Subset Of Cancer-associated Fibroblasts Research Articles

Interactions between cancer-associated fibroblasts and T-cells: functional crosstalk with targeting and biomarker potential.

Cancer-associated fibroblasts (CAFs) are a heterogeneous cell population recognized as a key component of the tumour microenvironment (TME). Cancer-associated fibroblasts are known to play an important role in maintaining and remodelling the extracellular matrix (ECM) in the tumour stroma, supporting cancer progression and inhibiting the immune system's response against cancer cells. This review aims to summarize the immunomodulatory roles of CAFs, particularly focussing on their T-cell suppressive effects. Cancer-associated fibroblasts have several ways by which they can affect the tumour's immune microenvironment (TIME). For example, their interactions with macrophages and dendritic cells (DCs) create an immunosuppressive milieu that can indirectly affect T-cell anticancer immunity and enable immune evasion. In addition, a number of recent studies have confirmed CAF-mediated direct suppressive effects on T-cell anticancer capacity through ECM remodelling, promoting the expression of immune checkpoints, cytokine secretion and the release of extracellular vesicles. The consequential impact of CAFs on T-cell function is then reflected in affecting T-cell proliferation and apoptosis, migration and infiltration, differentiation and exhaustion. Emerging evidence highlights the existence of specific CAF subsets with distinct capabilities to modulate the immune landscape of TME in various cancers, suggesting the possibility of their exploitation as possible prognostic biomarkers and therapeutic targets.

Senescence Defines a Distinct Subset of Myofibroblasts That Orchestrates Immunosuppression in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) therapeutic resistance is largely attributed to a unique tumor microenvironment embedded with an abundance of cancer-associated fibroblasts (CAF). Distinct CAF populations were recently identified, but the phenotypic drivers and specific impact of CAF heterogeneity remain unclear. In this study, we identify a subpopulation of senescent myofibroblastic CAFs (SenCAF) in mouse and human PDAC. These SenCAFs are a phenotypically distinct subset of myofibroblastic CAFs that localize near tumor ducts and accumulate with PDAC progression. To assess the impact of endogenous SenCAFs in PDAC, we used an LSL-KRASG12D;p53flox;p48-CRE;INK-ATTAC (KPPC-IA) mouse model of spontaneous PDAC with inducible senescent cell depletion. Depletion of senescent stromal cells in genetic and pharmacologic PDAC models relieved immune suppression by macrophages, delayed tumor progression, and increased responsiveness to chemotherapy. Collectively, our findings demonstrate that SenCAFs promote PDAC progression and immune cell dysfunction. Significance: CAF heterogeneity in PDAC remains poorly understood. In this study, we identify a novel subpopulation of senescent CAFs that promotes PDAC progression and immunosuppression. Targeting CAF senescence in combination therapies could increase tumor vulnerability to chemo or immunotherapy. See related article by Ye et al., p. 1302.

Cancer-associated fibroblast activation predicts progression, metastasis, and prognosis of cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer and the metastatic disease is associated with poor prognosis. Cancer-associated fibroblasts (CAFs) promote progression of cancer, but their role in cSCC is largely unknown. We examined the potential of CAF markers in the assessment of metastasis risk and prognosis of primary cSCC. We utilized multiplexed fluorescence immunohistochemistry for profiling CAF landscape in metastatic and non-metastatic primary human cSCCs, in metastases, and in premalignant epidermal lesions. Quantitative high-resolution image analysis was performed with two separate panels of antibodies for CAF markers and results were correlated with clinical and histopathological parameters including disease-specific mortality. Increased stromal expression of fibroblast activation protein (FAP), α-smooth muscle actin, and secreted protein acidic and rich in cysteine (SPARC) were associated with progression to invasive cSCC. Elevation of FAP and platelet-derived growth factor receptor-β (PDGFRβ) expression was associated with metastasis risk of primary cSCCs. High expression of PDGFRβ and periostin correlated with poor prognosis. Multimarker combination defined CAF subset, PDGFRα-/PDGFRβ+/FAP+, was associated with invasion and metastasis, and independently predicted poor disease-specific survival. These results identify high PDGFRβ expression alone and multimarker combination PDGFRα-/PDGFRβ+/FAP+ by CAFs as potential biomarkers for risk of metastasis and poor prognosis.

Cancer-associated fibroblasts expressing fibroblast activation protein and podoplanin in non-small cell lung cancer predict poor clinical outcome.

Cancer-associated fibroblasts (CAFs) are a dominant cell type in the stroma of non-small cell lung cancer (NSCLC). Fibroblast heterogeneity reflects subpopulations of CAFs, which can influence prognosis and treatment efficacy. We describe the subtypes of CAFs in NSCLC. Primary human NSCLC resections were assessed by flow cytometry and multiplex immunofluorescence for markers of fibroblast activation which allowed identification of CAF subsets. Survival data were analysed for our NSCLC cohort consisting of 163 patients to understand prognostic significance of CAF subsets. We identified five CAF populations, termed CAF S1-S5. CAF-S5 represents a previously undescribed population, and express FAP and PDPN but lack the myofibroblast marker αSMA, whereas CAF-S1 populations express all three. CAF-S5 are spatially further from tumour regions then CAF-S1 and scRNA data demonstrate an inflammatory phenotype. The presence of CAF-S1 or CAF-S5 is correlated to worse survival outcome in NSCLC, despite curative resection, highlighting the prognostic importance of CAF subtypes in NSCLC. TCGA data suggest the predominance of CAF-S5 has a poor prognosis across several cancer types. This study describes the fibroblast heterogeneity in NSCLC and the prognostic importance of the novel CAF-S5 subset where its presence correlates to worse survival outcome.

