Cancer-associated Fibroblasts Subtypes Research Articles

Abstract 3645: 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 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, suggesting little contribution of epithelium to the CAF pool. To determine whether the bone marrow gives rise to CAFs, we transplanted donor bone marrow carrying a ubiquitously expressed GFP reporter allele to GFP-negative recipient mice. We found that only a small portion 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 TRFs 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 were persistent in subsets of CAFs in both the PDAC mouse model and human patient samples. In summary, we found that bone marrow contributes to a small portion of CAFs in PDAC, and the pancreatic epithelium contributes even less. 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. This study provides approaches to robustly target CAFs in vivo, and novel insights into CAF origin and heterogeneity in PDAC. Citation Format: Lu Han, Yongxia Wu, Melodie Parrish, Khushbu Patel, Tony Trimboli, Julia Lefler, Xuezhong Yu, 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 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3645.

Abstract 1563: Spatial transcriptomics reveals heterogeneity and pathway dependencies of cancer associated fibroblasts in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDA) is a deadly disease characterized by an immunosuppressive microenvironment and a dense stroma that encapsulates the tumor, making therapeutic targeting particularly challenging. One of the main cellular components of PDA tumor microenvironment (TME) is cancer associated fibroblasts (CAFs), whose function is to sustain tumor growth and provide structural support to the TME. Previously, we established the existence of three transcriptional subtypes of CAFs, myCAF, iCAF and apCAF, each with distinct functions and location in respect to cancer cells. Two of these subtypes, myCAF and iCAF, display a highly plastic potential in vitro and can transdifferentiate into one another upon activation of specific signaling pathways. The plasticity observed supports the existence of transitional CAF sub-states that have previously eluded observation due to technical limitations to their isolation. Through a combination of spatial transcriptomics and fluorescent in situ hybridization (FISH) on tumor samples obtained from KPC (KrasLSL-G12D; p53LSL-R172H; PDX-CRE) and FPC (KrasFrt-LSL-G12V-Frt; p53LSL-R172H; PDX-CRE; Rosa26FlpOERT2) mice, two PDA mouse models displaying a Kras G12D and G12V mutation respectively, we were able to confirm the presence of the previously identified CAF subtypes and two additional CAF sub-states. These sub-states are associated with the expression of specific markers and display different pathway enrichment. Notably, Kras G12D and G12V mutations generate similar organization of the stroma and heterogeneity of the CAF population. Subsequently, to understand the contribution of activating Kras mutation to these CAF phenotypes, we utilized the unique feature of the FPC mouse model which allows the excision of Kras G12V once the tumor has fully developed. Through FISH analysis, we observed deep reorganization of the stroma and a shift in marker expressions within the CAF populations upon Kras excision. Overall, the data here presented offers insight into the diversity of PDA CAFs and the role of mutated Kras in regulating CAF subtypes and stromal organization. Citation Format: Giuseppina Caligiuri, Jennifer Thalappillil, Juliene Hinds, Elise T. Courtois, William F. Flynn, Paul Robson, Alexander Dobin, Youngkyu Park, David A. Tuveson. Spatial transcriptomics reveals heterogeneity and pathway dependencies of cancer associated fibroblasts in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1563.

Abstract 3198: Validation and analysis of cancer associated fibroblast subtype markers in metastatic colorectal cancer

