Cancer-associated Fibroblasts Subpopulations Research Articles

Fibroblast heterogeneity in prostate carcinogenesis.

Our understanding of stromal components, specifically cancer-associated fibroblasts (CAF), in prostate cancer (PCa), has evolved from considering these cells as inert bystanders to acknowledging their significance as players in prostate tumorigenesis. CAF are multifaceted-they promote cancer cell growth, migration and remodel the tumor microenvironment. Although targeting CAF could be a promising strategy for PCa treatment, they incorporate a high but undefined degree of intrinsic cellular heterogeneity. The interaction between CAF subpopulations, with the normal and tumor epithelium and with other cell types is not yet characterized. Defining these interactions and the critical signaling nodes that support tumorigenesis will enable the development of novel strategies to control prostate cancer progression. Here we will discuss the origins, molecular and functional heterogeneity of CAF in PCa. We highlight the challenges associated with delineating CAF heterogeneity and discuss potential areas of research that would assist in expanding our knowledge of CAF and their role in PCa tumorigenesis.

Cytokine Interaction With Cancer-Associated Fibroblasts in Esophageal Cancer.

Esophageal cancer (EC) is a highly aggressive cancer with poor outcomes under current treatment regimens. More recent findings suggest stroma elements, specifically cancer-associated fibroblasts (CAFs), play a role in disease occurrence and progression. Cancer-associated fibroblasts are largely the product of converted fibroblasts, but a variety of other local cell types including epithelial cells, endothelial cells, and mesenchymal cells have also been shown to transform to CAFs under the correct conditions. Cancer-associated fibroblasts primarily function in the communication between the tumor microenvironment and cancer cells via cytokine and chemokine secretions that accentuate immunosuppression and cancer growth. Cancer-associated fibroblasts also pose issues for EC treatment by contributing to resistance of current chemotherapeutics like cisplatin. Targeting this cell type directly proves difficult given the heterogeneity between CAFs subpopulations, but emerging research provides hope that treatment is on the horizon. This review aims to unravel some of the complexities surrounding CAFs’ impact on EC growth and therapy.

Cancer-associated fibroblasts in intrahepatic cholangiocarcinoma progression and therapeutic resistance.

Cancer-associated fibroblasts (CAFs) are one of the most abundant stromal cell type in the tumor microenvironment (TME) of intrahepatic cholangiocarcinoma (iCCA), where they are actively involved in cancer progression through a complex network of interactions with other stromal cells. The majority of the studies investigating CAFs in iCCA have focused their attention on CAF tumor-promoting roles, remarking their potential as therapeutic targets. However, indiscriminate targeting of CAFs in other desmoplastic tumors has ended in failure with no effects or even accelerated cancer progression and reduced survival, indicating the urgent need to better understand the nuances and functions of CAFs to avoid deleterious effects. Indeed, recent single cell RNA sequencing studies have shown that heterogeneous CAF subpopulations coexist in the same tumor, some promoting- and other restricting- tumor growth. Moreover, recent studies have shown that in iCCA, diverse CAF subtypes interact differently with the cells of the TME, suggesting that CAFs may dynamically change their phenotypes during tumor progression, a field that remains uninvestigated. The characterization of heterogenous CAF subpopulations and their functionality, will provide a feasible and safer approach to facilitate the development of new therapeutic approaches aimed at targeting CAFs and their interactions with other stromal cells in the TME rather than solely tumor cells in iCCA. Here, we discuss the origin of CAFs, as well as their heterogeneity, plasticity, mechanisms and targeting strategies to provide a brief snapshot of the current knowledge in iCCA.

CD73-Mediated Immunosuppression Is Linked to a Specific Fibroblast Population That Paves the Way for New Therapy in Breast Cancer.