TSPAN8+ myofibroblastic cancer-associated fibroblasts promote chemoresistance in patients with breast cancer.

Cancer-associated fibroblasts (CAFs) are abundant stromal cells in the tumor microenvironment that promote cancer progression and relapse. However, the heterogeneity and regulatory roles of CAFs underlying chemoresistance remain largely unclear. Here, we performed a single-cell analysis using high-dimensional flow cytometry analysis and identified a distinct senescence-like tetraspanin-8 (TSPAN8)+ myofibroblastic CAF (myCAF) subset, which is correlated with therapeutic resistance and poor survival in multiple cohorts of patients with breast cancer (BC). TSPAN8+ myCAFs potentiate the stemness of the surrounding BC cells through secretion of senescence-associated secretory phenotype (SASP)-related factors IL-6 and IL-8 to counteract chemotherapy. NAD-dependent protein deacetylase sirtuin 6 (SIRT6) reduction was responsible for the senescence-like phenotype and tumor-promoting role of TSPAN8+ myCAFs. Mechanistically, TSPAN8 promoted the phosphorylation of ubiquitin E3 ligase retinoblastoma binding protein 6 (RBBP6) at Ser772 by recruiting MAPK11, thereby inducing SIRT6 protein destruction. In turn, SIRT6 down-regulation up-regulated GLS1 and PYCR1, which caused TSPAN8+ myCAFs to secrete aspartate and proline, and therefore proved a nutritional niche to support BC outgrowth. By demonstrating that TSPAN8+SIRT6low myCAFs were tightly associated with unfavorable disease outcomes, we proposed that the combined regimen of anti-TSPAN8 antibody and SIRT6 activator MDL-800 is a promising approach to overcome chemoresistance. These findings highlight that senescence contributes to CAF heterogeneity and chemoresistance and suggest that targeting TSPAN8+ myCAFs is a promising approach to circumvent chemoresistance.

Abstract 287: Analysis for the role of antigen-presenting cancer-associated fibroblasts in tumor microenvironment of colorectal cancer

Abstract A new subset of cancer associated fibroblasts (CAFs) called antigen presenting CAF (apCAF) was recently identified, and modulation of the tumor immunity might be associated with tumor progression. We examined the markers of apCAF, such as IRF5, HLA-DRA and HLA-DQA1 by bioinformatics analyses of human colorectal cancer (CRC) datasets. The expression of apCAF markers were upregulated in CAFs isolated from human CRCs and liver metastases compared with their normal counterparts. Kaplan-Meir analysis of 172 human CRC samples clarified that the patients with apCAF marker positive expression in the tumor stroma had worse prognosis than that with negative expression. In addition, the origin of apCAFs was reported to be mesothelial cells, therefore, we established cancer-associated mesothelial cells (CAmes) from ascites of human CRC patients with peritoneal dissemination. Consistently, the expression of antigen-presentation markers was highly upregulated in CAmes, however, allo-mixed lymphocyte reaction analysis demonstrated that their stimulating activity of CD4+ T lymphocytes was diminished. In an in vivo study, we applied a rectal orthotopic transplant model with mouse tumor organoid (MTO) to investigate the effect of apCAF on tumor progressions. Using fluorescence-activated cell sorting (FACS), we examined the ratios of apCAF, myCAF, and iCAF in orthotopic rectal tumors. The percentage of apCAF was highest in the 6-week-tumors and the percentage of myCAF was highest in the 9-week-tumors. Interestingly, the percentage of apCAF was higher in liver metastases than in primary tumor, consistent with bioinformatics analysis. The apCAF increased during the stage of tumor growth, and the percentage of myCAF increase in the later stage of tumor progression with fibrosis. In summary, apCAF might be associated with tumor progression in CRC. Citation Format: Yasuhiro Fukui, Hiroaki Kasashima, Zizhou Wang, Ken Yonemitsu, Kishu Kitayama, Yuichiro Miki, Mami Yoshii, Tatsunari Fukuoka, Tatsuro Tamura, Masatsune Shibutani, Takahiro Toyokawa, Hiroaki Tanaka, Shigeru Lee, Masakazu Yashiro, Kiyoshi Maeda. Analysis for the role of antigen-presenting cancer-associated fibroblasts in tumor microenvironment of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 287.

