Cancer-associated Fibroblasts Subtypes Research Articles

Identification of BGN positive fibroblasts as a driving factor for colorectal cancer and development of its related prognostic model combined with machine learning

BackgroundNumerous studies have indicated that cancer-associated fibroblasts (CAFs) play a crucial role in the progression of colorectal cancer (CRC). However, there are still many unknowns regarding the exact role of CAF subtypes in CRC.MethodsThe data for this study were obtained from bulk, single-cell, and spatial transcriptomic sequencing data. Bioinformatics analysis, in vitro experiments, and machine learning methods were employed to investigate the functional characteristics of CAF subtypes and construct prognostic models.ResultsOur study demonstrates that Biglycan (BGN) positive cancer-associated fibroblasts (BGN + Fib) serve as a driver in colorectal cancer (CRC). The proportion of BGN + Fib increases gradually with the progression of CRC, and high infiltration of BGN + Fib is associated with poor prognosis in terms of overall survival (OS) and recurrence-free survival (RFS) in CRC. Downregulation of BGN expression in cancer-associated fibroblasts (CAFs) significantly reduces migration and proliferation of CRC cells. Among 101 combinations of 10 machine learning algorithms, the StepCox[both] + plsRcox combination was utilized to develop a BGN + Fib derived risk signature (BGNFRS). BGNFRS was identified as an independent adverse prognostic factor for CRC OS and RFS, outperforming 92 previously published risk signatures. A Nomogram model constructed based on BGNFRS and clinical-pathological features proved to be a valuable tool for predicting CRC prognosis.ConclusionIn summary, our study identified BGN + Fib as drivers of CRC, and the derived BGNFRS was effective in predicting the OS and RFS of CRC patients.

Single-Cell Profiling Reveals Immune-Based Mechanisms Underlying Tumor Radiosensitization by a Novel Mn Porphyrin Clinical Candidate, MnTnBuOE-2-PyP5+ (BMX-001).

Manganese porphyrins reportedly exhibit synergic effects when combined with irradiation. However, an in-depth understanding of intratumoral heterogeneity and immune pathways, as affected by Mn porphyrins, remains limited. Here, we explored the mechanisms underlying immunomodulation of a clinical candidate, MnTnBuOE-2-PyP5+ (BMX-001, MnBuOE), using single-cell analysis in a murine carcinoma model. Mice bearing 4T1 tumors were divided into four groups: control, MnBuOE, radiotherapy (RT), and combined MnBuOE and radiotherapy (MnBuOE/RT). In epithelial cells, the epithelial-mesenchymal transition, TNF-α signaling via NF-кB, angiogenesis, and hypoxia-related genes were significantly downregulated in the MnBuOE/RT group compared with the RT group. All subtypes of cancer-associated fibroblasts (CAFs) were clearly reduced in MnBuOE and MnBuOE/RT. Inhibitory receptor-ligand interactions, in which epithelial cells and CAFs interacted with CD8+ T cells, were significantly lower in the MnBuOE/RT group than in the RT group. Trajectory analysis showed that dendritic cells maturation-associated markers were increased in MnBuOE/RT. M1 macrophages were significantly increased in the MnBuOE/RT group compared with the RT group, whereas myeloid-derived suppressor cells were decreased. CellChat analysis showed that the number of cell-cell communications was the lowest in the MnBuOE/RT group. Our study is the first to provide evidence for the combined radiotherapy with a novel Mn porphyrin clinical candidate, BMX-001, from the perspective of each cell type within the tumor microenvironment.

ScRNA-seq characterizing the heterogeneity of fibroblasts in breast cancer reveals a novel subtype SFRP4+ CAF that inhibits migration and predicts prognosis.