Abstract Background: Cancer Associated Fibroblasts (CAFs) are a significant component of tumor stroma, and have an important impact on immune infiltration in the tumor microenvironment (TME). Two major subtypes of CAFs have been previously identified by literature: myofibroblastic (myCAF) and inflammatory (iCAF). Our lab has identified subtype markers for each CAF phenotype and previously analyzed a sampling of 153 colorectal cancer (CRC) patients. Here, we validate these subtype markers and investigate CAF phenotypes in metastatic colorectal cancer patients. Methods: Dual immunofluorescence on formalin fixed paraffin embedded tissue sections was performed to analyze co-staining between combinations of myCAF markers, αSMA and TAGLN, and iCAF markers, PDPN and ICAM1. Slides were imaged using a fluorescent microscope. Also, tissue microarrays sampling 212 CRC patients spanning all stages of disease, 90 with matched metastatic cores, were stained via immunohistochemistry (IHC) for the CAF subtype markers then quantified on an intensity scale from 0-3+. iCAF and myCAF marker scores were averaged to get a composite score for each, then split into low (average score <2) and high (average score ≥2) groups. CD8 IHC stains were quantified as the number of tumor infiltrating lymphocytes (TILs) per high power field (HPF) in the epithelial compartment. Results: Significant co-staining was observed between iCAF markers PDPN and ICAM1, as well as myCAF markers αSMA and TAGLN. Co-staining did not occur, or was minimal, between combinations of myCAF and iCAF markers. There is not significant different in abundance of iCAFs or myCAFs in primary site cores of patients with metastatic versus non-metastatic disease (p = 0.67 for iCAF, p = 0.57 for myCAF). Of matched primary and metastatic samples able to be scored, 43.3% of samples had a decrease in iCAF score from primary to metastatic site while only 18.8% increased. Overall, 34.4% of samples had a decrease in score of more than 1 and only 2.2% of samples had an increase of more than 1. However, the percentage of samples that had a decrease in myCAF score was 32.2% while 22.2% increased. In all primary cores of patients with metastatic disease, there was higher average CD8+ TILs in those with high iCAF scores compared to those with low iCAF scores (12.0 vs 5.5, p=0.03). There was not a significant difference in average CD8+ TILs in those with high myCAF scores compared to those with low myCAF scores (9.3 vs 7.1, p=0.7). Conclusions: Here, we validate the myCAF markers TAGLN and αSMA, as well as, iCAF markers ICAM1 and PDPN by demonstrating co-staining between CAFs of the same subtype and exclusion between different subtypes. These data indicate that that CAF phenotype correlates with CD8 T cell infiltration into the TME. iCAFs correlate with immune infiltration and myCAFs with immune exclusion. Citation Format: Anna L. Lippert, Katherine A. Johnson, Cheri A. Pasch, Sean G. Kraus, Philip B. Emmerich, Linda Clipson, Kristina A. Matkowskyj, Wei Zhang, Dustin A. Deming. Validation and analysis of cancer associated fibroblast subtype markers in metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3198.

Comprehensive Integrated Single-Cell Whole Transcriptome Analysis Revealed the p-EMT Tumor Cells—CAFs Communication in Oral Squamous Cell Carcinoma

Cancer-associated fibroblasts (CAFs) and partial epithelial–mesenchymal transition (p-EMT) tumor cells are closed together and contribute to the tumor progression of oral squamous cell carcinoma (OSCC). In the present study, we deeply analyzed and integrated OSCC single-cell RNA sequencing datasets to define OSCC CAFs and p-EMT subpopulations. We highlighted the cell–cell interaction network of CAFs and p-EMT tumor cells and suggested biomarkers for the diagnosis and prognosis of OSCC during the metastasis condition. The analysis discovered four subtypes of CAFs: one p-EMT tumor cell population, and cycling tumor cells as well as TNFSF12-TNFRSF25/TNFRSF12A interactions between CAFs and p-EMT tumor cells during tumor metastasis. This suggests the prediction of therapeutically targetable checkpoint receptor–ligand interactions between CAFs and p-EMT tumor cells in OSCC regarding the metastasis status.

Cancer-associated fibroblasts in pancreatic cancer: new subtypes, new markers, new targets.

Cancer‐associated fibroblasts (CAFs) have conflicting roles in the suppression and promotion of cancer. Current research focuses on targeting the undesirable properties of CAFs, while attempting to maintain tumour‐suppressive roles. CAFs have been widely associated with primary or secondary therapeutic resistance, and strategies to modify CAF function have therefore largely focussed on their combination with existing therapies. Despite significant progress in preclinical studies, clinical translation of CAF targeted therapies has achieved limited success. Here we will review our emerging understanding of heterogeneous CAF populations in tumour biology and use examples from pancreatic ductal adenocarcinoma to explore why successful clinical targeting of protumourigenic CAF functions remains elusive. Single‐cell technologies have allowed the identification of CAF subtypes with a differential impact on prognosis and response to therapy, but currently without clear consensus. Identification and pharmacological targeting of CAF subtypes associated with immunotherapy response offers new hope to expand clinical options for pancreatic cancer. Various CAF subtype markers may represent biomarkers for patient stratification, to obtain enhanced response with existing and emerging combinatorial therapeutic strategies. Thus, CAF subtyping is the next frontier in understanding and exploiting the tumour microenvironment for therapeutic benefit. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The Role of Cancer-Associated Fibroblasts in Ovarian Cancer.

