Subset Of Cancer-associated Fibroblasts Research Articles

Single-cell RNA Sequencing Analysis Reveals the Role of Cancerassociated Fibroblasts in Skin Melanoma.

Mechanism of fibroblasts in skin melanoma (SKME) revealed by single-cell RNA sequencing data. SKME is responsible for more than 80% of skin-related cancer deaths. Cancer-associated fibroblasts (CAFs) generate inflammatory factors, growth factors and extracellular matrix proteins to facilitate cancer cell growth, metastasis, drug resistance and immune exclusion. However, molecular mechanisms of CAFs in SKME are still lacking. Our goal was to reveal the role of CAFs in SKME. We downloaded the single-cell RNA sequencing (scRNA-seq) dataset from the Gene Expression Omnibus (GSE215120) database. Then, the Seurat package was applied to analyze the single-cell atlas of SKME data, and cell subsets were annotated with the CellMarker database. The molecular mechanisms of CAFs in SKME were disclosed via differential gene expression and enrichment analysis, Cellchat and SCENIC methods. Using scRNA-seq data, three SKME cases were used and downscaled and clustered to identify 11 cell subgroups and 5 CAF subsets. The enrichment of highly expressed genes among the 5 CAF subsets suggests that cell migration-inducing hyaluronan-binding protein (CEMIP) + fibroblasts and naked cuticle homolog 1 (NKD1)+ fibroblasts were closely associated with epithelial to mesenchymal transition. Cellchat analysis revealed that CAF subpopulations promoted melanocyte proliferation through Jagged1 (JAG1)-Notch homolog 1 (NOTCH1), JAG1-NOTCH3 and migration through pleiotrophin (PTN)-syndecan-3 (SDC3) receptor-ligand pairs. The SCENIC analysis identified that most of the transcription factors in each CAF subpopulation played a certain role in the metastasis of melanoma and were highly expressed in metastatic SKME samples. Specifically, we observed that CEMIP+ fibroblasts and NKD1+ fibroblasts had potential roles in participating in immune therapy resistance. Collectively, we uncovered a single-cell atlas of SKME and revealed the molecular mechanisms of CAFs in SKME development, providing a base for immune therapy and prognosis assessment. Our study reveals that 5 CAFs in SKME have a promoting effect on melanocyte proliferation and metastasis. More importantly, CEMIP+ fibroblasts and NKD1+ fibroblasts displayed close connections with immune therapy resistance. These findings help provide a good basis for future immune therapy and prognosis assessment targeting CAFs in SKME.

Abstract B007: Identification of Asporin as a HER3 ligand exposes a therapeutic vulnerability in metastatic prostate cancer