Cancer-associated fibroblasts (CAFs) are a diverse group of cells that significantly impact the tumor microenvironment and therapeutic responses in breast cancer (BC). Despite their importance, the comprehensive profile of CAFs in BC remains to be fully elucidated. To address this gap, we utilized single-cell RNA sequencing (scRNA-seq) to delineate the CAF landscape within 14 BC normal-tumor paired samples. We further corroborated our findings by analyzing several public datasets, thereby validating the newly identified CAF subtype. Additionally, we conducted coculture experiments with BC cells to assess the functional implications of this CAF subtype. Our scRNA-seq analysis unveiled eight distinct CAF subtypes across five tumor and six adjacent normal tissue samples. Notably, we discovered a novel subtype, designated as SFRP4+ CAFs, which was predominantly observed in normal tissues. The presence of SFRP4+ CAFs was substantiated by two independent scRNA-seq datasets and a spatial transcriptomics dataset. Functionally, SFRP4+ CAFs were found to impede BC cell migration and the epithelial-mesenchymal transition (EMT) process by secreting SFRP4, thereby modulating the WNT signaling pathway. Furthermore, we established that elevated expression levels of SFRP4+ CAF markers correlate with improved survival outcomes in BC patients, yet paradoxically, they predict a diminished response to neoadjuvant chemotherapy in cases of triple-negative breast cancer. This investigation sheds light on the heterogeneity of CAFs in BC and introduces a novel SFRP4+ CAF subtype that hinders BC cell migration. This discovery holds promise as a potential biomarker for refined prognostic assessment and therapeutic intervention in BC.

Exploring the association of POSTN+ cancer-associated fibroblasts with triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with a poor prognosis. Cancer-associated fibroblasts (CAFs) play a critical role in regulating TNBC tumor development. This study aimed to identify and characterize a specific subtype of CAFs associated with TNBC. Initially, using high-throughput bulk transcriptomic data in two cohorts, we identified three CAF-related subtypes (CS1, CS2, CS3) in TNBC samples. These three CAFs subtypes were closely linked to the tumor microenvironment. The CS1 subtype exhibited a relatively immune-rich microenvironment and a favourable prognosis, whereas the CS3 subtype displayed an immune-deprived tumor microenvironment and an unfavourable prognosis. Through WGCNA analysis, POSTN was identified as a key biomarker for CAFs associated with TNBC. Then, POSTN+CAFs was identified and characterized. Both POSTN and POSTN+CAFs showed significant positive correlations with stromal molecules HGF and MET at both the transcriptional and protein levels. Specifically co-localized with CAFs in the tumor stromal area, POSTN, produced by POSTN+CAFs, could modulate the HGF-MET axis, serving as a bypass activation pathway to regulate tumor cell proliferation in response to EGFR inhibitor and MET inhibitor. This study underscores the significance of POSTN and POSTN+CAFs as crucial targets for the diagnosis and treatment of TNBC.

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.

Cellular plasticity in the breast cancer ecosystem.

The complex interplay between genetically diverse tumor cells and their microenvironment significantly influences cancer progression and therapeutic responses. This review highlights recent findings on cellular plasticity and heterogeneity within the breast cancer ecosystem, focusing on the roles of cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). We discuss evidence suggesting that breast cancer cells exhibit phenotypic plasticity driven by both intrinsic genetic factors and external microenvironmental cues, impacting treatment responses and disease recurrence. Moreover, single-cell RNA sequencing studies reveal diverse subtypes of CAFs and TAMs, each with distinct functional gene expression programs and spatial organization within the tumor microenvironment. Understanding the hierarchical relationships and niche cues governing cellular phenotypes offers new opportunities for targeted therapeutic interventions. By elucidating the organizational principles of the tumor ecosystem, future therapies may target phenotypic states or entire cellular niches, advancing precision medicine approaches in breast cancer treatment.

Integration of Single-Cell and Bulk RNA-seq Data to Identify the Cancer-Associated Fibroblast Subtypes and Risk Model in Glioma.