Simple SummaryOvarian cancer is a lethal gynecologic tumor and is generally resistant to conventional treatments. Stable cancer-associated fibroblasts (CAFs) are important cellular components in the ovarian cancer tumor microenvironment and may provide novel resources for future treatment strategies. Different subtypes of CAFs display specific functions in tumor pathogenesis and various CAF markers suggest potential treatment targets. Several clinical or preclinical trials have targeted stromal fibroblasts and focused on the properties of CAFs to enhance ovarian cancer treatment efficacy. This review concentrates on the origins, subtypes, and activation of CAFs, as well as specific roles of CAFs in regulating tumor development and drug resistance, and aims to provide potential and prospective targets for improving the therapeutic efficacy of ovarian cancer treatment.Ovarian cancer is a lethal gynecologic tumor and is generally resistant to conventional treatments. Stable cancer-associated fibroblasts (CAFs) are important cellular components in the ovarian cancer tumor microenvironment and may provide novel resources for future treatment strategies. Different subtypes of CAFs display specific functions in tumor pathogenesis and various CAF markers suggest potential treatment targets, such as FAP and GPR77. Both autocrine and paracrine cytokines play important roles in the CAF activation process and regulate tumor progression. Downstream mediators and pathways, including IL-6, TGF-β, NF-κB, mitogen-activated protein kinase (MAPK), and AKT/mTOR/(p70S6K), play important roles in the initiation, proliferation, invasiveness, and metastasis of ovarian cancer cells and also participate in angiogenesis, therapeutic resistance, and other biological processes. Several clinical or preclinical trials have targeted stromal fibroblasts and focused on the properties of CAFs to enhance ovarian cancer treatment outcomes. This review concentrates on the origins, subtypes, and activation of CAFs, as well as specific roles of CAFs in regulating tumor development and drug resistance, and aims to provide potential and prospective targets for improving the therapeutic efficacy of ovarian cancer treatment.

Tumor-Stroma Interaction in PDAC as a New Approach for Liquid Biopsy and its Potential Clinical Implications.

The extremely poor prognosis for patients with pancreatic ductal adenocarcinoma (PDAC) has remained unchanged for decades. As a hallmark of PDAC histology, the distinct desmoplastic response in the tumor microenvironment is considered a key factor exerting pro- and antitumor effects. Increasing emphasis has been placed on cancer-associated fibroblasts (CAFs), whose heterogeneity and functional diversity is reflected in the numerous subtypes. The myofibroblastic CAFs (myCAFs), inflammatory CAFs (iCAFs) and antigen presenting CAFs (apCAFs) are functionally divergent CAF subtypes with tumor promoting as well as repressing effects. Precise knowledge of the underlying interactions is the basis for a variety of treatment approaches, which are subsumed under the term antistromal therapy. Clinical implementation is still pending due to the lack of benefit—as well as paradoxical preclinical findings. While the prominent significance of CAFs in the immediate environment of the tumor is becoming clear, less is known about the circulating (c)CAFs. cCAFs are of particular interest as they seem not only to be potential new liquid biopsy biomarkers but also to support the survival of circulating tumor cells (CTC) in the bloodstream. In PDAC, CTCs correlate with an unfavorable outcome and can also be employed to monitor treatment response, but the current clinical relevance is limited. In this review, we discuss CTCs, cCAFs, secretomes that include EVs or fragments of collagen turnover as liquid biopsy biomarkers, and clinical approaches to target tumor stroma in PDAC.

Abstract A029: Identification of tumor microenvironment components in patient-derived pancreatic ductal adenocarcinoma organoids