Abstract 6970: identification of spatially enriched cancer cell- and immune cell-regulatory multi-marker-defined prognostic cancer associated fibroblast subsets of human colon cancer

Abstract Introduction: Subsets of cancer-associated fibroblasts (CAFs) in colorectal cancer remain poorly characterized preventing their use as biomarkers and drug targets. Results: Integrated scRNA seq profiling and multiplex staining identified four human CAF subsets These were A1 (PDGFRA+/FAP-/TF), PDGFRAhighA2 (PDGFRA+/FAP+/TF-), PDGFRAlowA2 (PDGFRA-/FAP+/TF-) and A3 (PDGFRA+/FAP-/TF+) (TF: Tissue Factor).Spatial analyses identified spatial associations indicative of specific functional interactions of fibroblast subsets with cancer cells or immune cells. Explorative analyses in six CRC cases suggested, in general, favorable effects of FAP-negative subsets (A1 and A3) on immune features and cancer cell proliferation, whereas FAP-positive subsets PDGFRAhighA2 and PDGFRAlowA2 overall showed pro-tumoral associations. To extend findings, niches were quantitated in U-CAN cohort of approximately 200 stage I-IV cases with around 80% stage II/III. Spatial screening generated more than 1600 different multi-marker-defined tissue metrics. Analyses identified a higher average of A1 density in areas surrounding proliferating vs non-proliferating T-cells, and a higher average PDGFRAlowA2 peri-T-cell density in the low proliferating T-cell group. Regarding cancer cell proliferation, high proliferating cancer islands showed lower peri-epithelial density of A3 and PDGFRAlowA2 cells as compared to low proliferating cancer islands. As for T-cell cancer island infiltration, high peri-cancer island density of A1 and PDGFRAhighA2 was associated with high T-cell cancer island infiltration. Additional analyses related CAF metrics to stage and survival. Density of PDGFRAlow A2 cells increased with stage (I-III), whereas density of A1 showed an opposite pattern with an inverse association with stage (I-III). Regarding survival associations, these were in general stronger for metrics collected in peripheral regions of tumors. Among prognosis signals related to niches referred to above, it was noted that A1 density in peri-cancer regions was significantly associated with good prognosis. Interestingly, this association was not detected when A1 density was scored in total stroma. Among all metrics, the strongest metrics were A1 density in 20μm A1 expansion areas (good prognosis) and T-reg density in the 10μm A3 expansion areas (bad prognosis) (P<0.001; univariate Cox-regression); both in peripheral regions. Conclusion: Four novel CRC CAF subsets, defined by PDGFRalpha, FAP and TF, was identified by integrated single cell RNA seq analyses and multiplex staining with associated digital image analyses. Non-random spatial distribution of these subsets and compartment-specific prognosis associations are compatible with distinct and different functions of these subsets. Citation Format: Linglong Huang, Mercedes Herrera, Jonas Sjölund, Vladimir Chocoloff, Simon Joost, Rasul M. Tabiev, Lina Wik Leiss, Carina Strell, Luis Nunes, Artur Mezheyeuski, David Edler, Anna Martling, Fredrik Pontén, Bengt Glimelius, Tobias Sjöblom, Maria Kasper, Kristian Pietras, Arne Östman. identification of spatially enriched cancer cell- and immune cell-regulatory multi-marker-defined prognostic cancer associated fibroblast subsets of human colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6970.

Abstract 5485: Spatial characterization of the immunosuppressive tumor microenvironment in estrogen receptor positive breast cancer at single cell level

Abstract We characterized immune cell exclusion in estrogen receptor positive (ER+) breast cancer through the study of spatial relationships among various subsets of cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), and other immune cells using a novel in-situ spatial transcriptomic technology. ER+ breast cancer is the most common breast cancer subtype. While endocrine therapy and immune checkpoint inhibitors have considerably reduced relapse and mortality for breast cancer patients, tumor resistance to treatment remains a major challenge. The tumor microenvironment (TME) plays an important role in this mechanism of resistance. Our previous spatial proteomic analysis of ER+ breast FFPE samples demonstrated the intricate relationship between tumor and immunosuppressive cells such as CAFs and TAMs via the CXCR4-CXCL12 axis associated with the exclusion of CD8 T cells from the TME. However, the limited number of antibodies in the panel made it challenging to investigate the diversity of those CAF and TAM populations and better understand their contribution to T cell exclusion. For this purpose, we further characterized the TME of 4 human ER+ breast cancer FFPE cores using the Xenium in-situ transcriptomic platform with a breast cancer-focused panel of 280 genes from 10x Genomics plus 100 additional genes selected from literature and publicly available single cell datasets to delineate CAF and TAM sub-populations. Image analysis enabled the quantification and spatial mapping of the target genes within each cell boundary. Cells were manually annotated through unsupervised clustering of over 1 million cells. The cell phenotyping analysis allowed us to annotate major cell types including tumor cells, macrophages, T cells and fibroblasts. With our curated panel, we further characterized CAF and TAM sub-populations by identifying myofibroblastic CAFs (myCAFs), inflammatory CAFs (iCAFs), and antigen-presenting CAFs (apCAFs) in addition to M1 and M2-like macrophages as reported in the literature. We then performed spatial neighborhood analysis based on the similarity of the cell type composition in the vicinity of each cell. The results showed that myCAFs and M2-like macrophages are found in proximity to tumor cells and may play a role in T-cell exclusion. In addition, iCAFs mainly express CXCL12, a chemokine known to attract immunosuppressive cells such as regulatory T cells and M2-like macrophages as well as repel cytotoxic T cells from infiltrating the tumor via the CXCR4-CXCL12 axis. Overall, our work shows the potential of spatial transcriptomics to elucidate the spatial distribution of cells and their contribution to the immunosuppressive TME, which may enable researchers to improve understanding of tumor biology and drug target discovery efforts. Citation Format: Julien Tessier, Sandra Curras-Alonso, Joon Sang Lee, Fabien Delahaye, Hong Ma, Katie Malley, Dinesh Bangari, Donald Jackson, Angela Hadjipanayis. Spatial characterization of the immunosuppressive tumor microenvironment in estrogen receptor positive breast cancer at single cell level [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5485.