Simple SummaryRecent findings have revealed the contribution of cancer-associated fibroblasts (CAF) in immune escape in breast cancer. Still, how to specifically target immunosuppressive CAF remains an unmet medical question. Here, we provide a promising therapeutic strategy by highlighting the role of CD73 in immunosuppressive CAF. By studying cohorts of breast cancer patients and performing functional assays, our study uncovers how CD73 contributes to immunosuppression by acting in a specific CAF subpopulation (referred to as CAF-S1) in breast cancer. In addition, we validate that using an anti-CD73 antibody significantly reduces CAF-S1-mediated immunosuppression, thereby highlighting a new interesting therapeutic strategy for breast cancer patients.Background: Cancer-associated fibroblasts (CAF) are heterogeneous with multiple functions in breast cancer. Recently, we identified a specific CAF subpopulation (referred to as CAF-S1), which promotes immunosuppression and immunotherapy resistance. Methods and Results: Here, by studying a large collection of human samples, we highlight the key function of CD73/NT5E in CAF-S1-mediated immunosuppression in breast cancer. We first reveal that CD73 protein level specifically accumulates in CAF-S1 in breast cancer patients. Interestingly, infiltration of regulatory T lymphocytes (Tregs) is significantly correlated with CD73 expression in stroma but not in epithelium, indicating that CD73 contributes to immunosuppression when expressed in CAF-S1 and not in tumor cells. By performing functional assays based on relevant systems using primary CAF-S1 isolated from patients, we demonstrate that CAF-S1 increase the content in both PD-1+ and CTLA-4+ Tregs. Importantly, the use of a blocking anti-CD73 antibody on CAF-S1 reduces CAF-S1-mediated immunosuppression by preventing expression of these immune checkpoints on Tregs. Conclusions: Our data support the potential clinical benefit of using both anti-CD73 and immune-checkpoint inhibitors in breast cancer patients for inhibiting CAF-S1-mediated immunosuppression and enhancing anti-tumor immune response.

Breast Cancer CAFs: Spectrum of Phenotypes and Promising Targeting Avenues.

Activation of the tumor-associated stroma to support tumor growth is a common feature observed in different cancer entities. This principle is exemplified by cancer-associated fibroblasts (CAFs), which are educated by the tumor to shape its development across all stages. CAFs can alter the extracellular matrix (ECM) and secrete a variety of different molecules. In that manner they have the capability to affect activation, survival, proliferation, and migration of other stromal cells and cancer cell themselves. Alteration of the ECM, desmoplasia, is a common feature of breast cancer, indicating a prominent role for CAFs in shaping tumor development in the mammary gland. In this review, we summarize the multiple roles CAFs play in mammary carcinoma. We discuss experimental and clinical strategies to interfere with CAFs function in breast cancer. Moreover, we highlight the issues arising from CAFs heterogeneity and the need for further research to identify CAFs subpopulation(s) that can be targeted to improve breast cancer therapy.

Abstract 669: A comprehensive single cell analysis of cancer-associated fibroblasts (CAFs) reveals subtypes with prognostic impact in pancreatic ductal adenocarcinoma

Abstract Introduction The presence of heterogeneity in cancer-associated fibroblasts (CAFs) of pancreatic ductal adenocarcinoma (PDAC) is well-known. The presence of mouse inflammatory CAFs (iCAFs) and myofibroblastic CAFs (myCAFs) were published earlier. Recently, we identified the first patient-derived sub-populations of CAFs (pCAFassigner) with different phenotypic and pathological characteristics. We demonstrated the presence of intra-tumoral heterogeneity in patients, differential sensitivity of CAF subtypes in co-cultures with cancer cells to existing PDAC treatment options and prognostic impact of these subtypes. The presence of antigen-presenting CAFs (apCAFs) in humans and mouse was introduced recently. However, a comprehensive, unbiased analysis is required to reconcile different CAF classifications and understand the prognostic significance of these subtypes for future clinical translation. Methods Mouse and human PDAC single-cell RNA sequencing (scRNA-seq)-based suite of analyses were performed to assign CAFs into pCAFassigner and other subtypes. Enrichment (hypergeometric) and differential gene expression (DGE) analyses were performed to identify sample- and gene-wise similarities between classifications, as well as develop and validate a new pCAFassigner gene signature. For prognostic impact, a published single sample classifier was used to identify a dominant CAF subtype in 1071 whole tumor samples, for which clinical information was available for 618 samples. The overall survival (OS) analysis was performed using Kaplan-Meier survival analysis. Results As expected, our analysis revealed the synonymity between subtype-A and myCAF (91 %), subtypes-B and -C with iCAF (69 %) and subtype D with apCAF (91 %, FDR < 0.001). We revised our pCAFassigner gene signature from a prediction analysis of microarrays (PAM) centroid to an 82 gene signature unique to each subtype. Given the similarities between these separate classifications, we sought to identify association of iCAF (subtype-B/C), apCAF (subtype-D) and myCAF (subtype-A) with survival. We observed a significant difference in OS with apCAF- and myCAF-dominant samples having the poorest prognosis, while patients with dominant iCAF had prolonged survival, questioning the understanding of iCAFs being pro-tumorigenic given its secretory nature and consequently, its presumed association with poor survival. Conclusions Our results indicate that while methodologies to derive each CAF classification is distinct, ultimately, they have proven to be similar. Further, we reveal prognostic impact of iCAFs, apCAFs and myCAFs, supporting the use of these CAF subtypes as independent prognostic indicators for resected PDAC and will aid better management of the disease. Citation Format: Aasia Hussain, Jiahui Ji, Prashant K. Srivastava, Anguraj Sadanandam. A comprehensive single cell analysis of cancer-associated fibroblasts (CAFs) reveals subtypes with prognostic impact in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 669.