Cancer-associated fibroblasts (CAFs) are an important component of the stroma. Studies showed that CAFs were pivotally in glioma progression which have long been considered a promising therapeutic target. Therefore, the identification of prognostic CAF markers might facilitate the development of novel diagnostic and therapeutic approaches. A total of 1333 glioma samples were obtained from the TCGA and CGGA datasets. The EPIC, MCP-counter, and xCell algorithms were used to evaluate the relative proportion of CAFs in glioma. CAF markers were identified by the single-cell RNA-seq datasets (GSE141383) from the Tumor Immune Single-Cell Hub database. Unsupervised consensus clustering was used to divide the glioma patients into different distinct subgroups. The least absolute shrinkage and selection operator regression model was utilized to establish a CAF-related signature (CRS). Finally, the prognostic CAF markers were further validated in clinical specimens by RT‒qPCR. Combined single-cell RNA-seq analysis and differential expression analysis of samples with high and low proportions of CAFs revealed 23 prognostic CAF markers. By using unsupervised consensus clustering, glioma patients were divided into two distinct subtypes. Subsequently, based on 18 differentially expressed prognostic CAF markers between the two CAF subtypes, we developed and validated a new CRS model (including PCOLCE, TIMP1, and CLIC1). The nomogram and calibration curves indicated that the CRS was an accurate prognostic marker for glioma. In addition, patients in the high-CRS score group had higher immune infiltration and tumor mutation burden levels. Moreover, the CRS score had the potential to predict the response to immune checkpoint blockade (ICB) therapy and chemotherapy. Finally, the expression profiles of three CAF markers were verified by RT‒qPCR. In general, our study classified glioma patients into distinct subgroups based on CAF markers, which will facilitate the development of individualized therapy. We also provided insights into the role of the CRS in predicting the response to ICB and chemotherapy in glioma patients.

Abstract 6524: Single-cell landscape of cancer-associated fibroblasts in immunotherapy resistance of hepatocellular carcinoma

Abstract Introduction: The advent of immune checkpoint blockade (ICB) therapy has revolutionized the treatment approach for hepatocellular carcinoma (HCC). However, the response rate to ICB therapy remains low (below 20%). This limited response rate can be attributed to the considerable heterogeneity observed in HCC cells and the tumor microenvironment (TME). Recent research has highlighted the pivotal role of cancer-associated fibroblasts (CAFs) in orchestrating various components within the TME of HCC. Although the significance of lipid metabolism-associated CAFs in the efficacy of ICB therapy has been demonstrated in HCC murine models [1], the functions and contributions of other CAF subtypes remain largely unknown. Therefore, identifying CAF heterogeneity could advance our comprehension of HCC TME and potentially augment ICB efficacy. Methods: We performed single-cell RNA sequencing on tumor biopsy specimens obtained from patients with advanced HBV-related HCC who had participated in a pembrolizumab (anti-PD1) phase II clinical trial (NCT03419481). Our objective was to examine the dynamic alterations in the HCC TME before and during anti-PD1 treatment (upon 2-cycle). We utilized the Seurat and Cellchat packages for bioinformatics analysis which enabled us to investigate the associations between changes in the proportions of CAF subtypes and the patients' clinical responses, as well as the potential cell-cell interactions involving CAF and other TME components. Results: We identified four CAF subtypes within the TME: cycling, inflammatory, matrix, and vascular CAFs in our anti-PD1 HCC patient cohort. Among the anti-PD1 non-responders, there was a significant increase in the proportions of cycling CAFs (cCAFs) and matrix CAFs (matCAFs) compared to the responders. Notably, matCAFs exhibited a high enrichment of Gene Ontology (GO) Terms related to the extracellular matrix (ECM), whose higher proportion correlated with poor prognosis of patients. Moreover, a distinct collagen pathway signal was found between matCAFs and two immunosuppressive cell types, namely regulatory T cells and M2 macrophages in the non-responders. Discussion and Conclusion: The presence of matCAFs within the HCC tumor may associate with the development of an immunosuppressive TME and unfavorable prognosis for patients. Targeting this specific CAF subtype holds promise as a potential strategy to overcome resistance to ICB therapy. Acknowledgments: The CUHK Strategic Seed Funding for Collaborative Research Scheme and The Li Ka Shing Foundation.

Abstract 291: Unveiling heterogeneity of cancer-associated fibroblasts (CAFs) across multiple solid cancer types