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is highly drug resistant with no change in therapeutic approach for the last decade. The PDAC tumor microenvironment (TME) is highly desmoplastic and associated with altered treatment response. Our group previously identified 9 distinct cell types in human PDAC tumors using single-cell RNA-sequencing, demonstrating a diverse TME. However, the TME of in vitro organoid PDAC models remains underexplored. We initiated characterization of the TME from PDAC patient derived organoids (PDOs) to establish a working model for accurate drug sensitivity testing. Methods: We performed immunofluorescence (IF) staining on whole-mount early passage (<P4) PDO lines and corresponding paraffin-embedded primary PDAC specimens to evaluate for activated cancer-associated fibroblasts (CAFs) using antibodies against α-smooth muscle actin (α-SMA) and fibroblast activated protein (FAP); and to evaluate for T-regulatory (Treg) cells with antibodies against interleukin-2 receptor α (IL-2Rα) and forkhead box protein P3 (FOXP3). Results: CAFs promote an immunosuppressive TME in PDAC and have recently been subclassified into inflammatory fibroblasts and myofibroblasts, which can both restrain and promote tumor progression. In this study, we identified three distinct subtypes of CAFs in PDOs, which are consistent with the subtypes found in matched clinical tumor specimens: α-SMA+, which has been reported to be indicative of pancreatic stellate cell activation and associated with increased desmoplasia; FAP+, which influences cancer cell motility, invasion, and cycle progression, extracellular matrix deposition, and angiogenesis within the TME; and α-SMA+/FAP+, which is a myofibroblast subtype. We additionally identified Treg populations in PDOs with peripheral IL-2Rα and central FOXP3 expression in the organoid spheres. Tregs have previously been shown to be overexpressed in the PDAC TME, making this finding clinically relevant. Conclusions: In this preliminary study, we show that PDAC PDOs demonstrate TME components that mirror primary tumor specimens, including CAFs and Tregs. The demonstration of a tumor-matched, immunocompetent TME in PDAC PDOs provides a more clinically relevant in vitro tumor model for patient-matched drug sensitivity testing. Citation Format: Mei Gao, Charles J. Bailey, Megan M. Harper, Michael J. Cavnar, Prakash Pandalai, Shadi A. Qasem, Joseph Kim. Identification of tumor microenvironment components in patient-derived pancreatic ductal adenocarcinoma organoids [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A029.

Refining colorectal cancer classification and clinical stratification through a single-cell atlas

BackgroundColorectal cancer (CRC) consensus molecular subtypes (CMS) have different immunological, stromal cell, and clinicopathological characteristics. Single-cell characterization of CMS subtype tumor microenvironments is required to elucidate mechanisms of tumor and stroma cell contributions to pathogenesis which may advance subtype-specific therapeutic development. We interrogate racially diverse human CRC samples and analyze multiple independent external cohorts for a total of 487,829 single cells enabling high-resolution depiction of the cellular diversity and heterogeneity within the tumor and microenvironmental cells.ResultsTumor cells recapitulate individual CMS subgroups yet exhibit significant intratumoral CMS heterogeneity. Both CMS1 microsatellite instability (MSI-H) CRCs and microsatellite stable (MSS) CRC demonstrate similar pathway activations at the tumor epithelial level. However, CD8+ cytotoxic T cell phenotype infiltration in MSI-H CRCs may explain why these tumors respond to immune checkpoint inhibitors. Cellular transcriptomic profiles in CRC exist in a tumor immune stromal continuum in contrast to discrete subtypes proposed by studies utilizing bulk transcriptomics. We note a dichotomy in tumor microenvironments across CMS subgroups exists by which patients with high cancer-associated fibroblasts (CAFs) and C1Q+TAM content exhibit poor outcomes, providing a higher level of personalization and precision than would distinct subtypes. Additionally, we discover CAF subtypes known to be associated with immunotherapy resistance.ConclusionsDistinct CAFs and C1Q+ TAMs are sufficient to explain CMS predictive ability and a simpler signature based on these cellular phenotypes could stratify CRC patient prognosis with greater precision. Therapeutically targeting specific CAF subtypes and C1Q + TAMs may promote immunotherapy responses in CRC patients.

MRNA Subtype of Cancer-Associated Fibroblasts Significantly Affects Key Characteristics of Head and Neck Cancer Cells.