Abstract 7471: Cancer-associated fibroblasts drive T cell infiltration and resistance to immunotherapy in non-small cell lung cancer with mature tertiary lymphoid structures

Abstract Background: The presence of mature tertiary lymphoid structures (mTLS) within the tumor microenvironment (TME) has been linked to the effectiveness of PD-L1 blockade in non-small cell lung cancer (NSCLC). However, only a subset of patients experiencing clinical benefit. Our study aims to delve into the determinants of immunotherapy response in mTLS-positive NSCLC. Methods: We included 77 TLS-positive NSCLC from the BIP study (NCT02534649). First, we employed the GeoMx whole-transcriptome atlas (WTA) assay to spatially profiled the expression of >18,000 protein-coding in patients exhibiting extreme responses (3 responders [R] and 3 non-responders [NR]). To target regions of interest (ROIs), we selected areas that encompassed TLS (CD3+/CD20+) as entire areas of illumination (AOIs). Additionally, we segmented areas outside TLS into ‘tumor’ (PanCK+/CD45−) vs ‘stroma’ (PanCK−/CD45+) AOIs. To validate our findings, we conducted multiplex immunofluorescence (mIF) and complemented our results with bulk gene expression analysis using the HTG whole human transcriptome panel. Results: Spatial transcriptomic analysis pinpointed significant differences in gene expression within the stromal compartment between R and NR. Notably, a subset of cancer-associated fibroblasts (CAFs) expressing both FAP and αSMA markers emerged as the most differentially expressed genes. Subsequently, we used FFPE tissue mIF to delineate the histological profile CAF subsets and the spatial distribution of T cells. Notably, the stromal density of FAP+αSMA+ CAFs and MYH11+αSMA+ CAFs were significantly higher in NR compared to R. Correspondingly, tumors enriched in FAP+αSMA+ CAFs and MYH11+αSMA+ CAFs exhibited significantly lower overall response rates (ORR) and progression-free survival (PFS) than those with a low abundance of CAFs. Interestingly, tumors with elevated FAP+αSMA+ CAFs and MYH11+αSMA+ CAFs were associated with a higher infiltration of CD8+ T cells. To delve deeper into the CD8+ T cell infiltrate, we conducted a comparative bulk transcriptomic analysis. Both FAP+αSMA+ CAFs High and MYH11+αSMA+ CAFs High demonstrated significant associations with regulatory T cells (Treg), exhaustion signatures, and inflammatory processes. To validate the CD8+ T cell phenotype, we performed complementary mIF. Consistently, the CD8+ T cell infiltrate in tumors featuring FAP+αSMA+ CAFs High and MYH11+αSMA+ CAFs High exhibited higher levels of exhaustion markers (PD1, LAG3, TIM3, TIGIT) and Treg infiltration, underscoring an immunosuppressive TME. Conclusions: FAP+αSMA+ CAFs and MYH11+αSMA+ CAFs play a pivotal role in driving T cell exhaustion and Treg cell infiltration, contributing to the resistance to immune checkpoint inhibition. Targeting these specific CAF subsets may hold significant promise in overcoming resistance in mature TLS-positive NSCLC. Citation Format: Florent Peyraud, Jean-Philippe Guegan, Lucile Vanhersecke, Christophe Rey, Oren Lara, Ophélie Odin, Emma Clot, Maxime Brunet, Thomas Grellety, Marie Del Castillo, Sylvestre Le Moulec, François Le Loarer, Alban Bessede, Antoine Italiano. Cancer-associated fibroblasts drive T cell infiltration and resistance to immunotherapy in non-small cell lung cancer with mature tertiary lymphoid structures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7471.

Abstract 2914: AI/ML-driven discovery of CTHRC1, collagen triple helix repeat-containing 1, a novel proteoglycan for stroma + tumor targeting and delivery of 4-1BB costimulation