Role of cancer-associated fibroblast subpopulations in immune infiltration, as a new means of treatment in cancer.

The tumor microenvironment (TME) has been identified as one of the driving factors of tumor progression and invasion. Within this microenvironment, cancer‐associated fibroblasts (CAF) have multiple tumor‐promoting functions and play key roles in drug resistance, through multiple mechanisms, including extracellular matrix (ECM) remodeling, production of growth factors, cytokines, and chemokines, and modulation of metabolism and angiogenesis. More recently, a growing body of evidence has shown that CAF also modulate immune cell activity and suppress anti‐tumor immune response. In this review, we describe the current knowledge on CAF heterogeneity in terms of identity and functions. Moreover, we analyze how distinct CAF subpopulations differentially interact with immune cells, with a particular focus on T lymphocytes. We address how specific CAF subsets contribute to cancer progression through induction of an immunosuppressive microenvironment. Finally, we highlight potential therapeutic strategies for targeting CAF subpopulations in cancer.

In-Depth Characterization of Stromal Cells within the Tumor Microenvironment Yields Novel Therapeutic Targets.

Simple SummaryThis up-to-date and in-depth review describes fibroblast-derived cells and their role within the tumor microenvironment for tumor progression. Moreover, targets for future antitumor therapies are summarized and potential aspects for future translational research are outlined. Furthermore, this review discusses the challenges and possible obstacles related to certain treatment targets.Cells within the tumor stroma are essential for tumor progression. In particular, cancer-associated fibroblasts (CAF) and CAF precursor cells (resident fibroblasts and mesenchymal stromal cells) are responsible for the formation of the extracellular matrix in tumor tissue. Consequently, CAFs directly and indirectly mediate inflammation, metastasis, immunomodulation, angiogenesis, and the development of tumor chemoresistance, which is orchestrated by complex intercellular cytokine-mediated crosstalk. CAFs represent a strategic target in antitumor therapy but their heterogeneity hinders effective treatment regimes. In-depth understanding of CAF subpopulations and knowledge of specific functions in tumor progression will ultimately result in more specific and effective cancer treatments. This review provides a detailed description of CAFs and CAF precursor cells and summarizes possible treatment strategies as well as molecular targets of these cells in antitumor therapies.

Abstract PO051: Fibroblast lineage tracing in metastatic and pre-metastatic microenvironments