Abstract Background: Stromal cells, including CAFs, are a heterogeneous group of cells in the tumor microenvironment. Many models describe their heterogeneity like the classification into 3 main subtypes in breast cancer: myCAF (myofibroblasts), iCAF (inflammatory CAF), and PVL (perivascular-like) cells. However, a pan-cancer model has not been established to exhaustively characterize stromal cell heterogeneity, undermining efforts in targeting CAFs with anti-tumor therapies. We investigated the heterogeneity of stromal cells in diverse diagnoses with single-cell RNA sequencing (scRNA-seq) data and bulk RNA-seq of sorted stromal cells to define consistent subtypes. Design: First, a meta-cohort of 11 scRNA-seq open-source datasets (>500,000 cells, >100 patients, 6 diagnoses) was analyzed. We also generated a scRNA-seq dataset of CD90+ cells derived from tumor tissue of an ovarian cancer patient. Additionally, bulk RNA-seq of 6 PVL, 2 iCAF, and 4 myCAF sorted cell samples across four solid cancers was performed. scRNA-seq data was analyzed with the SCANPY Python package. Results: Stromal cell clusters were defined as positive for PDGFRB, THY1 (CD90), and COL1A1 expression, and negative for the expression of known non-stromal and endothelial cell markers, by separately analyzing the open-source scRNA-seq datasets. Analysis of stromal cell heterogeneity revealed that the CAFs were consistently classified into 3 subtypes across all datasets: myCAF (high expression: FAP and ACTA2), PVL (high expression: MCAM (CD146) and ACTA2), and iCAF (high expression: APOD and cytokines IL6, CXCL12, CXCL2). Differential gene expression analysis of the scRNA-seq meta-cohort allowed us to develop functional gene expression signatures to define each subtype. All scRNA-sequenced CD90+ cells from ovarian cancer tissue were classified into the CAF subtypes. The myCAF and PVL signatures were specifically upregulated in the corresponding cell types (p<0.01, Mann-Whitney U test). While the iCAF signature was enriched in both iCAFs and myCAFs, myCAF and PVL signatures were downregulated in iCAFs. Finally, we developed a fluorescence-activated cell sorting (FACS) panel to separate CAF subtypes, using negative markers (CD45, CD31, EPCAM), a positive marker for stromal cells (CD90) and cell-type surface markers (CD146, FAP, CD91, CD44) for myCAF, iCAF and PVL cells. The high expression of CAF-specific genes in the sorted CAF populations denoted high sample purity. Importantly, the 3 CAF subpopulations separated by gating based on different levels of cell-type surface markers showed expression patterns congruent with the developed signatures across diagnoses. Summary: Altogether, the consistent classification of stromal cells with our cell-specific expression profiles demonstrated that the 3 CAF subtypes are conserved in many cancer types, offering groundwork for a pan-cancer model of stromal cell heterogeneity. Citation Format: Andrey Kravets, Boris Shpak, Anastasia Zotova, Maria Savchenko, Sofya Kust, Olga Golubeva, Mary Abdou, Fedor Grigoryev, Irina Bulusheva, Beniamin Sargsyan, Polina Pavlovich, Vlad Maximov, Vladimir Kushnarev, Michael Goldberg, Nikita Kotlov, Alexander Bagaev. Unveiling heterogeneity of cancer-associated fibroblasts (CAFs) across multiple solid cancer types [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 291.

Abstract 1578: RAS-GTP inhibition modulates Hedgehog signaling, suppressing myCAFs and promoting iCAFs in pancreatic ductal adenocarcinoma

