypes. CAFs express phenotypic markers like fibroblast activation protein (FAP), vimentin, S100A4, α-smooth muscle actin (αSMA), and functional markers such as MMP9. This study aimed to evaluate the protein expression of vimentin, S100A4, αSMA, FAP, and MMP9 in mesenchymal stem cells (MSC)-CAF cells, as well as in cervical cancer samples. MSC cells were stimulated with HeLa and SiHa tumor cell supernatants, followed by protein evaluation and cytokine profile to confirm differentiation towards a CAF phenotype. In addition, automated immunohistochemistry (IHQa) was performed to evaluate the expression of these proteins in CC samples at different stages. Our findings revealed a high expression of FAP in stimulated MSC cells, accompanied by the secretion of pro/anti-inflammatory cytokines. In the other hand, CC samples were observed to have high expression of FAP, vimentin, αSMA, and MMP9. Most importantly, there was a high expression of their activation proteins αSMA and FAP during the different stages. In the early stages, a myofibroblast-like phenotype (CAFs αSMA+ FAP+), and in the late stages a protumoral phenotype (CAF αSMA- FAP+). In summary, FAP has a crucial role in the activation of CAFs during cervical cancer progression.

Abstract 1577: Cancer associated fibroblast - tumor cell crosstalk enhances epithelial to mesenchymal transition and promotes a classical to basal switch in human pancreatic ductal adenocarcinoma

Abstract Introduction: Cancer associated fibroblasts (CAF) drive a complex tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) through incompletely understood interactions. Two major CAF subtypes, myofibroblastic (myCAF) and inflammatory (iCAF), dominate the tumor landscape and have been defined by gene expression profiles. There is significant plasticity between subtypes and alterations in CAF behavior likely drive changes in tumorigenicity and immune evasion. Additionally, PDAC tumor cells usually present with a predominant classical or basal phenotype that is associated with clinical outcomes. We hypothesized that CAF phenotype impacts tumor cell gene expression and alters the clinically-relevant tumor subtyping. Methods: We interrogated cellular crosstalk in the PDAC TME using a novel three-dimensional, patient-matched coculture system of patient-derived organoids (PDO) and CAFs, established from 12 patients undergoing surgical resection. Molecular characterization included bulk RNA sequencing, proteome profiling (107 factor), flow cytometry, multiplex RNA Scope, imaging mass cytometry (IMC), and RT-qPCR. Results: PDO gene expression analysis from bulk RNAseq demonstrated CAF coculture induced phenotypic shifts in the Moffitt subtype from a classical to a basal phenotype. Further, CAF coculture enhanced epithelial to mesenchymal transition (EMT) across all patients, even those with a baseline basal phenotype. We hypothesized this could be due to both cell-cell crosstalk and secreted factors. We therefore treated PDO with CAF-conditioned media (CM) and demonstrated that CM is sufficient to drive the observed classical-to-basal shift by RT-qPCR. Secretome analysis of CM derived from 3 CAF lines identified HGF, FGF19, and IL-8 as factors contributing to this transcriptional change. Next, we used spatial - omics (transcriptomics and proteomics) to examine the validity of these findings in source patient tissue. Using RNA Scope, we discovered distinct tumor neighborhoods expressing basal mRNAs KRT17 and KRT6a surrounded by CAFs compared to those expressing classical genes TFF1 and GATA6. We then used IMC to further explore CAF subtypes in patient tumors and found FAP+ myCAF regions corresponded to basal tumor regions, while CXCL12+ iCAFs were found in classical regions. Conclusions: PDAC tumor biology is likely driven by interactions between cancer cells and CAFs. Potential mediators of these interactions include both cell-cell contact and secreted factors. We introduce a novel approach combining in vitro coculture with tissue-based assays, demonstrating that modern culture techniques preserve cell type heterogeneity and plasticity. Together, these data demonstrate that a CAF-rich TME drives tumor cell plasticity not only towards EMT but also a more basal disease phenotype. Citation Format: Samantha Guinn, Brayan Perez, Jignasha Patel, Jae Lee, Daniel Zabransky, Won Ho, Elana J. Fertig, Emma Kartalia, Joseph Tandurella, Richard Burkhart, Jacquelyn W. Zimmerman, Elizabeth Jaffee. Cancer associated fibroblast - tumor cell crosstalk enhances epithelial to mesenchymal transition and promotes a classical to basal switch in human pancreatic ductal adenocarcinoma [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 1577.