Abstract Metastatic breast cancer is one of the leading causes of cancer-related mortality among women. However, the complex process of tumor cell dissemination and colonization of distant tissues is incompletely understood and largely incurable using existing therapies. Metastatic disease is not solely driven by tumor cell-intrinsic properties but is instead a consequence of dynamic interactions between cancer cells and other cell types in the tumor microenvironment (TME) including cancer-associated fibroblasts (CAFs). However, CAFs are known to display paradoxical functions across different cancer types, suggesting that heterogeneous CAF subpopulations play diverse or opposing roles in the TME. It was recently shown that periostin, a TGFβ-induced matricellular protein important for collagen cross-linking and extracellular matrix stiffening, can distinguish CAF subtypes in breast cancer. This specific CAF subpopulation is not well characterized, and their functional properties and contributions to disease progression have not been well-studied in vivo. Here, we have used in vivo genetic labelling of periostin+ subpopulations (Postn-Cre:ZSGreenl/s/l) and their progeny to assess their expansion and function during mammary tumor growth and metastasis. We find that ZSGreen-labelled periostin-expressing CAFs arise from mesenchymal-lineage cells and are more abundant in primary tumors, metastatic, and pre-metastatic sites of high-metastatic tumors versus their low-metastatic counterparts. We also find differential collagen organization in high- versus low-metastatic tumors: high-metastatic tumors display longer, aligned collagen fibers whereas low-metastatic tumors display shorter, disorganized fibers. Knocking down periostin in primary human breast CAFs inhibits tumor cell invasion through collagen in a CAF/tumor cell co-culture spheroid assay, indicating that periostin-expressing CAFs may remodel the extracellular matrix via collagen cross-linking to facilitate cell invasion. Finally, we show that periostin/ZSGreen+ cells in highly metastatic tumors are larger and more spindle-shaped, and that periostin knockdown in primary human breast CAFs reduces their size and ability to migrate, suggesting that periostin expression associates with CAF biomechanical properties such as spreading and motility. Our results indicate that highly-metastatic cancer cells mobilize periostin-expressing CAFs in the TME, which may promote collagen remodeling and collective migration of CAFs and cancer cells during tissue invasion. Citation Format: Jamie L. Null, Dae Joong Kim, Yu Zhang, James V. McCann, Thomas H. Barker, Andrew C. Dudley. Fibroblast lineage tracing in metastatic and pre-metastatic microenvironments [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO051.

Adipose-derived mesenchymal stem cells differentiate into heterogeneous cancer-associated fibroblasts in a stroma-rich xenograft model

Cancer-associated fibroblasts (CAFs) are the key components of the densely proliferated stroma in pancreatic ductal adenocarcinoma (PDAC) and contribute to tumor progression and drug resistance. CAFs comprise heterogeneous subpopulations playing unique and vital roles. However, the commonly used mouse models have not been able to fully reproduce the histological and functional characteristics of clinical human CAF. Here, we generated a human cell-derived stroma-rich CDX (Sr-CDX) model, to reproduce the clinical tumor microenvironment. By co-transplanting human adipose-derived mesenchymal stem cells (AD-MSCs) and a human PDAC cell line (Capan-1) into mice, the Sr-CDX model recapitulated the characteristics of clinical pancreatic cancer, such as accelerated tumor growth, abundant stromal proliferation, chemoresistance, and dense stroma formed from the heterogeneous CAFs. Global RNA sequencing, single-cell based RNA sequencing, and histological analysis of CAFs in the Sr-CDX model revealed that the CAFs of the Sr-CDX mice were derived from the transplanted AD-MSCs and composed of heterogeneous subpopulations of CAF, including known and unknown subtypes. These lines of evidences suggest that our new tumor-bearing mouse model has the potential to address an open question in CAF research, that is the mechanism of CAF differentiation.

Cancer-Associated Fibroblasts Suppress Cancer Development: The Other Side of the Coin.

Cancer-associated fibroblasts (CAFs) are the main stromal components of cancer, representing a group of heterogeneous cells. Many studies indicate that CAFs promote tumor development. Besides, evidence of the tumor suppression effects of CAFs keeps on merging. In the tumor microenvironment, multiple stimuli can activate fibroblasts. Notably, this does not necessarily mean the activated CAFs become strong tumor promoters immediately. The varying degree of CAFs activation makes quiescent CAFs, tumor-restraining CAFs, and tumor-promoting CAFs. Quiescent CAFs and tumor-restraining CAFs are more present in early-stage cancer, while comparatively, more tumor-promoting CAFs present in advanced-stage cancer. The underlying mechanism that balances tumor promotion or tumor inhibition effects of CAFs is mostly unknown. This review focus on the inhibitory effects of CAFs on cancer development. We describe the heterogeneous origin, markers, and metabolism in the CAFs population. Transgenetic mouse models that deplete CAFs or deplete CAFs activation signaling in the tumor stroma present direct evidence of CAFs protective effects against cancer. Moreover, we outline CAFs subpopulation and CAFs derived soluble factors that act as a tumor suppressor. Single-cell RNA-sequencing on CAFs population provides us new insight to classify CAFs subsets. Understanding the full picture of CAFs will help translate CAFs biology from bench to bedside and develop new strategies to improve precision cancer therapy.

Periostin+ cancer-associated fibroblasts promote lymph node metastasis by impairing the lymphatic endothelial barriers in cervical squamous cell carcinoma.

Lymph node metastasis (LNM), a critical prognostic determinant in cancer patients, is critically influenced by the presence of numerous heterogeneous cancer-associated fibroblasts (CAFs) in the tumor microenvironment. However, the phenotypes and characteristics of the various pro-metastatic CAF subsets in cervical squamous cell carcinoma (CSCC) remain unknown. Here, we describe a CAF subpopulation with elevated periostin expression (periostin+ CAFs), located in the primary tumor sites and metastatic lymph nodes, that positively correlated with LNM and poor survival in CSCC patients. Mechanistically, periostin+ CAFs impaired lymphatic endothelial barriers by activating the integrin-FAK/Src-VE-cadherin signaling pathway in lymphatic endothelial cells and consequently enhanced metastatic dissemination. In contrast, inhibition of the FAK/Src signaling pathway alleviated periostin-induced lymphatic endothelial barrier dysfunction and its related effects. Notably, periostin- CAFs were incapable of impairing endothelial barrier integrity, which may explain the occurrence of CAF-enriched cases without LNM. In conclusion, we identified a specific periostin+ CAF subset that promotes LNM in CSCC, mainly by impairing the lymphatic endothelial barriers, thus providing the basis for potential stromal fibroblast-targeted interventions that block CAF-dependent metastasis.

Adipose-derived mesenchymal stem cells differentiate into pancreatic cancer-associated fibroblasts invitro.

Cancer-associated fibroblasts (CAFs) are key components of the dense, proliferating stroma observed in pancreatic ductal adenocarcinoma (PDAC), and CAF subpopulations drive tumor heterogeneity and play a major role in PDAC progression and drug resistance. CAFs consist of heterogenous subpopulations such as myoblastic CAF (myCAF) and inflammatory CAF (iCAF), and each has distinct essential roles. However, it is not clear how CAF subpopulations are formed in PDAC. Adipose-derived MSCs (AD-MSCs), which possess a high multilineage potential and self-renewal capacity, are reported to be one of the invivo CAF sources. Here, we aimed to investigate whether AD-MSCs can act as precursors for CAFs invitro. We recorded morphological features and collected omics data from two invitro co-culture models for recapitulating clinical PDAC. Additionally, we tested the advantages of the co-culture model in terms of accurately modeling morphology and CAF heterogeneity. We showed that AD-MSCs differentiate into two distinct CAF subpopulations: Direct contact co-culture with PDAC cell line Capan-1 induced differentiation into myCAFs and iCAFs, while indirect co-culture induced differentiation into only iCAFs. Using these co-culture systems, we also identified novel CAF markers that may be helpful for elucidating the mechanisms of CAFs in the tumor microenvironment (TME). In conclusion, AD-MSCs can differentiate into distinct CAF subtypes depending on the different co-culture conditions invitro, and the identification of potential CAF markers may aid in future investigations of the mechanisms underlying the role of CAFs in the TME.

Cancer-Associated Fibroblasts: Versatile Players in the Tumor Microenvironment

Simple SummaryCancer-associated fibroblasts (CAFs) are key players in the tumor microenvironment. They are responsible for potentiating growth and metastasis through versatile functions, including maintenance of the extracellular matrix, blood vessel formation, modulation of tumor metabolism, suppression of antitumor immunity, and promotion of chemotherapy resistance. As such, CAFs are associated with poor prognosis and have emerged as a focus of anticancer research. In this review, we discuss the origins of CAFs, their heterogenous subtypes and their properties. We then detail the current state of preclinical and clinical research targeting CAF activities. We believe the limited efficacy of current cancer therapeutic approaches is driven by an incomplete understanding of CAF functions and by a nonstandardized CAF classification system. Therefore, we suggest a unified CAF classification based on specific functions to develop a new class of therapies that will focus on targeting the pro-tumorigenic properties of CAFs during tumor progression.Cancer-associated fibroblasts (CAFs) are indispensable architects of the tumor microenvironment. They perform the essential functions of extracellular matrix deposition, stromal remodeling, tumor vasculature modulation, modification of tumor metabolism, and participation in crosstalk between cancer and immune cells. In this review, we discuss our current understanding of the principal differences between normal fibroblasts and CAFs, the origin of CAFs, their functions, and ultimately, highlight the intimate connection of CAFs to virtually all of the hallmarks of cancer. We address the remarkable degree of functional diversity and phenotypic plasticity displayed by CAFs and strive to stratify CAF biology among different tumor types into practical functional groups. Finally, we summarize the status of recent and ongoing trials of CAF-directed therapies and contend that the paucity of trials resulting in Food and Drug Administration (FDA) approvals thus far is a consequence of the failure to identify targets exclusive of pro-tumorigenic CAF phenotypes that are mechanistically linked to specific CAF functions. We believe that the development of a unified CAF nomenclature, the standardization of functional assays to assess the loss-of-function of CAF properties, and the establishment of rigorous definitions of CAF subpopulations and their mechanistic functions in cancer progression will be crucial to fully realize the promise of CAF-targeted therapies.