Abstract Background: While checkpoint inhibitors have demonstrated efficacy in a number of solid tumor indications, those with high stromal presence have been difficult to treat with minimal objective responses observed. Phenomic has developed a proprietary machine learning/artificial intelligence platform to identify novel stromal targets with superior expression profiles that enable selective targeting of immune activating agents that will relieve these immunosuppressive barriers in difficult to treat indications. Methods: Using our single cell RNA Atlas, we assessed cancer-associated fibroblasts (CAFs) in several solid tumor indications for identification of novel targets, including proteoglycans. Our Atlas was also used to identify immune activating payloads whose cognate receptors were present in indications of interest. Antibodies were generated, and lead clones who demonstrated potent ligand binding and cell staining were used to generate fusion proteins to immune activating ligands. Efficacy and PD were assessed in multiple syngeneic tumor models. Results: Bioinformatic analysis identified a unique subset of pathogenic CAFs, which are TGF beta responsive, secrete several ECM proteins, and their presence tracks with poor outcome and resistance to immunotherapy in several solid tumor types. CTHRC1 was identified as a novel matrix protein highly expressed in this CAF subtype as well as tumor epithelium and is highly selective in a range of tumor types such as ovarian cancer, triple negative breast cancer, and pancreatic ductal adenocarcinoma. While secreted, CTHRC1 is complexed on the cell surface and affords the opportunity to drug this target in a variety of ways. We identified an absence of 4-1BBL expression across indications of interest, and fusion proteins were generated to deliver 4-1BBL via CTHRC1 targeting. In checkpoint-resistant tumor models, we observed significant increases in CD8 T cells and robust anti-tumor activity with anti-CTHRC1-targeted 4-1BBL. Biodistribution studies were conducted using our targeting mAb and demonstrate uptake only in sites of primary and metastatic tumors, even at doses 20-fold higher than those that achieve maximal therapeutic activity, suggesting the potential to minimize the toxicity that has been observed with other 4-1BB agonists. Conclusions: We have identified CTHRC1 as a novel proteoglycan expressed by both pathogenic CAFs and tumor cells that is highly selective for tumors, enabling the therapeutic targeting of immune activating payloads with the potential for safely delivering payloads while limiting toxicity. Given the specificity and selectivity afforded by CTHRC1 expression, ADC and CD3 engager approaches are also being pursued. These data represent novel approaches aimed at breaking down stromal barriers in tumors previously unresponsive to immunotherapies. Citation Format: Christopher Harvey, Elizabeth Koch, Amanda Hanson, Lindsey Rice, Amy Berkley, Kerry White, Reza Saberianfar, Nikolai Suslov, Sam Cooper, Michael Briskin. AI/ML-driven discovery of CTHRC1, collagen triple helix repeat-containing 1, a novel proteoglycan for stroma + tumor targeting and delivery of 4-1BB costimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2914.

Abstract 7642: Activated FAP+ cancer-associated fibroblasts in non-muscle invasive bladder cancer are associated with progression to muscle-invasive disease

Abstract The majority of patients with bladder cancer initially present with non-muscle-invasive disease (NMIBC). While NMIBC may be cured with transurethral resection +/- intravesical therapies, approximately 20% of patients develop progression to muscle-invasive bladder cancer (MIBC) which requires life-altering treatments such as cystectomy and has a high risk of lethal metastatic recurrence. Current risk stratification for NMIBC relies on clinicopathologic features that overlook the potential role of the tumor microenvironment (TME) in bladder cancer pathogenesis. We sought to delineate the stromal and immune landscape alterations within the TME upon bladder cancer progression and to identify TME-related features that could enhance current prognostic approaches. Analyzing by single-cell RNA sequencing and multiplexed imaging of 22 untreated NMIBC and MIBC specimens, coupled with publicly available datasets, we discovered significant transcriptional variability among cancer-associated fibroblasts (CAFs) and macrophages across patients. This inter-patient variability was attributable to the extensive remodeling of these cell types during cancer progression. Notably, a specific subset of CAF, characterized by ITGA8+F3+ markers, was found to be highly enriched in NMIBC lesions and transcriptionally related to sub-urothelial fibroblasts of the healthy bladder, whereas a FAP+ CAF subset enriched for core matrisome related genes was dominant in MIBC lesions. We developed a FAP+ CAF signature which was then projected onto a large public dataset of bulk-RNAseq data from patients with NMIBC demonstrating that the FAP+ CAF signature was significantly associated with progression to MIBC independently of contemporary risk stratification scores. The presence of FAP+ CAFs in a subset of NMIBC specimens was orthogonally validated using immunohistochemistry. Importantly, areas with FAP staining were observed in close contact with individual or small groups of tumor cells exhibiting an invasive phenotype and partial epithelial-mesenchymal transition, likely explaining the higher risk of progression to MIBC. Our study improves our understanding of fibroblasts remodeling during bladder cancer progression and highlights the potential of integrating CAF-derived biomarkers into NMIBC risk stratification models. Citation Format: Abdenour Abbas, Shrisha Maskey, Evan Cheng, Ziao Li, Koushik Ganesh, Sudeh Izadmehr, Serena Janho dit Hreich, Benjamin Demaille, Jeremy Mesple, Philemon Sirven, Clementine Krucker, Jacqueline Fontugne, Isabelle Bernard-Pierrot, Yves Allory, Miriam Merad, Julien Adam, John Sfakianos, Ephraim Kenigsberg, Matthew Galsky, Helene Salmon. Activated FAP+ cancer-associated fibroblasts in non-muscle invasive bladder cancer are associated with progression to muscle-invasive disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7642.