Abstract Background: Rates of prostate cancer incidence and advanced-stage diagnoses are increasing. Despite curative-intent therapies, nearly all patients with advanced disease progress to metastatic castration-resistant prostate cancer (mCRPC), a lethal form of the disease. There is an urgent unmet need to understand the mechanisms driving prostate cancer progression and identify alternative therapies for patients with mCRPC. It is well-established that cancer-associated fibroblasts (CAF) in the tumor microenvironment secrete factors that enable cancer cells to establish metastases. Asporin (ASPN) is an extracellular protein secreted by a subset of CAF and is associated with worse oncologic outcomes, yet the mechanisms remain poorly understood. In this study, we sought to identify the molecular interactions between the CAF-secreted ASPN and metastatic prostate cancer cells. Methods: ASPN-induced HER2/HER3 activation and downstream signaling was identified by RNA-sequencing and then confirmed by immunoblotting in multiple human metastatic prostate cancer cell lines. Binding interactions were assessed computationally by AlphaFOld2 with Rosetta refinement and substantiated by co-immunoprecipitation assays. CRISPR-cas9 targeting of HER2 and HER3 and therapeutic inhibition of HER2 with Tucatinib were used to determine the role of HER2/HER3 in ASPN-induced signaling and migration. The efficacy of Tucatinib and antibody-drug conjugates (ADC) targeting HER2 (Trastuzumab-deruxtecan) and HER3 (Patritumab-deruxtecan) were assessed in multiple metastatic prostate cancer cell lines in vitro. Trastuzumab-deruxtecan was assessed in PC3 xenografts in vivo. Prostate cancer metastases from 33 patients were assessed by IHC for HER2 and HER3 and by RNAscope for ASPN. Results: We report that ASPN is a novel ligand of HER3 and induces HER3 heterodimerization with its preferred dimerization partner, HER2. ASPN activates established ErbB-associated pathways including Phosphoinositide 3-kinase (PI3K), Mitogen-activated protein kinase (MAPK), and calcium signaling, in multiple metastatic prostate cancer cell lines to promote cell migration. Genetic and molecular inhibition of HER2/HER3 mitigates ASPN-induced signaling and cell migration, suggesting these receptors are required for paracrine activation by ASPN. Importantly, small molecule and ADC therapies targeting HER2/HER3 significantly diminished prostate cancer cell growth in vitro, with enzalutamide-resistant cells showing increased sensitivity. Trastuzumab-deruxtecan nearly resolved tumors in an in vivo xenograft model. Lastly, ASPN+ CAF in the TME of HER2/HER3-expressing metastatic prostate cancer is frequently observed in patient samples, supporting the clinical relevance of these findings. Conclusions: Collectively, these findings indicate ASPN functions as a HER3 ligand to induce cellular migration, and inhibition with anti-HER2/HER3 therapies highlights potential clinical utility for patients with HER2-expressing metastatic prostate cancer. Citation Format: Amanda B. Hesterberg, Hong Yuen Wong, Jorgen Jackson, Monika Antunovic, Brenda L. Rios, Evan Watkins, Riley E. Bergman, Brad A. Davidson, Sarah E. Ginther, Diana Graves, Elliott F. Nahmias, Jared A. Googel, Lillian B. Martin, Violeta Sanchez, Paula Gonzalez Ericsson, Quanhu Sheng, Benjamin P. Brown, Jens Meiler, Ben H. Park, Kerry R. Schaffer, Jennifer B. Gordetsky, Paula J. Hurley. Identification of Asporin as a HER3 ligand exposes a therapeutic vulnerability in metastatic prostate cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B007.

High-resolution subtyping of fibroblasts in gastric cancer reveals diversity among fibroblast subsets and an association between the MFAP5-fibroblast subset and immunotherapy.

Gastric cancer (GC) remains a global health threat due to frequent treatment failures caused by primary or acquired resistance. Although cancer-associated fibroblasts (CAFs) have been implicated in this process, it is still unclear which specific subtype(s) of CAFs hinder T-cell infiltration and promote resistance to immunotherapy. We analyzed the GC fibroblast atlas in detail by combining 63,955 single cells from 14 scRNA-seq datasets. We also performed RNA-seq data in a local GC cohort and examined 13 bulk RNA-seq datasets to understand the biological and clinical roles of different CAF subsets. Additionally, we conducted in vitro experiments to study the role of specific proteins in GC development. We identified a total of 17 fibroblast subsets in gastric cancer, nine of which did not fit into the existing CAFs classification. These subsets exhibited significant heterogeneity in distribution and biological characteristics (metabolism, cell-cell interactions, differentiation state), as well as clinical functions such as prognosis and response to immunotherapy. In particular, cluster 6 stood out for its high expression of MFAP5, CFD, and PI16; it was found to be negatively associated with both overall survival and response to immunotherapy in GC. This association was linked to an immunosuppressive microenvironment characterized by an increase in M2 macrophages but higher levels of T cell dysfunction and exclusion-a feature shared by tumors expressing MFAP5. Furthermore, the addition of human recombinant MFAP5 promoted proliferation and migration of HGC-27 cells by inducing the MFAP5/NOTCH1/HEY1 signaling pathway. We introduce a high-resolution GC fibroblast atlas. The 17 identified fibroblast clusters provide valuable opportunities for gaining deeper biological insights into the relationship between fibroblasts and GC development. Particularly, cluster 6 and its specific marker MFAP5 could serve as prognostic factors in GC and form a foundation for personalized therapeutic combinations to address primary resistance to ICIs.