Abstract 5719: Tumor-suppressive stromal activity of pro-fibrogenic microRNA-21 in initiation and progression of K-Ras-driven mouse models of pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a recalcitrant and lethal disease with an overall 5-year survival rate of less than 8%. The abundance and heterogeneity of cancer-associated fibroblasts (CAFs) in a highly desmoplastic tumor microenvironment are unique features of PDAC. Therapeutic strategies aimed at indiscriminately eliminating CAFs have failed in clinical trials. The opposing tumor-restraining or tumor-promoting role of distinct CAFs subpopulations of has emerged as a new paradigm in PDAC basic and translational research. Understanding how the composition of these distinct CAF subpopulations is affected by and interferes with current treatments is a key question to solve in order to obtain more effective and durable clinical responses. Genetic studies in K-Ras-driven genetically engineered mouse models (GEMMs) indicate that α-smooth muscle actin-expressing (SMA+) CAFs have a predominant tumor-restraining role. Here, we studied global loss of pro-fibrotic non-coding regulatory microRNA-21 (miR-21) in PDAC GEMMs. We generated mouse strains carrying wild type or knockout alleles of Mir-21 in a well-characterized K-Ras-driven, p53-deleted PDAC model (LSL-KrasG12D; p53lox/+;Pdx1-Cre, KPC). Even though miR-21 expression was upregulated in cancer cells and cancer-associated fibroblasts in KPC tumors, global loss of miR-21 activity did not inhibit tumor growth. Strikingly, loss of miR-21 accelerated tumor initiation via mucinous cystic neoplastic lesions and progression to locally advanced invasive carcinoma from which animals precipitously succumbed at an early age. We uncovered a cell-autonomous requirement of miR-21 activity for induction of tumor-restraining SMA+ CAFs, whose absence led to a profound remodeling of the stroma and massive infiltrate of tumor-promoting immune cells. Low miR-21 expression negatively correlated with a fibroblast gene expression signature and positively with an immune cell gene expression signature in TCGA PDAC data set (n = 156) mirroring findings in GEMMs. In vitro and in vivo isograft experiments showed that miR-21 activity was dispensable for cancer cell growth, but required for complete execution of TGF-β-mediated programs. Our results suggest that modulation of miR-21 activity may provide a novel therapeutic opportunity to affect the composition of CAF subpopulations. We will present our genetic and pharmacological approaches to modulate stromal activity of miR-21 in established disease in order to enhance current chemotherapy and/or immunotherapy treatments. Citation Format: Josh Schipper, Brooke Jackson, Ian Beddows, Elizabeth Kenyon, Katie Powell, Neil Robertson, Galen Hostetter, Matti Kiupel, Anna Moore, Jose Conejo-Garcia, Lorenzo sempere. Tumor-suppressive stromal activity of pro-fibrogenic microRNA-21 in initiation and progression of K-Ras-driven mouse models of pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5719.

Abstract 2757: Analysis of stromal myofibroblasts identifies secreted proteins that promote pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most incurable types of cancer. Cancer that develops in the acinar cells of the pancreas is typically not diagnosed until later stages, such as stage 3 or 4. As such, this form of cancer is particularly lethal with only 9% of patients reaching 5-year survival. PDAC is known to have a particularly dense extracellular matrix composed of fibroblasts, which have been previously shown to play an important role in promoting resistance to drug therapy. Characterization of the stromal networks involved in PDAC tumor development as well as protein markers of fibroblast subpopulations within the tumor stroma are critical to developing new fibroblast-targeted therapeutic approaches as well as understanding key signaling molecules that ultimately promote tumor progression and drug resistance. Single-cell RNA sequencing (scRNAseq) was utilized to analyze cancer-associated fibroblasts (CAFs) from KPC mouse-derived, MT3 subcutaneous murine allografts along with two fibroblast lines derived from human PDAC tumors: CRC-811 and IA-1340. Single cell gene expression profiling and subsequent analysis of MT3-derived CAFs resulted in the identification of three CAF subpopulations including a myofibroblast subpopulation that expressed high levels of smooth muscle actin (Acta2), which was also observed in both human samples. Fibroblast subpopulations enriched in Acta2 expression, expressed high levels of Wnt5a along with several other secreted factors including Tgfb1, Tgfb2 and Ctgf. Wnt5a is a secreted protein that activates non-canonical Wnt signaling pathways and is known to regulate normal developmental processes, including proliferation, differentiation, migration, adhesion and polarity. However Wnt5a is not natively expressed in MT3 cancer cells derived from syngeneic tumors, but potentially in the stroma. We hypothesize it is exclusively derived from fibroblasts. Previously it has been shown that Wnt5a inhibition suppressed gastric cancer metastasis, therefore further validation of the role of myofibroblast-derived Wnt5a on PDAC disease progression is warranted. This study received funding by LDRD 19-SI-003. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). IM Release Number: LLNL-ABS-798442. Citation Format: Kelly A. Martin, Aimy Sebastian, Nicholas R. Hum, Stephen Byers, Elizabeth K. Wheeler, Matthew A. Coleman, Gabriela G. Loots. Analysis of stromal myofibroblasts identifies secreted proteins that promote pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2757.

Cancer-associated fibroblast compositions change with breast cancer progression linking the ratio of S100A4+ and PDPN+ CAFs to clinical outcome.

Tumors are supported by cancer-associated fibroblasts (CAFs). CAFs are heterogeneous and carry out distinct cancer-associated functions. Understanding the full repertoire of CAFs and their dynamic changes as tumors evolve could improve the precision of cancer treatment. Here we comprehensively analyze CAFs using index and transcriptional single-cell sorting at several time points along breast tumor progression in mice, uncovering distinct subpopulations. Notably, the transcriptional programs of these subpopulations change over time and in metastases, transitioning from an immunoregulatory program to wound-healing and antigen-presentation programs, indicating that CAFs and their functions are dynamic. Two main CAF subpopulations are also found in human breast tumors, where their ratio is associated with disease outcome across subtypes and is particularly correlated with BRCA mutations in triple-negative breast cancer. These findings indicate that the repertoire of CAF changes over time in breast cancer progression, with direct clinical implications.

Single-Cell Transcriptomic Analysis of Tumor-Derived Fibroblasts and Normal Tissue-Resident Fibroblasts Reveals Fibroblast Heterogeneity in Breast Cancer.

Cancer-associated fibroblasts (CAFs) are a prominent stromal cell type in solid tumors and molecules secreted by CAFs play an important role in tumor progression and metastasis. CAFs coexist as heterogeneous populations with potentially different biological functions. Although CAFs are a major component of the breast cancer stroma, molecular and phenotypic heterogeneity of CAFs in breast cancer is poorly understood. In this study, we investigated CAF heterogeneity in triple-negative breast cancer (TNBC) using a syngeneic mouse model, BALB/c-derived 4T1 mammary tumors. Using single-cell RNA sequencing (scRNA-seq), we identified six CAF subpopulations in 4T1 tumors including: 1) myofibroblastic CAFs, enriched for α-smooth muscle actin and several other contractile proteins; 2) ‘inflammatory’ CAFs with elevated expression of inflammatory cytokines; and 3) a CAF subpopulation expressing major histocompatibility complex (MHC) class II proteins that are generally expressed in antigen-presenting cells. Comparison of 4T1-derived CAFs to CAFs from pancreatic cancer revealed that these three CAF subpopulations exist in both tumor types. Interestingly, cells with inflammatory and MHC class II-expressing CAF profiles were also detected in normal breast/pancreas tissue, suggesting that these phenotypes are not tumor microenvironment-induced. This work enhances our understanding of CAF heterogeneity, and specifically targeting these CAF subpopulations could be an effective therapeutic approach for treating highly aggressive TNBCs.

Cancer-associated fibroblasts in cholangiocarcinoma.

To give a state-of-art knowledge regarding cancer-associated fibroblasts (CAF) in cholangiocarcinoma (CCA) based both on direct evidence and studies on other desmoplastic cancers. High contingency of CAF characterizes CCA, a tumor with a biliary epithelial phenotype that can emerge anywhere in the biliary tree. Current treatments are very limited, the surgical resection being the only effective treatment but restricted to a minority of patients, whereas the remaining patients undergo palliative chemotherapy regimens. In cancer, CAF shape the tumor microenvironment, drive cancer growth and progression, and contribute to drug resistance. All these functions are accomplished through an interplay network between CAF and surrounding cells including tumor and other stromal cells, i.e. immune and endothelial cells. Several studies have pointed out the existence of CAF sub-populations carrying out several and opposite functions, cancer-promoting or cancer-restraining as shown in pancreatic cancer, another prototypic desmoplastic tumor in which heterogeneity of CAF is well demonstrated. New CAF functions are now emerging in pancreatic and breast cancers like the modulation of immune responses or tumor metabolism, opening new area for treatments.

Abstract PL1: Stromal Cells and Matrix Remodeling as Essential Regulators of the Tumor Microenvironment

Abstract Carcinomas represent complex neo-organs in which extensive communication networks between surrounding non-transformed endothelial, neural, inflammatory and immune cells provide biochemical signals that govern the behavior of malignant epithelial cells. Moreover, the initiation, progression and metastatic spread of malignant cells is in many human carcinomas associated with desmoplasia. Desmoplasia is typically associated with poor prognosis and in many contexts, confers resistance to therapy. Desmoplasia is characterized by activation of mesenchymal-derived stromal cells such as adipocytes and cancer associated fibroblasts (CAFs) and dynamic remodeling of the extracellular matrix that underlies the dramatic physical differences of tumor tissue compared to corresponding normal tissue that result in further alternations in biochemical signals, contribute to a pro-tumorigenic metabolic milieu as well as alterations in biomechanical signals. Recent studies indicate that fibroblasts also play a critical role in regulating inflammatory and immune cell infiltration, intra-tumoral migration and function in primary tumors and metastatic disease. Recent studies have established that the stromal cell component is heterogeneous with subpopulations of CAFs and specific CAF-mediated pathways that either restrain or promote tumor progression. Genetic and immune-based therapy approaches have identified a subset of Fibroblast Activation Protein (FAP)-expressing tumor promoting CAFs in pre-clinical models of multiple types of carcinoma and FAP -expression correlates with worse survival of patients with a variety of carcinomas including breast cancer. Studies demonstrating that FAP+ CAFs promote tumorigenesis through both immune-dependent and immune-independent mechanisms in pre-clinical models will be presented that support efforts to develop FAP+ cell-targeted therapies in combination with cancer cell targeted therapies. In an effort to refine our approach to therapeutically targeting stroma-dependent mechanisms from a cellular to a molecular level, we have also identified the proteolytic activity of FAP, a cell surface serine protease, as a critical molecular pathway by which FAP+ cells exert their pro-tumorogenic effects. We previously demonstrated in models of lung, colon, and pancreatic cancer, that FAP-null mice are protected from tumorigenesis, progression and metastasis. We recently expanded these results to pre-clinical models of breast cancer and investigated whether stroma-dependent mechanisms contribute to the increased risk of breast cancer incidence and/or progression associated with obesity. Using the spontaneous MMTV-neu murine breast cancer model, we found that FAP-null mice showed delayed primary tumorigenesis. In vitro spheroid formation assays revealed a role for FAP+/+ cells in supporting tumorigenesis, while experiments using primary stromal cell-derived matrices (CDMs) also established a role for FAP-mediated matrix remodeling in promoting cancer cell growth. Taken together, these data suggest that stromal cell-expressed FAP has multiple pro-tumorigenic functions in the context of early breast cancer. Citation Format: E Pure. Stromal Cells and Matrix Remodeling as Essential Regulators of the Tumor Microenvironment [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PL1.