Abstract 217: Establishment of a spatially defined co-culture model of non-small cell lung cancer organoids and cancer-associated fibroblasts to investigate phenotypic heterogeneity

Abstract Background: Cancer-associated fibroblasts (CAFs) are heterogeneous, with CAF subtypes being localized in spatially defined areas of tumors. However, it remains unclear which CAF subsets specifically play a role in fundamental processes such as tumor growth, invasion, and response to therapy. This study aims to establish a novel in vitro model that recapitulates the in vivo tumor-stroma spatial relationship to investigate CAF heterogeneity and its impact on tumor cell growth in non-small cell lung cancer (NSCLC). Methods: The in vitro model was established using NSCLC patient-derived CAFs and tumor organoids as a 6:1 ratio. Tumor cells originating from xenograft-derived organoids (XDOs) were embedded in a hydrogel containing CAFs (Day 0) and co-cultured for 5 days. Confocal microscopy was utilized to characterize and measure the proliferation of the XDO tumor cells in the co-culture setup, and the proliferation rate was defined as differences in tumor spheroid area between Day 1 and 5. Single-cell RNA-sequencing (scRNAseq) and gene set enrichment analysis (GSEA) were then used to assess the gene expression and signaling pathways dominant in mono- and co-culture to characterize CAF heterogeneity in this model. Results: Initially, the culture media formulations and the matrix for the growth of both CAFs and tumor cells were screened to assess the compatibility of different culture elements. Both CAFs and XDO tumor cells proliferated well in reduced organoid media (M27RD). This reduced medium and the novel matrix composition (50% Matrigel/50% Rat Tail type I collagen) also allowed for maintaining CAF contractility and XDO proliferation. Utilizing these harmonized media and matrix conditions, a spatially defined co-culture model, which incorporated XDOs as a central cluster surrounded by CAFs, was generated. In confocal microscopy, the proliferation rate of tumor cells was significantly higher in co-cultures with CAFs (adjusted p-value < 0.01), while not significant in mono-culture of tumor cells. The GSEA using the Hallmark gene sets on scRNAseq data generated in three co-cultures identified the heterogeneity among CAFs: two CAFs showed significantly higher effect on tumor cell proliferation with the most highly differential expressed genes (DEGs) enriched with inflammation and mitosis and the top high DEGs associated with epithelial-mesenchymal transition, while the other CAF has no effect. Conclusion: The harmonized co-culture conditions for NSCLC organoids and CAFs enabled the characterization of both cell phenotypes and the building of an informed model architecture to study tumor-stroma communication. The presence of CAFs increased tumor cell growth with enrichment in secreted proteins involved in epithelial-mesenchymal transition. Citation Format: Cheol-Kyu Park, Roya Navab, Elisa D'Arcangelo, Quan Li, Hiroyuki Ogawa, Nikolina Radulovich, Nhu-An Pham, Ming-Sound Tsao. Establishment of a spatially defined co-culture model of non-small cell lung cancer organoids and cancer-associated fibroblasts to investigate phenotypic heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 217.

Abstract 5480: Single cell and spatial transcriptomic profile of appendiceal tumour peritoneal disease

Abstract Introduction: Appendiceal tumors (AT) are rare and often develop peritoneal disease (PD), with 5 year survival of 15-63% for high grade adenocarcinomas (HG) and 46-96% for low grade mucinous neoplasms (LG). While the mainstay of treatment is cytoreductive surgery and heated intraperitoneal chemotherapy (CRS/HIPEC), the role of systemic chemotherapy is less clear with limited biological characterization of AT and its tumor microenvironment (TME). The purpose of this study was to evaluate the TME of AT PD. Here we present an atlas of AT PD cells alongside a spatially resolved transcriptomic profile comparing HG and LG. Methods: We collected tissue from 29 patients with AT PD during CRS prior to HIPEC (18 LG, nine HG and two no tumor control). We performed comprehensive single-cell RNA sequencing (38224 cells) on fresh tissue specimens from nine patients (four HG, five LG), and whole transcriptome spatial analysis (>18000 genes) using the GeoMx Digital Spatial Profiler on formalin-fixed paraffin-embedded tissue from 22 patients (five HG, 13 LG). In the spatial profiling, we used anti-CK20, anti-CD45 and SYTO 83 as morphology markers to select areas of interest (AOIs): tumor (CK20), immune (CD45) and stroma (non-CK20/CD45). Analyses were performed with R and a false discovery rate threshold of 0.05. Results: Single-cell RNA sequencing showed that both LG and HG have a preponderance of T-lineage cells including CD8, CD4 and NK T-cells. Both LG and HG tumors display association of mesothelial cells with cancer-associated fibroblasts (CAF), and CAF subsets expressing genes of epithelial-mesenchymal transition (EMT). LG and HG tumors display different patterns of mucin gene expression - epithelial cells of HG express the secreted mucin MUC20 in addition to MUC2 and MUC5B also expressed in LG; a subset of mesothelial cells in LG but not HG express the transmembrane mucin genes MUC1, MUC3A, MUC12 and MUC16. Spatial analysis of tumor AOIs revealed HG compared to LG had increased expression of genes associated with MYC targets, oxidative phosphorylation, anti-tumor immune pathways (IFNα/γ response, intestinal immune network for IGA production), cell cycle pathways (E2F targets, G2M checkpoint) and EMT. LG compared to HG had increased expression of genes associated with inflammation-mediated disease (TNFα signaling via NFKB, inflammatory response), regulation of tumor growth (P53), hypoxia and early cell cycle (estrogen response) and glycosylation pathways. Immune and stromal AOIs did not have significant transcriptomic differences between LG and HG. Conclusion: This study provides novel insights into the biological profile of AT PD by describing TME cell types, immune function, tumor growth and cell cycle pathways. These findings may be used to identify clinically relevant biomarkers and new therapeutic targets. Citation Format: Madeleine C. Strach, Nicole Yeung, Eva Apostolov, Tony Wang, Hui-Ming Lin, Nabila Ansari, Cherry Koh, Joo-Shik Shin, James Kench, Alexander Swarbrick, Lisa Horvath, Kate L. Mahon. Single cell and spatial transcriptomic profile of appendiceal tumour peritoneal disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5480.