Simple SummaryThe interplay between cancer-associated fibroblasts (CAFs) and head and neck cancer cells and its effect on the metabolic state, mechanobiology, and aggressiveness of cancer cells is described. In vitro coculture techniques were used. Not all CAFs support tumours–the right kind of cooperation between cancer cells and CAFs is needed for tumour growth and progression, and only specific mRNA subtypes of CAFs can support the growth of primary cancer cells or metastases carrying different mutational statuses.Head and neck squamous cell carcinomas (HNSCC) belong among severe and highly complex malignant diseases showing a high level of heterogeneity and consequently also a variance in therapeutic response, regardless of clinical stage. Our study implies that the progression of HNSCC may be supported by cancer-associated fibroblasts (CAFs) in the tumour microenvironment (TME) and the heterogeneity of this disease may lie in the level of cooperation between CAFs and epithelial cancer cells, as communication between CAFs and epithelial cancer cells seems to be a key factor for the sustained growth of the tumour mass. In this study, we investigated how CAFs derived from tumours of different mRNA subtypes influence the proliferation of cancer cells and their metabolic and biomechanical reprogramming. We also investigated the clinicopathological significance of the expression of these metabolism-related genes in tissue samples of HNSCC patients to identify a possible gene signature typical for HNSCC progression. We found that the right kind of cooperation between cancer cells and CAFs is needed for tumour growth and progression, and only specific mRNA subtypes can support the growth of primary cancer cells or metastases. Specifically, during coculture, cancer cell colony supporting effect and effect of CAFs on cell stiffness of cancer cells are driven by the mRNA subtype of the tumour from which the CAFs are derived. The degree of colony-forming support is reflected in cancer cell glycolysis levels and lactate shuttle-related transporters.

Identification of Cancer-Associated Fibroblast Subtype of Triple-Negative Breast Cancer

Background There is limited knowledge about the role of cancer-associated fibroblasts (CAF) in the tumor microenvironment of triple-negative breast cancer (TNBC). Methods Three hundred and thirty-five TNBC samples from four datasets were retrieved and analyzed. In order to determine the CAF subtype by combining gene expression profiles, an unsupervised clustering analysis was adopted. The prognosis, enriched pathways, immune cells, immune scores, and tumor purity were compared between CAF subtypes. The genes with the highest importance were selected by bioinformatics analysis. The machine learning model was built to predict the TNBC CAF subtype by these selected genes. Results TNBC samples were classified into two CAF subtypes (CAF+ and CAF-). The CAF- subtype of TNBC was linked to the longer overall survival and more immune cells than the CAF+ subtype. CAF- and CAF+ were enriched in immune-related pathways and extracellular matrix pathways, respectively. Bioinformatics analysis identified 9 CAF subtype-related markers (ADAMTS12, AEBP1, COL10A1, COL11A1, CXCL11, CXCR6, EDNRA, EPPK1, and WNT7B). We constructed a robust random forest model using these 9 genes, and the area under the curve (AUC) value of the model was 0.921. Conclusion The current study identified CAF subtypes based on gene expression profiles and found that CAF subtypes have significantly different overall survival, immune cells, and immunotherapy response rates.

Research Status of Tumor-associated Fibroblasts Regulating Immune Cells

肿瘤相关成纤维细胞（cancer-associated fibroblasts, CAFs）和肿瘤浸润性免疫细胞是肿瘤微环境（tumor microenvironment, TME）中重要的组成成分。二者在肿瘤微环境中相互通信，在肿瘤的发生和发展中发挥着重要作用。CAFs具有很大的异质性，不同的CAFs亚群存在着不同的功能。同时其又可以通过分泌多种细胞因子、趋化因子等方式实现对肿瘤浸润性免疫细胞的调节，从而进一步影响肿瘤的发生、发展、侵袭、转移等生物学行为。本文对国内外近年来有关CAFs在TME中对浸润性免疫细胞调节作用的研究现状作一综述。

Subpopulations of cancer-associated fibroblasts link the prognosis and metabolic features of pancreatic ductal adenocarcinoma.