Abstract More than 90% of human pancreatic ductal adenocarcinoma (PDAC) cases are driven by activating mutations in KRAS. RMC-7977 is a potent inhibitor of GTP-bound RAS proteins (RAS(ON), including both wild type and mutant variants of KRAS, NRAS, and HRAS. The related investigational agent, RMC-6236, is a first-in-class, potent, orally bioavailable, RASMULTI(ON) inhibitor currently in Phase 1/1b clinical trials (NCT05379985). We performed preclinical studies in a range of models of PDAC, including the highly chemoresistant K-rasLSL.G12D/+, p53LSL.R172H/+, Pdx1Cretg/+ (KPC) genetically engineered mouse. RMC-7977 exhibited broad anti-tumor activity at tolerable doses, provoking radiographic responses in KPC pancreatic tumors and extending overall survival by 3-fold, the largest response observed yet in this model. RMC-7977 dosing produced a metronomic effect on RAS signaling in tumor and normal tissues. This yielded tumor-selective effects on apoptosis and proliferation, consistent with RAS oncogene addition in PDAC. PDAC tumors are characterized by an expansive stroma that harbors multiple subtypes of cancer associated fibroblasts (CAFs) that manufacture a fibrotic, inflammatory extracellular matrix. CAF proliferation is driven by signals emanating from the malignant epithelial cells, such as Sonic Hedgehog (SHH), a secreted ligand that drives downstream Hedgehog pathway signaling in myofibroblastic CAFs (myCAFs). To understand the impact of RAS-GTP inhibition on paracrine signaling to CAFs in PDAC, To understand the dynamic responses of malignant epithelial cells following GTP-RAS inhibition, we performed single cell RNA sequencing (scRNAseq) on KPC pancreatic tumors treated with RMC-7977 or vehicle at multiple timepoints. Using the ARACNe and VIPER algorithms, we performed single cell regulatory network analysis on the expression profiles of >210,000 cells to calculate the activities of thousands of transcriptional regulatory protein in each cell. We found that the RAS-GTP inhibition blocked SHH expression in malignant epithelial cells, resulting in loss of downstream Hh pathway activity in CAFs within 24 hours of treatment. By one week of treatment, myCAFs were significantly depleted whereas inflammatory CAFs (iCAFs) were significantly accumulated. Strikingly, CAF heterogeneity was restored in KPC pancreatic tumors that acquired resistance to RMC-7977, despite continued suppression of Hedgehog pathway signaling. Consistent with prior studies of Smoothened inhibitors, the depletion of myCAFs from RAS-GTP inhibition was associated with the rapid opening and hypersprouting of intra-tumoral blood vessels. Ongoing studies using the mutant-selective KRASG12D inhibitor RM-044 should discriminate between direct effects of wild type RAS inhibition in CAFs versus the paracrine consequences of mutant RAS inhibition in malignant epithelial cells. Citation Format: Marie Hasselluhn, Lorenzo Tomassoni, Urszula Wasko, Alvaro Curiel-Garcia, Tanner Dalton, Steve A. Sastra, Carmine Palermo, Andrea Califano, Kenneth P. Olive. RAS-GTP inhibition modulates Hedgehog signaling, suppressing myCAFs and promoting iCAFs in pancreatic ductal adenocarcinoma [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 1578.

Abstract 283: Fibroblast diversity and immune microenvironment in pseudoangiomatous stromal hyperplasia (PASH): Associations with breast cancer risk

Abstract Benign breast disease (BBD) affects over one million women annually in the United States and is diagnosed in nearly half of all women during their lifetime. Pseudoangiomatous stromal hyperplasia (PASH), a common BBD subtype, is characterized by an overaccumulation of tissue fibroblasts. Current research suggests a pivotal role of the immune microenvironment in progression of BBD to breast cancer, suggesting its composition as a determinant of breast cancer risk. Our study evaluates whether fibroblast variations and immune microenvironment differences in BBD biopsies containing PASH lesions are associated with subsequent breast cancer development. We evaluated BBD biopsies from women who subsequently developed breast cancer (cases, N=5) or remained cancer-free (controls, N=5). Using Opal-based multiplex immunofluorescence (mIF) assays, we mapped expression of fibroblast markers linked to breast cancer associated fibroblasts (CAFs), including vimentin (VIM), smooth muscle actin (ACTA2), fibroblast specific protein-1 (S100A4/FSP1), fibroblast associated protein (FAP), integrin beta-1 (CD29/ITGB1), and platelet derived growth factor receptor beta (PDGFRβ) and markers of key immune cells (T cells: CD4, CD8; dendritic cells: CD11c; B cells: CD20; and macrophages: CD68) along with MKI67 as a proliferation marker. Our results revealed marked differences in fibroblast marker expression and immune cell populations between these groups in the normal lobules in the BBD biopsies. PASH cases showed elevated PDGFRβ and CD29/ITGB1 expression in VIM-positive fibroblasts, markers characteristic of CAF subtypes known to accumulate in more aggressive tumor microenvironments. Conversely, a higher percentage of FSP1+/VIM+ cells, similar to that of a different CAF subtype, was noted in the lobules of PASH controls. This difference in biomarker expression, along with corresponding variations in immune cell counts, suggests a complex interplay between fibroblast composition and immune response that ultimately modulate cancer risk in PASH-containing BBD tissues. Additionally, increased MKI67 expression in PASH cases potentially signals the higher likelihood of cancer development. This work advances our understanding of fibroblast heterogeneity in BBD and its implications for breast cancer risk and progression, and paves the way for innovative strategies to improve clinical outcomes for BBD patients to reduce their incidence of breast cancer. Citation Format: Nicole Cruz-Reyes, Matilde Rossi, