Cancer-associated Fibroblasts Heterogeneity Research Articles

ScRNA-seq to examine heterogeneity of transcriptional regulation of CAFs between left-sided and right-sided CRC.

e15548 Background: Cancer-associated fibroblasts (CAFs) play prominent and diverse tumor-promoting function roles in tumor microenvironment (TME) and influence cancer hallmarks, but without systematic investigation between left-sided and right-sided CRC (colorectal cancer). Methods: We collected a public scRNA-seq dataset (Pelka et al., 2021) with 21 samples from left-sided CRC and 79 from right-sided CRC. Only CAFs were extracted for downstream analysis. Seurat (Stuart et al., 2019), Monocle2 (den Berge et al., 2020) and pySCENIC (Aibar et al., 2017) were used to perform dimensionality reductions, differential analysis, pseudo-time trajectory analysis, and the transcriptional regulation analysis, respectively. Results: Firstly, we performed differential analysis on 2,049 cells from 62 of 100 samples that contained CAFs. The results showed that highly expressed genes including IGFBP1, IGFBP2, LCN2, ALDH1A1 and CASP1 in left-sided CRC were mainly enriched in immune response pathway, while up-regulated genes in right-sided CRC were neutrophil-migration-chemotaxis-related such as PPBP1, PF4V1, CCL7, CXCL3 and CXCL8. Then, we classified 2,049 CAFs into four subclusters based on the markers of CCL8+ CAF, CXCL14+ CAF, GREM1+ CAF and MMP3+ CAF. The proportion of CXCL14+ CAF was higher in left-sided CRC while MMP3+ CAF was preferentially enriched in right-sided CRC, which was also independently validated by 33 TCGA cohorts. Further investigation on those subclusters showed that CXCL14+ CAF was myCAF, while the rest (CCL8+ CAF, MMP3+ CAF and GREM1+ CAF) were iCAF. After investigating the heterogeneity of CAFs, we focused on the transcriptional regulatory networks (TRNs). The results showed that the dominant regulons in left-sided CRC ( HOXD9, HSF2, MYBL1, HES6, XRCC4, and ZNF585B) and right-sided CRC ( CREB5, ZNF274, IRF7, TFEC, NR2F1, and PITX3) were distinct, which highlighted the heterogeneity and plasticity of CAF-based TRNs between left-sided and right-sided CRC. Conclusions: Collectively, although further experimental verification is required to establish the functional roles of each CAF cluster and the diverse origins of CAFs, this study may facilitate better understanding molecular mechanism of CAF-targeted therapy strategies in CRC.

Fibroblasts in pancreatic cancer: molecular and clinical perspectives.

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

The Tango between Cancer-Associated Fibroblasts (CAFs) and Immune Cells in Affecting Immunotherapy Efficacy in Pancreatic Cancer.

The lack of response to therapy in pancreatic ductal adenocarcinoma (PDAC) patients has contributed to PDAC having one of the lowest survival rates of all cancer types. The poor survival of PDAC patients urges the exploration of novel treatment strategies. Immunotherapy has shown promising results in several other cancer types, but it is still ineffective in PDAC. What sets PDAC apart from other cancer types is its tumour microenvironment (TME) with desmoplasia and low immune infiltration and activity. The most abundant cell type in the TME, cancer-associated fibroblasts (CAFs), could be instrumental in why low immunotherapy responses are observed. CAF heterogeneity and interactions with components of the TME is an emerging field of research, where many paths are to be explored. Understanding CAF-immune cell interactions in the TME might pave the way to optimize immunotherapy efficacy for PDAC and related cancers with stromal abundance. In this review, we discuss recent discoveries on the functions and interactions of CAFs and how targeting CAFs might improve immunotherapy.

Epigenetic regulation of cancer-associated fibroblast heterogeneity.

Cancer-associated fibroblasts (CAFs), a significant component of the tumor microenvironment (TME), contribute to cancer progression through the secretion of extracellular matrix (ECM), growth factors, and metabolites. It is now well recognized that CAFs are a heterogenous population with ablation experiments leading to reduced tumor growth and single-cell RNA sequencing demonstrating CAF subgroups. CAFs lack genetic mutations yet substantially differ from their normal stromal precursors. Here, we review epigenetic changes in CAF maturation, focusing on DNA methylation and histone modifications. DNA methylation changes in CAFs have been demonstrated globally, while roles of methylation at specific genes affect tumor growth. Further, loss of CAF histone methylation and gain of histone acetylation has been shown to promote CAF activation and tumor promotion. Many CAF activating factors, such as transforming growth factor β (TGFβ), lead to these epigenetic changes. MicroRNAs (miRNAs) serve as targets and orchestrators of epigenetic modifications that influence gene expression. Bromodomain and extra-terminal domain (BET), an epigenetic reader, recognizes histone acetylation and activates the transcription of genes leading to the pro-tumor phenotype of CAFs.

Abstract 5847: The splanchnic mesenchyme is the main tissue origin of fibroblasts in the pancreas during homeostasis and tumorigenesis

Abstract In pancreatic ductal adenocarcinoma (PDAC), cancer associated fibroblasts (CAFs) play critical and complex roles in the tumor microenvironment. CAFs are also a major cell type in the desmoplastic stroma in PDAC and may account for half of the entire tumor tissue. Multiple subtypes of CAFs have been suggested, but the tissue origin(s) of CAF subtypes are unknown and genetic tools to robustly target them in vivo are lacking. Here we aimed to examine three potential tissue sources of CAFs: the pancreatic epithelium (through epithelium-to-mesenchyme transition), the bone marrow (through circulation), and the pancreatic mesenchyme or tissue resident fibroblasts (TRFs) in the normal pancreas (through proliferation). We utilized a genetically engineered mouse model of PDAC, where Kras and p53 mutations were engineered in the pancreatic epithelium using an Flp-Frt system. To determine whether the pancreatic epithelium gives rise to CAFs, we permanently labeled the pancreatic epithelium with a GFP reporter and traced their cell descendants by GFP expression. Despite robust GFP labeling of the epithelium, GFP expression was rarely identified in CAFs. To determine whether the bone marrow gives rise to CAFs, we transplanted donor bone marrow carrying a ubiquitously expressed GFP reporter to GFP-negative recipient mice. We found that minimal proportion of pancreatic CAFs were tagged with GFP. Lastly, to determine whether pancreatic TRFs give rise to CAFs, we used an inducible CreER-LoxP system to allow for permanent Tomato labeling in TRF progenitors, the splanchnic mesenchyme, during mid-gestation. Lineage tracing in PDAC showed that the vast majority of CAFs were labeled with Tomato expression, suggesting their splanchnic origin. Furthermore, certain splanchnic gene expression signatures persisted in subsets of CAFs in both the PDAC mouse model and human patient samples. Deletion of one of the splanchnic genes, Gata6, in CAFs resulted in increased tumor burden in the pancreas, suggesting a tumor-restraining role of Gata6 in CAFs. In summary, we found that the pancreatic epithelium and bone marrow contributes to a minimal proportion of CAFs in PDAC. Meanwhile, pancreatic TRFs are derived from the splanchnic mesenchyme during fetal development and they expand to contribute to the vast majority of CAFs in PDAC. Moreover, the persistence of splanchnic signature defines subtypes of CAFs, with a potential tumor-suppressing function. This study provides genetic approaches to robustly target CAFs in vivo, and novel insights into CAF origin, heterogeneity and function in PDAC. Citation Format: Lu Han, Yongxia Wu, Kun Fang, Sean Sweeney, Ulyss Roesner, Melodie Parrish, Khushbu Patel, Tom Walter, Julia Piermattei, Anthony Trimboli, Julia Lefler, Cynthia Timmers, Xue-Zhong Yu, Victor Jin, Michael Zimmermann, Angela Mathison, Raul Urrutia, Michael Ostrowski, Gustavo Leone. The splanchnic mesenchyme is the main tissue origin of fibroblasts in the pancreas during homeostasis and tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5847.