BackgroundCancer-associated fibroblasts (CAFs) are a vital constituent of the tumor microenvironment (TME) and have several activities, but the effect of CAF heterogeneity on the molecular features and clinical outcomes of pancreatic ductal adenocarcinoma (PDAC) remains unknown.MethodsAn algorithm “scFrac” based on single-cell sequencing data from the Gene Expression Omnibus was introduced to emulate the enrichment of CAF subtypes in a TCGA-PDAC cohort and their prognostic influence, and confirmed by an external validation group (66 patients with PDAC) with multiplex immunohistochemistry staining. A comprehensive analysis including metabolic profile and transcription factor regulon activity was carried out among CAF subtypes.ResultsThree distinct CAF populations were confirmed: myofibroblast (myCAF), inflammatory CAF (iCAF), and antigen-presenting CAF (apCAF). These subtypes expressed distinct metabolic profiles and transcriptional regulon activity. KEGG pathway annotation demonstrated that complement and coagulation cascades, as well as cytokine-cytokine receptor interaction were dominant in iCAFs, and pathways related to focal adhesion, and ECM-receptor interaction showed dominance in myCAFs, while antigen processing and presentation were the top enriched pathways in apCAFs. iCAFs trended to glycolysis with CREB3L1, EGR2 and SOX4 activation, whereas myCAFs depend on the tricarboxylic acid cycle and its derivatives with NRF2, CEBPD and YBX1 activation. iCAF is a protective factor associated with an inflammatory phenotype, but myCAF is an important factor in the poor prognosis of PDAC.ConclusionsWe identified distinct molecular characteristics of 3 CAF subtypes in PDAC and plotted their metabolism profile. We introduced a novel algorism, scFrac, for exploring how CAF subgroups dysregulate cancer biology, and also shed a new therapeutic light on targeting the CAF subtype in TME.

Heterogeneity of CAFfeinated Tumors: Sweet Targeting Perspectives.

The tumor microenvironment (TME) shows heterogeneity within a tumor. An important element of the TME is cancer-associated fibroblasts (CAF). In this issue of Cancer Research, Bouchard and colleagues investigate the heterogeneity of CAFs from spatially different zones of lung adenocarcinoma resection specimens. Multiomics analysis revealed changes in the O-glycoproteome, unique to CAFs from the tumor edge, an effect reinforced by contact with cancer cells. This O-glycoprotein signature offers unique targeting perspectives that reciprocally affect cancer cell epithelial-to-mesenchymal plasticity, a key mechanism of cancer progression. Deeper understanding of the cancer-stimulating and cancer-inhibiting role of CAF subtypes will facilitate the development of CAF-directed therapeutic approaches. See related article by Bouchard et al., p. 648.

Cancer-associated fibroblasts as accomplices to confer therapeutic resistance in cancer.

The "seed and soil" concept has reformed paradigms for cancer treatment in the past decade. Accumulating evidence indicates that the intimate crosstalk between cancer cells and stromal cells plays a tremendous role in tumor progression. Cancer-associated fibroblasts (CAFs), the largest population of stroma cells, influence therapeutic effects through diverse mechanisms. Herein, we summarize the recent advances in the versatile functions of CAFs regarding their heterogeneity, and we mainly discuss the pro-tumorigenic functions of CAFs which promote tumorigenesis and confer therapeutic resistance to tumors. Targeting CAFs is emerging as one of the most appealing strategies in anticancer therapies. The endeavors to target or reprogram the specific subtypes of CAFs provide great cancer treatment opportunities, which may provide a better clinical benefit to cancer patients.

Integrative analysis deciphers the heterogeneity of cancer-associated fibroblast and implications on clinical outcomes in ovarian cancers

Accumulating evidence has recognized that cancer-associated fibroblasts (CAFs) are major players in the desmoplastic stroma of ovarian cancer, modulating tumor progression and therapeutic response. However, it is unclear regarding the signatures of CAFs could be utilized to predict the clinical outcomes of ovarian cancer patients. To fill in this gap, we explored the intratumoral compartment of ovarian cancer by analyzing the single-cell RNA-sequencing (scRNA-seq) datasets of ovarian carcinoma samples, and identified two distinct CAFs (tumor-promoting CAF_c1 subtype and myofibroblasts-like CAF_c2 subtype). The clinical significance of CAF subtypes was further validated in The Cancer Genomics Atlas (TCGA) database and other independent immunotherapy response datasets, and the results revealed that the patients with a higher expression of CAF_c1 signatures had a worse prognosis and showed a tendency of resistance to immunotherapy. This work uncovered the signatures of the CAF_c1 subtype that could serve as a novel prognostic indicator and predictive marker for immunotherapy.

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.

Targeting Tumor-Stromal Interactions in Pancreatic Cancer: Impact of Collagens and Mechanical Traits.