AKAP12 positive fibroblast determines immunosuppressive contexture and immunotherapy response in patients with TNBC by promoting macrophage M2 polarization

BackgroundTriple-negative breast cancer (TNBC) is a molecular subtype of breast cancer with high aggressiveness and poor prognosis. Cancer-associated fibroblasts (CAFs) are major components of the TNBC microenvironment and play an...

8109 Cancer-Associated Fibroblasts Correlate with Aggressive Thyroid Cancer Behavior: Insights from Four Large Patient Cohorts

Abstract Disclosure: M.A. Loberg: None. X. George: None. P.J. Courtney: None. H.C. Eric: None. A. Golding: None. B. David: None. M. Alshalalfa: Employee; Self; Veracyte, Inc.. Stock Owner; Self; Veracyte, Inc. Y. Hao: Employee; Self; Veracyte, Inc.. Stock Owner; Self; Veracyte, Inc. J.P. Klopper: Employee; Self; Veracyte, Inc.. Stock Owner; Self; Veracyte, Inc. R.T. Kloos: Employee; Self; Veracyte, Inc.. Stock Owner; Self; Veracyte, Inc.. V. Weiss: None. Thyroid tumor molecular analysis often utilizes fine-needle aspirate (FNA) samples to predict malignancy with high sensitivity. An outstanding question is if molecular sequencing can provide prognostic information to predict disease aggression, potentially informing management. Recently, the composition of the tumor microenvironment, in particular the presence of tumor-promoting fibroblasts, has been associated with aggressive thyroid cancer. However, fibroblast gene expression has not previously been analyzed in thyroid tumor FNA samples. In this work, we detect cancer-associated fibroblast (CAF) gene signatures across 4 distinct bulk RNA sequencing cohorts and confirm detection in FNA samples. Associations between CAF gene signatures and tumor characteristics were explored in: Afirma Genomic Sequencing Classifier (GSC) cohort from FNA (n=47,695), a Memorial Healthcare System (MHS) retrospective FNA cohort with pathology outcome data (n=318), TCGA papillary thyroid cancers (n=496), and a Vanderbilt University Medical Center-University of Washington (VUMC-UW) resection cohort (n=312). Published breast CAF gene sets were used to generate normal fibroblast and CAF subtype scores. Novel thyroid cancer specific CAF gene sets were generated from thyroid cancer single-cell RNA sequencing data. Scores for each gene set were calculated as the average Z-score. Across all cohorts, multiple CAF subsets were enriched in samples with BRAFV600E and papillary histology. The strongest enrichment was in an SFRP2+ CAF subset identified in thyroid cancer single-cell sequencing data. In Afirma GSC FNA samples, SFRP2+ CAFs were enriched in GSC-suspicious and Bethesda V/VI relative to GSC-benign nodules (Wilcoxon p value < 2e-[1]6 for all). In TCGA, SFRP2+ CAFs were associated with extrathyroidal extension, advanced disease stage, and aggressive histology. In the MHS cohort of 318 malignant FNA samples, SFRP2+ CAFs were significantly higher in patients with vascular invasion or lymph node metastasis (LNM) (OR:2.75 [95% CI 1.63-4.75], p=4e-5). In the VUMC-UW cohort, SFRP2+ CAFs were associated with shorter progression-free survival (PFS) (p=0.0033). In summary we created a novel thyroid cancer-specific CAF gene signature that is elevated in BRAFV600E+ and aggressive thyroid cancers across multiple large patient cohorts. We also demonstrated the ability to detect these CAF signatures in thyroid FNAs using the Afirma GSC molecular testing platform, with enrichment in suspicious and malignant samples. Amongst the CAF scores tested, thyroid-specific CAF subsets had the highest prognostic potential, and SFRP2+ CAFs, specifically, were associated with shorter PFS, tumor invasion and LNM. Together, these results highlight the potential of interrogating the thyroid tumor stroma in FNA using RNA-based assays to inform prognosis and possibly management. Presentation: 6/3/2024