Abstract 5850: Development and characterization of patient-derived pancreatic fibroblasts to study CAF heterogeneity

Abstract Pancreatic ductal adenocarcinoma (PDAC) has complex tumor microenvironment (TME), characterized by phenotypically and functionally heterogenous cancer-associated fibroblasts (CAFs). Different CAF subtypes have been identified that evidently contribute to desmoplasia, immunosuppression, and therapy response variably. The lack of models to study their plasticity and heterogeneity reproducibly poses a major limitation. We, thus, describe the development and characterization of a panel of immortalized patient-derived pancreatic fibroblasts (iPDPFs), studying their impact on PDAC cell lines in vitro and in vivo. Method: We developed six iPDPF lines using human telomerase reverse transcriptase (hTERT) from cancer-adjacent normal pancreas (9-26-NP), chronic pancreatitis (CPP1), and PDAC CAFs (9-17-P, 10-15-P, 10-03-P, & 10-32-P). Immunoblotting, bulk-RNAseq, and qPCR were performed for their molecular characterization. Functional characterization was done in vitro using collagen contraction assay and co-culture studies with COLO357. The impact of iPDPFs on the tumor cell behavior in vivo was studied by their co-implantation with COLO357 in athymic nude mice. Results: The iPDPFs exhibited variable expression of activation, inflammatory, and ECM markers indicative of heterogeneity across the panel. While CAFs expressed high levels of αSMA (alpha-smooth muscle actin) and fibroblast-specific protein-1 (FSP1), the expression of these markers was low in 9-26 NP & CPP-1. Also, the iPDPFs variably expressed TGFβ, PDGFRβ, VDR, COL1A, and small GTPases. High expression of inflammatory markers was observed in 9-26 NP, CPP-1, & 10-32-P suggesting their iCAF type while 9-17-P, 10-03-P, and 10-15-P were of myCAF type. These observations were recapitulated in the transcriptomic analysis. Interestingly, the marker expression changed with passage suggesting existence of plasticity in the iPDPFs. 9-17-P induced highest collagen contraction (47.9%) while 10-15-P induced the least contraction (25.89%). Co-culture of iPDPFs with COLO357 enhanced the expression of ROCK-1. In vivo, iPDPFs resulted in stroma-rich tumors; however, the extent of stroma and growth rate were variable across iPDPF lines and implantation sites. 9-17-P xenograft tumors exhibited the highest percent αSMA (14.5%), and FSP1 (30.7%) positive cells. Limited analysis showed that iPDPF co-implanted tumors exhibited variable vascularity and ECM composition suggesting that the heterogeneity observed in vitro was manifested in vivo. Conclusion: Overall, the iPDPFs recapitulate pancreatic fibroblasts heterogeneity, reflecting the overlapping nature of CAF subtypes. Also, the iPDPFs are functionally variable and plastic in vitro. Importantly, these fibroblasts exert variable effects on the tumor cells in vivo. The iPDPFs can thus serve as a useful model to reproducibly study CAF heterogeneity and plasticity in PDAC. Citation Format: Nidhi V. Dwivedi, Shailendra K. Gautam, Satya Rachagani, Ramakanth Venkata, Vipin Dalal, Maneesh Jain, Surinder K. Batra. Development and characterization of patient-derived pancreatic fibroblasts to study CAF heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5850.

CAFrgDB: a database for cancer-associated fibroblasts related genes and their functions in cancer

As one of the most essential components of the tumor microenvironment (TME), cancer-associated fibroblasts (CAFs) interact extensively with cancer cells and other stromal cells to remodel TME and participate in the pathogenesis of cancer, which earmarked themselves as new promising targets for cancer therapy. Numerous studies have highlighted the heterogeneity and versatility of CAFs in most cancer types. Thus, the identification and appropriate use of CAF-related genes (CAFGenes) in the context of specific cancer types will provide critical insights into disease mechanisms and CAF-related therapeutic targets. In this study, we collected and curated 5421 CAFGenes identified from small- or large-scale experiments, encompassing 4982 responsors that directly or indirectly participate in cancer malignant behaviors managed by CAFs, 1069 secretions that are secreted by CAFs and 281 regulators that contribute in modulating CAFs in human and mouse, which covered 24 cancer types. For these human CAFGenes, we performed gene expression and prognostic marker-based analyses across 24 cancer types using TCGA data. Furthermore, we provided annotations for CAF-associated proteins by integrating the knowledge of protein-protein interaction(s), drug-target relations and basic annotations, from 9 public databases. CAFrgDB (CAF related Gene DataBase) is free for academic research at http://caf.zbiolab.cn and we anticipate CAFrgDB can be a useful resource for further study of CAFs.

Cancer-Associated Fibroblasts: Heterogeneity and Bimodality in Oncogenesis

Cancer-associated fibroblasts (CAFs) often form a major component of the tumor microenvironment (TMA), providing conditions for cancer cells to thrive. CAFs may contribute to tumor growth, invasion, metastasis, and resistance to therapy. However, clinical trials of treatment strategies targeting CAFs have largely failed. Moreover, there is evidence that CAFs are capable of inhibiting tumor development. The review considers the current data on the functional heterogeneity of CAFs and their bimodality in tumor development and progression. Understanding the tumor-promoting and tumor-inhibiting activities of CAFs can help to develop new diagnostic and therapeutic approaches.

Cancer-associated fibroblasts: The chief architect in the tumor microenvironment.

Stromal heterogeneity of tumor microenvironment (TME) plays a crucial role in malignancy and therapeutic resistance. Cancer-associated fibroblasts (CAFs) are one of the major players in tumor stroma. The heterogeneous sources of origin and subsequent impacts of crosstalk with breast cancer cells flaunt serious challenges before current therapies to cure triple-negative breast cancer (TNBC) and other cancers. The positive and reciprocal feedback of CAFs to induce cancer cells dictates their mutual synergy in establishing malignancy. Their substantial role in creating a tumor-promoting niche has reduced the efficacy of several anti-cancer treatments, including radiation, chemotherapy, immunotherapy, and endocrine therapy. Over the years, there has been an emphasis on understanding CAF-induced therapeutic resistance in order to enhance cancer therapy results. CAFs, in the majority of cases, employ crosstalk, stromal management, and other strategies to generate resilience in surrounding tumor cells. This emphasizes the significance of developing novel strategies that target particular tumor-promoting CAF subpopulations, which will improve treatment sensitivity and impede tumor growth. In this review, we discuss the current understanding of the origin and heterogeneity of CAFs, their role in tumor progression, and altering the tumor response to therapeutic agents in breast cancer. In addition, we also discuss the potential and possible approaches for CAF-mediated therapies.

Single-cell analysis reveals the COL11A1+ fibroblasts are cancer-specific fibroblasts that promote tumor progression.

Background: Cancer-associated fibroblasts (CAFs) promote tumor progression through extracellular matrix (ECM) remodeling and extensive communication with other cells in tumor microenvironment. However, most CAF-targeting strategies failed in clinical trials due to the heterogeneity of CAFs. Hence, we aimed to identify the cluster of tumor-promoting CAFs, elucidate their function and determine their specific membrane markers to ensure precise targeting. Methods: We integrated multiple single-cell RNA sequencing (scRNA-seq) datasets across different tumors and adjacent normal tissues to identify the tumor-promoting CAF cluster. We analyzed the origin of these CAFs by pseudotime analysis, and tried to elucidate the function of these CAFs by gene regulatory network analysis and cell-cell communication analysis. We also performed cell-type deconvolution analysis to examine the association between the proportion of these CAFs and patients' prognosis in TCGA cancer cohorts, and validated that through IHC staining in clinical tumor tissues. In addition, we analyzed the membrane molecules in different fibroblast clusters, trying to identify the membrane molecules that were specifically expressed on these CAFs. Results: We found that COL11A1+ fibroblasts specifically exist in tumor tissues but not in normal tissues and named them cancer-specific fibroblasts (CSFs). We revealed that these CSFs were transformed from normal fibroblasts. CSFs represented a more activated CAF cluster and may promote tumor progression through the regulation on ECM remodeling and antitumor immune responses. High CSF proportion was associated with poor prognosis in bladder cancer (BCa) and lung adenocarcinoma (LUAD), and IHC staining of COL11A1 confirmed their specific expression in tumor stroma in clinical BCa samples. We also identified that CSFs specifically express the membrane molecules LRRC15, ITGA11, SPHK1 and FAP, which could distinguish CSFs from other fibroblasts. Conclusion: We identified that CSFs is a tumor specific cluster of fibroblasts, which are in active state, may promote tumor progression through the regulation on ECM remodeling and antitumor immune responses. Membrane molecules LRRC15, ITGA11, SPHK1 and FAP could be used as therapeutic targets for CSF-targeting cancer treatment.