Metabolic targeting of cancer associated fibroblasts overcomes T-cell exclusion and chemoresistance in soft-tissue sarcomas

T cell-based immunotherapies have exhibited promising outcomes in tumor control; however, their efficacy is limited in immune-excluded tumors. Cancer-associated fibroblasts (CAFs) play a pivotal role in shaping the tumor microenvironment and modulating immune infiltration. Despite the identification of distinct CAF subtypes using single-cell RNA-sequencing (scRNA-seq), their functional impact on hindering T-cell infiltration remains unclear, particularly in soft-tissue sarcomas (STS) characterized by low response rates to T cell-based therapies. In this study, we characterize the STS microenvironment using murine models (in female mice) with distinct immune composition by scRNA-seq, and identify a subset of CAFs we termed glycolytic cancer-associated fibroblasts (glyCAF). GlyCAF rely on GLUT1-dependent expression of CXCL16 to impede cytotoxic T-cell infiltration into the tumor parenchyma. Targeting glycolysis decreases T-cell restrictive glyCAF accumulation at the tumor margin, thereby enhancing T-cell infiltration and augmenting the efficacy of chemotherapy. These findings highlight avenues for combinatorial therapeutic interventions in sarcomas and possibly other solid tumors. Further investigations and clinical trials are needed to validate these potential strategies and translate them into clinical practice.

Bioengineered Hydrogels Recapitulate Fibroblast Heterogeneity in Cancer.

Recently mapped transcriptomic landscapes reveal the extent of heterogeneity in cancer-associated fibroblasts (CAFs) beyond previously established single-gene markers. Functional analyses of individual CAF subsets within the tumor microenvironment are critical to develop more accurate CAF-targeting therapeutic strategies. However, there is a lack of robust preclinical models that reflect this heterogeneity in vitro. In this study, single-cell RNA sequencing datasets acquired from head and neck squamous cell carcinoma tissues to predict microenvironmental and cellular features governing individual CAF subsets are leveraged. Some of these features are then incorporated into a tunable hyaluronan-based hydrogel system to culture patient-derived CAFs. Control over hydrogel degradability and integrin adhesiveness enabled derivation of the predominant myofibroblastic and inflammatory CAF subsets, as shown through changes in cell morphology and transcriptomic profiles. Last, using these hydrogel-cultured CAFs, microtubule dynamics are identified, but not actomyosin contractility, as a key mediator of CAF plasticity. The recapitulation of CAF heterogeneity in vitro using defined hydrogels presents unique opportunities for advancing the understanding of CAF biology and evaluation of CAF-targeting therapeutics.

PDGFRα+ITGA11+ fibroblasts foster early-stage cancer lymphovascular invasion and lymphatic metastasis via ITGA11-SELE interplay

Cancer-associated fibroblasts (CAFs) exhibit considerable heterogeneity in advanced cancers; however, the functional annotation and mechanism of CAFs in early-stage cancers remain elusive. Utilizing single-cell RNA sequencing and spatial transcriptomic, we identify a previously unknown PDGFRα+ITGA11+ CAF subset in early-stage bladder cancer (BCa). Multicenter clinical analysis of a 910-case cohort confirms that PDGFRα+ITGA11+ CAFs are associated with lymphovascular invasion (LVI) and poor prognosis in early-stage BCa. These CAFs facilitate LVI and lymph node (LN) metastasis in early-stage BCa, as evidenced in a PDGFRα+ITGA11+ CAFs-specific deficient mouse model. Mechanistically, PDGFRα+ITGA11+ CAFs promote lymphangiogenesis via recognizing ITGA11 surface receptor SELE on lymphatic endothelial cells to activate SRC-p-VEGFR3-MAPK pathway. Further, CHI3L1 from PDGFRα+ITGA11+ CAFs aligns the surrounding matrix to assist cancer cell intravasation, fostering early-stage BCa LVI and LN metastasis. Collectively, our study reveals the crucial role of PDGFRα+ITGA11+ CAFs in shaping metastatic landscape, informing the treatment of early-stage BCa LVI.

Cancer-associated Fibroblast-specific Expression of the Matricellular Protein CCN1 Coordinates Neovascularization and Stroma Deposition in Melanoma Metastasis.