Pancreatic ductal adenocarcinoma (PDAC) has one of the worst outcomes among cancers with a 5-years survival rate of below 10%. This is a result of late diagnosis and the lack of effective treatments. The tumor is characterized by a highly fibrotic stroma containing distinct cellular components, embedded within an extracellular matrix (ECM). This ECM-abundant tumor microenvironment (TME) in PDAC plays a pivotal role in tumor progression and resistance to treatment. Cancer-associated fibroblasts (CAFs), being a dominant cell type of the stroma, are in fact functionally heterogeneous populations of cells within the TME. Certain subtypes of CAFs are the main producer of the ECM components of the stroma, with the most abundant one being the collagen family of proteins. Collagens are large macromolecules that upon deposition into the ECM form supramolecular fibrillar structures which provide a mechanical framework to the TME. They not only bring structure to the tissue by being the main structural proteins but also contain binding domains that interact with surface receptors on the cancer cells. These interactions can induce various responses in the cancer cells and activate signaling pathways leading to epithelial-to-mesenchymal transition (EMT) and ultimately metastasis. In addition, collagens are one of the main contributors to building up mechanical forces in the tumor. These forces influence the signaling pathways that are involved in cell motility and tumor progression and affect tumor microstructure and tissue stiffness by exerting solid stress and interstitial fluid pressure on the cells. Taken together, the TME is subjected to various types of mechanical forces and interactions that affect tumor progression, metastasis, and drug response. In this review article, we aim to summarize and contextualize the recent knowledge of components of the PDAC stroma, especially the role of different collagens and mechanical traits on tumor progression. We furthermore discuss different experimental models available for studying tumor-stromal interactions and finally discuss potential therapeutic targets within the stroma.

Review: Challenges of In Vitro CAF Modelling in Liver Cancers

Simple SummaryLiver cancer and tumours spreading from other organs to the liver are associated with high death rates. Current treatments include surgical removal of the tumour and chemotherapy. Unfortunately, patients are often re-diagnosed with liver nodules in the years after cessation of the treatment. Therefore, scientists are looking for alternative treatment strategies, and these include targeting the tumour environment. The tumour environment includes the cancer-associated fibroblasts, which could be an interesting target for therapy in combination with current strategies. In this review paper we summarize the current models to investigate the effect of the tumour on the cancer-associated fibroblasts. Not many studies focus on the cancer-associated fibroblasts in non-animal models and this should improve in order to better understand the role of the cancer-associated fibroblasts and to evaluate the potential of cancer-associated fibroblast-directed therapies.Primary and secondary liver cancer are the third cause of death in the world, and as the incidence is increasing, liver cancer represents a global health burden. Current treatment strategies are insufficient to permanently cure patients from this devastating disease, and therefore other approaches are under investigation. The importance of cancer-associated fibroblasts (CAFs) in the tumour microenvironment is evident, and many pre-clinical studies have shown increased tumour aggressiveness in the presence of CAFs. However, it remains unclear how hepatic stellate cells are triggered by the tumour to become CAFs and how the recently described CAF subtypes originate and orchestrate pro-tumoural effects. Specialized in vitro systems will be needed to address these questions. In this review, we present the currently used in vitro models to study CAFs in primary and secondary liver cancer and highlight the trend from using oversimplified 2D culture systems to more complex 3D models. Relatively few studies report on the impact of cancer (sub)types on CAFs and the tumour microenvironment, and most studies investigated the impact of secreted factors due to the nature of the models.

Abstract PR-013: The splanchnic mesenchyme during fetal development is the major source of pancreatic cancer associated fibroblasts

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, suggesting little contribution of epithelium to the CAF pool. To determine whether the bone marrow gives rise to CAFs, we transplanted donor bone marrow carrying a ubiquitously expressed GFP reporter allele to GFP-negative recipient mice. We found that only a small proportion of pancreatic CAFs were tagged with GFP, suggesting their bone marrow origin. Lastly, to determine whether pancreatic TRFs give rise to CAFs, we used an inducible CreER-LoxP system to allow for permanent Tomato labeling in TRFs 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 were persistent in subsets of CAFs in both the PDAC mouse model and human patient samples. In summary, we found that bone marrow contributes to a small proportion of CAFs in PDAC, and the pancreatic epithelium contributes even less. 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. This study provides approaches to robustly target CAFs in vivo and novel insights into CAF heterogeneity in PDAC. Citation Format: Lu Han, Yongxia Wu, Melodie Parrish, Khushbu Patel, Xuezhong Yu, Michael Ostrowski, Gustavo Leone. The splanchnic mesenchyme during fetal development is the major source of pancreatic cancer associated fibroblasts [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-013.