Functional heterogeneity of cancer-associated fibroblasts with distinct neoadjuvant immunotherapy plus chemotherapy response in esophageal squamous cell carcinoma

Novel neoadjuvant immunotherapy combined with chemotherapy (neoICT) has improved outcomes for patients with esophageal squamous-cell carcinoma (ESCC), but challenges persist in low response rates and therapy resistance. Little is known about the intra-tumoral heterogeneity in the ESCC tumor microenvironment (TME) that underlies differential responses to neoadjuvant therapy. We applied single-cell RNA sequencing (scRNA-seq) profiling and multiplexed immunofluorescence staining to thoroughly decipher the TME in ESCC specimens from a neoadjuvant anti-PD1 combination therapy clinical trial. The cancer-associated fibroblasts (CAFs) population showed the significant alteration in abundance following neoadjuvant therapy. Specifically, IL6 + CCL2 + immunomodulatory CAFs and a novel CD248 + mechanoresponsive CAFs subset exhibited increasing infiltration. Mechanistically, CD248 + mechanoresponsive CAFs approached and lined the tumor nest to physically block the infiltration of CD8 + T cells and drug delivery, while IL6 + CCL2 + immunomodulatory CAFs induced therapeutic resistance with distinct IL-6 expression. Among patients treated with neoICT, we observed prominent CAF-T cell interactions. In particular, the NECTIN2-TIGIT ligand-receptor pair was enriched in treated samples, and TIGIT was identified as the major inhibitory checkpoint of T cells. Our findings demonstrate distinct alterations in TME constituent responses to neoadjuvant immunotherapy and identify functional phenotypes of CAFs associated with unfavorable therapeutic responses in patients. This provides potential targets to enhance responses to neoadjuvant therapy in ESCC.

Abstract A060: Dysregulation of PTEN expression in a subset of Cancer Associated Fibroblasts by Tumor Secreted Factors in PDAC

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is a deadly malignancy. Cancer associated fibroblasts (CAFs) play a complex role in the PDAC tumor microenvironment (TME), with a subset contributing significantly to tumor progression. Phosphatase and tensin homolog (PTEN) is a tumor suppressor and its loss is associated with increased tumor aggressiveness. Our group previously demonstrated PTEN loss in SMA+ positive CAFS correlated with worsened outcomes in patients and in mouse PDAC models. Our group previously demonstrated the loss of PTEN in CAFS led to activation of Stat3, resulting in an immunosuppressive microenvironment (Lefler et al, 2022 PMID: 35803738). However, little is known of the signals produced by tumor cells or other cell types in the TME that trigger loss of PTEN in CAFs. This study aims to to elucidate the molecular mechanisms underlying PTEN loss in CAFs in PDAC. To address this gap, we employed single cell RNA sequencing of tumors from a mouse PDAC model and identified a (PDRGFRa+Lys6+Stat3+Pten-low) CAF subpopulation. Comparing normal pancreas epithelial cells to tumor cells in this data set, we identified the IL6 family of cytokines, (IL6; Oncostatin-M, OSM; and Leukemia Inhibitory Factor, LIF) and Transforming Growth Factor beta 1 (TGFb1) as potential ligands that regulated PTEN, while myeloid cells were a source of the IL6 family member Oncostatin M (OSM). Treatment of isolated CAFs with these factors revealed no significant difference in PTEN mRNA expression. Thus, we developed an in vitro reporter system in which a PTEN-GFP fusion protein is expressed in CAFs. Treating these CAFs with IL6, OSM and TGFb1 (but not LIF) led to a significant decrease in GFP fluorescence. Analysis of endogenous PTEN by western blot confirmed these results. We posited that expression of PTEN E3 ligases might be increased by treatment with these factors, leading to destruction of PTEN. In support of this hypothesis, the expression of E3 ligases WWP1 and WWP2 was increased by treatment with IL6 and OSM. Additionally, preliminary data has revealed that PTEN is oxidized in isolated CAFs, which causes loss of PTEN activity and potentially increased protein turnover. Current experiments are aimed at testing these potential mechanisms. To conclude, PTEN is dysregulated post-transcriptionally in CAFs. The mechanism of PTEN dysregulation could be via increased expression of E3 ligases that target PTEN and/or PTEN oxidation. Future efforts will be aimed at testing whether inhibition of these pathways can restore PTEN expression in mouse PDAC models. These pathways and interactions could be further exploited for therapeutic benefit in curbing PDAC progression. Citation Format: Ivo N. Woogeng, Lu Han, Samaneh Saberi, Cameron Bumbleburg, Joseph Beaudet, Sudarshana Sharma, Michael Ostrowski. Dysregulation of PTEN expression in a subset of Cancer Associated Fibroblasts by Tumor Secreted Factors in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A060.

Abstract B071: Antigen-presenting cancer-associated fibroblasts are required for ICI-sensitivity in pancreatic ductal adenocarcinoma

Abstract Cancer-associated fibroblasts (CAFs) are a hallmark of many cancers and are particularly prevalent in pancreatic ductal adenocarcinoma (PDAC). This highly fibrotic environment is thought to play a role in resistance to therapy; however, attempts to target CAFs have been unsuccessful in clinical trials. Deeper insights into CAF biology have revealed that these cells are heterogeneous in origin, gene expression, and function. RNA sequencing studies have identified a subset of CAFs, antigen-presenting CAFs (apCAFs), which are defined by MHC-II and interact with CD4 T cells. Given the importance of CD4 T cells in the antitumor response in PDAC, understanding apCAF biology could demonstrate ways to promote their differentiation and enhance T cell activity.Methods: Two clones differing in sensitivity to ICIs, sKPC (sensitive) and rKPC (resistant), derived from the autochthonous KPC murine PDAC model, were implanted orthotopically into the pancreas of syngeneic C57BL/6 mice. Mice were untreated, treated with dual checkpoint inhibition, or immunized with a KRAS peptide vaccine. Tumors were harvested for TME characterization, functional assays, single-cell RNA sequencing (scRNAseq), and imaging mass cytometry (IMC). apCAFs, CD11c+, and CD4+ T cells were isolated for functional assays. Peptide-pulsed apCAFs and CD11c+ cells were cultured with naïve OT-II CD4+ T cells or sKPC tumor infiltrating CD4+ T cells. apCAF KO mice were generated by crossing PDGFRαCre and MHC-IIflx mice.Results: Immunosensitive sKPC tumors are characterized by higher immune cell and apCAF infiltration than rKPC tumors. These apCAFs activate tumor-infiltrating CD4 T cells and induce regulatory T cell (Treg) differentiation, but not production of immunosuppressive cytokines. IMC analysis shows greater proximity of apCAFs to CD4 T cells in sKPC tumors compared with rKPC tumors. Remarkably, apCAF KO mice implanted with sKPC tumors lose sensitivity to ICI. scRNAseq has uncovered a unique transcriptome of apCAFs depending on tumor of origin and time following tumor implantation, revealing potential targets by which to quantitatively and qualitatively modulate apCAFs and enhance the antitumor response. Furthermore, scRNAseq of CD4 T cells cocultured with apCAFs has shown a unique pattern of CD4 T cell gene upregulation depending on the TME from which apCAFs were isolated, which could yield strategies to modulate not only apCAFs but also CD4 T cell activity.Conclusions: apCAFs are more abundant and more proximal to CD4 T cells in immunosensitive tumors, and apCAF KO sKPC tumor-bearing mice lose sensitivity to ICI. Together these data implicate apCAFs as essential mediators of the antitumor immune response. The unique transcriptome of apCAFs and cocultured CD4 T cells depending on tumor of origin suggests multiple strategies to modulate their interactions. Although a small proportion of the TME, modulating apCAF number or function could provide a solution to the currently unmet need of new and more effective treatments for PDAC patients. Citation Format: Saumya Maru, Meredith Wetzel, Jacob T Mitchell, Nicole Gross, Lalitya Andaloori, Kathryn Howe, Emma Kartalia, Guanglan Mo, James Leatherman, Won Jin Ho, Luciane Kagohara, Elana Fertig, Edward J Pearce, Elizabeth M Jaffee. Antigen-presenting cancer-associated fibroblasts are required for ICI-sensitivity in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B071.