In human patients, the expression of proangiogenic matricellular protein CCN1 in CAFs correlates positively with expression of stroma and angiogenic markers and progressive disease/resistance to checkpoint inhibitor therapy. In an animal model, loss of CCN1 from CAFs impaired metastasis of melanoma cells, neovascularization, and collagen deposition, emphasizing that CAFs coordinate cellular behavior in a tumor microenvironment and that CCN1 may be a novel target.

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

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

Abstract C036: Restoration of T effector cells function by targeting senescent cancer-associated fibroblast in tumor microenvironment of stroma-rich cancers

Abstract The stroma that surrounds the tumor in many solid cancers exhibits potent immunosuppressive activity to promote tumor progression and confer resistance to immune-based therapies. Cancer associated fibroblasts (CAFs) encompassing functionally distinct populations are key components of the stroma that are emerging as a key cell type in regulating an immune response. However, the mechanisms by which CAFs can directly suppress the immune activity to promote tumor growth is not clear. Here, we identify that a subset of CAF in both rodent and human stromagenic cancers that displays characteristics of senescent fibroblasts and referred to as sCAFs. We show that sCAFs secrete a decoy protein that interferes with cytotoxic activity of activated T effector (Teff) cells. In mouse models for stroma-rich pancreatic cancer, administration of an antibody that blocked the decoy protein resulted in recalibration of the stromal fibroblast by promoted the formation of interferon-licensed-fibroblast cells at the expense of sCAFs. This switch not only enhanced infiltration of tumor-infiltrating T cells but also alleviated their direct suppression resulting in enhanced effector function and tumor regression. Collectively, these results point to the potent immunosuppressive activity of sCAFs mediated by decoy protein as a mechanism by which stroma can promote tumor progression. As stromagenic-cancers are resistance to current therapies, the development of treatments involving blocking of decoy protein may inhibit tumor growth and improve patient survival. Citation Format: Yao Wang, Hara Apostolopoulou, Arjun Sanyal, Hong Sun, Cynthia Sieland, Kavya Gupta, Jiayu Ye, David Gibbs, Paul Wong, Ntranos Vasilis, Sui Huang, James Gardner, Ajay Maker, Thea Tlsty, Anil Bhushan, Tamara Alliston. Restoration of T effector cells function by targeting senescent cancer-associated fibroblast in tumor microenvironment of stroma-rich cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C036.

Abstract B044: Targeting integrin alpha V beta 3 remodels the tumor microenvironment in pancreatic cancer

Abstract In pancreatic ductal adenocarcinoma (PDAC) the tumor microenvironment (TME) plays a crucial role in therapy resistance, metastasis, and recurrence. ProAgio is a novel therapeutic cytotoxin rationally designed to bind outside the RGD ligand pocket of the alphaV-beta3 integrin pair and then kill cells expressing this target. Pre-clinical studies demonstrated the safety and anti-tumor efficacy of ProAgio in animal models, and ProAgio is currently being tested in a Phase I clinical trial. The effect of ProAgio on TME cellular components has not been closely examined. We hypothesized that ProAgio remodels the PDAC TME to decrease stromal density and local immunosuppression by killing stromal cells thought to express the integrin pair, such as cancer associated fibroblasts (CAFs), proliferating endothelial cells, and tumor associated macrophages (TAMs). To identify potential cellular targets of ProAgio in the PDAC TME, we re-analyzed publicly available single cell RNA-seq datasets of human and mouse PDAC samples. We assessed integrin co-expression using flow cytometry, performed immunophenotyping and histopathological analysis to identify TME cellular and structural alterations induced by ProAgio. Integrins αV and β3 were both expressed at the RNA level in TAMs, myeloid cells, lymphocytes (T, B and NK), endothelial cells and CAFs. In the autochthonous KPC mouse model, we confirmed that granulocytes, macrophages, monocytes, CD4+ and CD8+T cells co-expressed integrin αVβ3 at the protein level. Amongst CAFs, inflammatory CAFs (iCAFs) had the highest percentage of cells co-expressing αV and β3 integrins. No anti-tumor effect was observed after 14 days of ProAgio treatment in a syngeneic KPC-derived PDAC orthotopic model, however, in the KPC autochthonous model the same treatment retarded tumor growth within 1 week. ProAgio treated autochthonous tumors displayed no changes in immune components or CAF subsets, but increased collagen area was observed. In addition, ProAgio treatment resulted in higher blood vessel area while integrin β3 protein levels remained stable. In conclusion, short-course ProAgio remodels the TME and delays tumor growth in an autochthonous PDAC model with mature stromal components, but does not deplete CAFs, TAMs or collagen area. Future studies will seek to refine our understanding of the cell subtypes affected by more prolonged ProAgio treatment and to determine the cellular and inflammatory mediators responsible for these changes. Citation Format: Mayrel Palestino Dominguez, Philip Homan, Xianyu Zhang, Sandra Navas Reyes, Theresa Guerin, Laura Bassel, Liu Zhi-ren, Serguei Kozlov, Christine Alewine. Targeting integrin alpha V beta 3 remodels the tumor microenvironment in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B044.