Cancer-Associated Fibroblasts: Lessons from Pancreatic Cancer

Cancer-associated fibroblasts (CAFs) are present in all malignancies. Arguably, in none are they as prevalent as they are in pancreatic ductal adenocarcinoma (PDAC), where they often outnumber cancer cells. The origin and function of CAFs are still not completely understood, and attempts to target this cell population as a component of combination therapy have so far not succeeded. Our understanding of pancreatic CAFs is in rapid evolution. Heterogeneity of CAFs is the key concept to understand this cell population. We discuss heterogeneity of origin, with recent findings challenging the notion that CAFs uniformly derive from pancreatic stellate cells, and instead suggesting that multiple types of resident fibroblasts contribute to CAF expansion. Heterogeneity in gene expression divides CAFs in different subpopulations. Most importantly, heterogeneity in function underlies the complexity of CAFs. CAFs deposit components of the extracellular matrix, contributing to the high interstitial pressure in pancreatic cancer. CAFs serve as “feeder” cells for cancer cells by providing metabolites, thus mitigating the effect of the low-nutrient environment of PDAC. At the same time, CAFs regulate the function of the immune system, inhibiting antitumor immune responses. Understanding the functional role of different CAF populations and the drivers of each of their functional roles is key to devising new ways to target this cell population in PDAC.

CD26-negative and CD26-positive tissue-resident fibroblasts contribute to functionally distinct CAF subpopulations in breast cancer

Cancer-associated fibroblasts (CAFs) are abundantly present in the microenvironment of virtually all tumors and strongly impact tumor progression. Despite increasing insight into their function and heterogeneity, little is known regarding the origin of CAFs. Understanding the origin of CAF heterogeneity is needed to develop successful CAF-based targeted therapies. Through various transplantation studies in mice, we show that CAFs in both invasive lobular breast cancer and triple-negative breast cancer originate from mammary tissue-resident normal fibroblasts (NFs). Single-cell transcriptomics, in vivo and in vitro studies reveal the transition of CD26+ and CD26- NF populations into inflammatory CAFs (iCAFs) and myofibroblastic CAFs (myCAFs), respectively. Functional co-culture experiments show that CD26+ NFs transition into pro-tumorigenic iCAFs which recruit myeloid cells in a CXCL12-dependent manner and enhance tumor cell invasion via matrix-metalloproteinase (MMP) activity. Together, our data suggest that CD26+ and CD26- NFs transform into distinct CAF subpopulations in mouse models of breast cancer.

Cancer-Associated Fibroblast Heterogeneity, Activation and Function: Implications for Prostate Cancer

The continuous remodeling of the tumor microenvironment (TME) during prostate tumorigenesis is emerging as a critical event that facilitates cancer growth, progression and drug-resistance. Recent advances have identified extensive communication networks that enable tumor-stroma cross-talk, and emphasized the functional importance of diverse, heterogeneous stromal fibroblast populations during malignant growth. Cancer-associated fibroblasts (CAFs) are a vital component of the TME, which mediate key oncogenic events including angiogenesis, immunosuppression, metastatic progression and therapeutic resistance, thus presenting an attractive therapeutic target. Nevertheless, how fibroblast heterogeneity, recruitment, cell-of-origin and differential functions contribute to prostate cancer remains to be fully delineated. Developing our molecular understanding of these processes is fundamental to developing new therapies and biomarkers that can ultimately improve clinical outcomes. In this review, we explore the current challenges surrounding fibroblast identification, discuss new mechanistic insights into fibroblast functions during normal prostate tissue homeostasis and tumorigenesis, and illustrate the diverse nature of fibroblast recruitment and CAF generation. We also highlight the promise of CAF-targeted therapies for the treatment of prostate cancer.

Cancer-associated fibroblasts in acute leukemia.

Although the prognosis for acute leukemia has greatly improved, treatment of relapsed/refractory acute leukemia (R/R AL) remains challenging. Recently, increasing evidence indicates that the bone marrow microenvironment (BMM) plays a crucial role in leukemogenesis and therapeutic resistance; therefore, BMM-targeted strategies should be a potent protocol for treating R/R AL. The targeting of cancer-associated fibroblasts (CAFs) in solid tumors has received much attention and has achieved some progress, as CAFs might act as an organizer in the tumor microenvironment. Additionally, over the last 10 years, attention has been drawn to the role of CAFs in the BMM. In spite of certain successes in preclinical and clinical studies, the heterogeneity and plasticity of CAFs mean targeting them is a big challenge. Herein, we review the heterogeneity and roles of CAFs in the BMM and highlight the challenges and opportunities associated with acute leukemia therapies that involve the targeting of CAFs.

Signaling pathways in cancer-associated fibroblasts: recent advances and future perspectives.

As a critical component of the tumor microenvironment (TME), cancer-associated fibroblasts (CAFs) play important roles in cancer initiation and progression. Well-known signaling pathways, including the transforming growth factor-β (TGF-β), Hedgehog (Hh), Notch, Wnt, Hippo, nuclear factor kappa-B (NF-κB), Janus kinase (JAK)/signal transducer and activator of transcription (STAT), mitogen-activated protein kinase (MAPK), and phosphoinositide 3-kinase (PI3K)/AKT pathways, as well as transcription factors, including hypoxia-inducible factor (HIF), heat shock transcription factor 1 (HSF1), P53, Snail, and Twist, constitute complex regulatory networks in the TME to modulate the formation, activation, heterogeneity, metabolic characteristics and malignant phenotype of CAFs. Activated CAFs remodel the TME and influence the malignant biological processes of cancer cells by altering the transcriptional and secretory characteristics, and this modulation partially depends on the regulation of signaling cascades. The results of preclinical and clinical trials indicated that therapies targeting signaling pathways in CAFs demonstrated promising efficacy but were also accompanied by some failures (e.g., NCT01130142 and NCT01064622). Hence, a comprehensive understanding of the signaling cascades in CAFs might help us better understand the roles of CAFs and the TME in cancer progression and may facilitate the development of more efficient and safer stroma-targeted cancer therapies. Here, we review recent advances in studies of signaling pathways in CAFs and briefly discuss some future perspectives on CAF research.

CCL18 signaling from tumor-associated macrophages activates fibroblasts to adopt a chemoresistance-inducing phenotype

The heterogeneity of cancer-associated fibroblasts (CAFs) might be ascribed to differences in origin. CD10 and GPR77 have been reported to identify a chemoresistance-inducing CAF subset in breast cancer. However, the precise mechanism for the formation of the CD10+GPR77+ CAFs remains unknown. In this study, we found that CCL18 expression was positively correlated with the density of CD10+GPR77+ CAFs in breast cancer and associated with a poor response to chemotherapy. Moreover, CCL18 secreted by tumor-associated macrophages (TAMs) activated a CD10+GPR77+ CAF phenotype in normal breast-resident fibroblasts (NBFs), which could then enrich cancer stem cells (CSCs) and induce chemoresistance in breast cancer cells. Mechanistically, CCL18 activated NF-κB signaling via PITPNM3 and thus enhanced the production of IL-6 and IL-8. Furthermore, intratumoral CCL18 injection significantly induced the activation of NBFs and the chemoresistance of xenografts in vivo. In addition, targeting CCL18 by anti-CCL18 antibody could inhibit the formation of CD10+GPR77+ CAFs and recover the chemosensitivity in vivo, leading to effective tumor control. Collectively, these findings reveal that inflammatory signaling crosstalk between TAMs and fibroblasts is responsible for the formation of the CD10+GPR77+ CAFs, suggesting CCL18–PITPNM3 signaling is a potential therapeutic target to block the activation of this specific CAF subtype and tumor chemoresistance.

442 Deciphering fibroblast heterogeneity in human skin cancer

Cancer-associated fibroblasts (CAFs) are a key component of the tumor microenvironment and play an important role in disease progression and treatment outcome. Here we used single-cell transcriptomics, CNV analysis, and in situ stainings to elucidate the yet unresolved intra-tumoral CAF heterogeneity in melanoma, basal and squamous cell carcinoma. We identified three major CAF subtypes, with their presence being indicative for tumor malignancy. RGS5+ CAFs, which share features with pericytes, are present in all tumors.

Abstract C084: Oncogenic KRAS signaling drives the activation of tissue-resident fibroblasts and is required to maintain CAF heterogeneity in pancreatic cancer

Abstract The complexity of the tumor microenvironment (TME) is one of the distinguishing features of pancreatic ductal adenocarcinoma (PDA) and is responsible for patients’ poor response to therapies. The heterogeneity of cancer-associated fibroblasts (CAFs) has been correlated to the key features of the stroma that contribute to making PDA the third-leading cause of cancer-related deaths in the United States. The recent development of FDA-approved drugs against oncogenic KRAS opened a new therapeutic avenue for the treatment of tumors that have KRAS as their driver oncogene, such as PDA. However, the precise mechanisms driving the development of the TME and the contribution of KRAS to these processes have yet to be elucidated. This poses a challenge for the prediction of the effects of KRAS inhibition on established PDA tumors. By employing spatial transcriptomic technologies on various murine models recapitulating different stages of tumor initiation and progression, from acute and chronic inflammation to PanIN and overt PDA, we were able to observe distinctive changes in the activation status of pancreatic fibroblasts. These resident activated fibroblasts (RAFs) display expression of discrete markers brought upon by inflammation (inflammatory RAFs, iRAFs) or specific to oncogenic KRAS activation (myofibroblastic RAFs, myRAFs). Importantly, these RAF populations are maintained in established tumors and are identifiable in human PDA. To assess the consequences of the disruption of KRAS signaling on CAFs and RAFs, we employed a PDA mouse model that allows for the irreversible excision of KrasG12V, the FPC model (KrasFrt-LSL-G12V-Frt; p53LSL-R172H; PDX-CRE; Rosa26FlpOERT2). Through a combination of single-cell RNA sequencing (scRNA-seq), spatial transcriptomics and fluorescent in situ hybridization combined with immunofluorescence (immunoFISH) on FPC mice before and after KrasG12V deletion, we demonstrate the profound impact of Kras ablation on the TME composition. A deep remodeling of the stroma as well as significant changes in the proportion of CAF subtypes were evident. Interestingly, we observed a decrease in CAFs with a concomitant increase in RAFs. Our results suggest that the activation of oncogenic Kras in pre-neoplastic lesions drives a unique paracrine signaling that shapes the TME and is required to maintain the CAF population in PDA. Citation Format: Giuseppina Caligiuri, Jennifer S. Thalappillil, Mojdeh Shakiba, Sandeep Nadella, Juliene Hinds, Elise Courtois, William F. Flynn, Brinda Alagesan, Georgi N. Yordanov, Benjamin Kaminow, Paul Robson, Pedro A. Perez-Mancera, Jonathan Preall, Alexander Dobin, Youngku Park, David A. Tuveson. Oncogenic KRAS signaling drives the activation of tissue-resident fibroblasts and is required to maintain CAF heterogeneity in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C084.

Abstract PR018: Hypoxia synergizes with IL1 to promote an inflammatory fibroblast state in the pancreatic tumor microenvironment

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by a prominent desmoplastic response created by cancer-associated fibroblasts (CAFs) that can acquire both tumor-promoting and -restraining functions. Recently, transcriptionally distinct subpopulations of CAFs have been identified in PDAC including tumor-restraining myofibroblastic CAFs (myCAFs) producing extracellular matrix and tumor-promoting inflammatory CAFs (iCAFs) secreting cytokines. While CAF heterogeneity can be driven in part by growth factor and cytokine gradients, other factors involved in shaping the CAF state in PDAC are largely unknown. Identifying and targeting such factors may help to drive CAFs from tumor-promoting into tumor-suppressive states. Hypoxia is a major environmental factor in the PDAC tumor microenvironment but its role in CAF plasticity is unknown. In this study, we use KPC organoid orthotopic transplantation and co-culture models to investigate the role of hypoxia in regulating the CAF state and functions. We find that iCAFs display a hypoxic gene expression and biochemical profile in vivo and are enriched in hypoxic regions of PDAC tumors. Hypoxia leads to acquisition of an inflammatory gene expression signature in fibroblasts and synergizes with cancer cell-derived IL1 to promote an iCAF phenotype in a HIF-1α dependent fashion. Furthermore, we show in PDAC organoid co-culture models that HIF-1α stabilization is sufficient to induce an iCAF phenotype in pancreatic stellate cells and to promote PDAC tumor growth in vivo in the absence of hypoxia. These data indicate Citation Format: Simon Schwoerer, Manon Ros, Kaloyan Tsanov, Francesco Cimino, Scott Lowe, Carlos Carmona-Fontaine, Craig Thompson. Hypoxia synergizes with IL1 to promote an inflammatory fibroblast state in the pancreatic tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR018.

Abstract C039: An EGFR-activated subset of myofibroblastic cancer-associated fibroblasts supports local metastatic dissemination in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDA) has a dismal prognosis and new therapies are needed. Cancer-associated fibroblasts (CAFs) can promote PDA progression and are recognized promising therapeutic targets. However, the ongoing characterization of PDA CAF heterogeneity and plasticity and the identification of potential tumor-restraining CAF populations have highlighted the need to further dissect the diverse biology of PDA CAFs to develop effective combination therapies. Using pancreatic organoid/fibroblast co-cultures and organoid-derived transplantation mouse models of PDA, we mechanistically investigated the signaling cascade downstream TGF-beta activation, which is a main driver of myofibroblastic CAF (myCAF) formation. We find that the EGFR/ERBB2 pathway is activated by TGF-beta in a subset of myCAFs, in both murine and human PDA, via amphiregulin induction and autocrine signaling. ERBB activation is important for the maintenance of this myCAF subset, with genetic and pharmacological inhibition of this pathway differentially affecting PDA CAF composition both in culture and mouse models. Importantly, analysis of lung cancer and breast cancer patient samples suggests that our observations in PDA are also relevant to these malignancies. Finally, we reveal a role of this EGFR-activated myCAF subset in promoting local metastatic dissemination in PDA and indicate new potential combination strategies. Our study highlights the need to better dissect PDA CAF heterogeneity also in populations typically considered to be tumor-restraining, in order to develop effective combination strategies for future clinical intervention. Our work also reveals how cancer-targeting therapies can directly affect the surrounding microenvironment with potential clinical implications for various malignancies. Citation Format: Gianluca Mucciolo, Joaquín Araos Henríquez, Sara Pinto Teles, Judhell Manansala, Wenlong Li, Eloise G. Lloyd, Priscilla Cheng, Giulia Biffi. An EGFR-activated subset of myofibroblastic cancer-associated fibroblasts supports local metastatic dissemination in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C039.