Cancer-associated fibroblast cell surface markers as potential biomarkers or therapeutic targets in lung cancer.

Cancer-associated fibroblasts (CAFs) are the vital constituent of the tumor microenvironment, and in communication with other cells, they contribute to tumor progression and metastasis. Fibroblasts are the proposed origin of CAFs, which are mediated by pro-inflammatory cytokines and the recruitment of immune cells akin to wound healing. Although various studies have identified different subpopulations of CAFs in lung cancer, the heterogeneity of CAFs, particularly in lung cancer, and their potential as a therapeutic target remain largely unknown. Notwithstanding CAFs were previously thought to have predominantly tumor-promoting features, their pro- or anti-tumorigenic properties may depend on various conditions and cell origins. The absence of distinct markers to identify CAF subpopulations presents obstacles to the successful therapeutic targeting and treatment of CAFs in cancer. Human clinical and animal studies targeting CAFs have shown that targeting CAFs exacerbates the disease progression, suggesting that subpopulations of CAFs may exert opposing functions in cancer progression. Therefore, it is essential to pinpoint specific markers capable of characterizing these subpopulations and revealing their mechanisms of function. The cell-specific surface markers of CAFs will serve as an initial step in investigating precise CAF subpopulations and their role in diagnosing and targeting therapy against cancer-promoting CAF subsets in lung cancer.

Pathomics and single-cell analysis of papillary thyroid carcinoma reveal the pro-metastatic influence of cancer-associated fibroblasts

BackgroundPapillary thyroid carcinoma (PTC) is globally prevalent and associated with an increased risk of lymph node metastasis (LNM). The role of cancer-associated fibroblasts (CAFs) in PTC remains unclear.MethodsWe collected postoperative pathological hematoxylin–eosin (HE) slides from 984 included patients with PTC to analyze the density of CAF infiltration at the invasive front of the tumor using QuPath software. The relationship between CAF density and LNM was assessed. Single-cell RNA sequencing (scRNA-seq) data from GSE193581 and GSE184362 datasets were integrated to analyze CAF infiltration in PTC. A comprehensive suite of in vitro experiments, encompassing EdU labeling, wound scratch assays, Transwell assays, and flow cytometry, were conducted to elucidate the regulatory role of CD36+CAF in two PTC cell lines, TPC1 and K1.ResultsA significant correlation was observed between high fibrosis density at the invasive front of the tumor and LNM. Analysis of scRNA-seq data revealed metastasis-associated myoCAFs with robust intercellular interactions. A diagnostic model based on metastasis-associated myoCAF genes was established and refined through deep learning methods. CD36 positive expression in CAFs can significantly promote the proliferation, migration, and invasion abilities of PTC cells, while inhibiting the apoptosis of PTC cells.ConclusionThis study addresses the significant issue of LNM risk in PTC. Analysis of postoperative HE pathological slides from a substantial patient cohort reveals a notable association between high fibrosis density at the invasive front of the tumor and LNM. Integration of scRNA-seq data comprehensively analyzes CAF infiltration in PTC, identifying metastasis-associated myoCAFs with strong intercellular interactions. In vitro experimental results indicate that CD36 positive expression in CAFs plays a promoting role in the progression of PTC. Overall, these findings provide crucial insights into the function of CAF subset in PTC metastasis.

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.