Abstract 6827: Myocardin and myocardin-related transcription factor as potential drivers of the myofibroblastic cancer-associated fibroblast phenotype in colorectal cancer

Abstract Background: Cancer-associated fibroblasts (CAFs) play a key role in modifying the tumor microenvironment (TME) and could be a future target for treatment. CAFs are typically categorized based on function: myofibroblastic CAFs (myCAFs) amplify extracellular matrix production that blocks therapy and immune cell infiltration while inflammatory CAFs (iCAFs) produce immuno-suppressive cytokines and chemokines. Here we sought to better understand the role and mechanisms of myCAFs in altering the TME by identifying their unique markers and potential drivers. Methods: Z-scores of ACTA2/TAGLN or ICAM1/PDPN were used to create datasets high in myCAF and iCAF markers, respectively, from TCGA data. A gene set enrichment analysis (GSEA) was performed on the resulting differential expression. Samples of patients were collected to create a cohort of colorectal cancers that were stained via immunohistochemistry (IHC) for alpha-smooth muscle actin (αSMA), transgelin (TAGLN), myocardin (MYOCD) and myocardin-related transcription factor A (MRTFA). Slides were evaluated for collagen using Masson’s trichrome stain. MYOCD and MRTFA nuclear expression was expressed as positively stained nuclei per high powered field (HPF; 400x). TAGLN, αSMA, and collagen were scored on a scale of 0-3+. Results: Of the 592 TCGA patients compiled across all disease stages, 23 (median age 63) formed the myCAF-high group (7 males, 16 females). Within this group, GSEA indicated an upregulation of myogenesis (q <0.001) with the following genes positively associated with myCAF markers: MYLK (q < 0.001, log2 fold change [FC] 1.52), DES (q < 0.001, 5.32), and MYH11 (q < 0.001, 3.57). The following were also upregulated in the myCAF high cohort: CNN1 (q < 0.001, 2.95) and MYOCD (q< 0.001, 2.27). These genes are targets of MYOCD and MRTFA transcription factors potentially driving the myCAF phenotype. To assess this potential relationship, we evaluated 306 tumor samples from 153 patients with CRC. The cohort consisted of 56 female patients and 97 male patients aged 13-90 years old across all disease stages. High amounts of nuclear MRTFA were associated with high scores for myCAF markers aSMA (average score 1.54 when MRTFA+ nuclei < 5 per HPF vs 2.06 when MRTFA + nuclei > 5 per HPF, p<0.001, Wilcoxon Rank Sum Test), TAGLN (1.37 vs 1.62, p = 0.043), and collagen (1.73 vs 2.10, p = 0.009). Likewise, for MYOCD, αSMA (1.79 vs 2.14, p = 0.001) and collagen (1.87 vs 2.15, p = 0.017) were positively correlated. Conclusions: Increased nuclear expression of MYOCD and MRTFA in stromal cells of CRC are positively correlated with myCAF gene expression, supporting our initial hypothesis of these being drivers of the phenotype. Our findings now motivate further analysis as to whether the inhibition of these transcription factors could lead to a reduction in the immune regulatory myCAF phenotype. Citation Format: Alec B. Cornelio, Katherine A. Johnson, Yousef Gadalla, Sharanya Nath, Cheri A. Pasch, Wei Zhang, Kristina A. Matkowskyj, Dustin A. Deming. Myocardin and myocardin-related transcription factor as potential drivers of the myofibroblastic cancer-associated fibroblast phenotype in colorectal 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 6827.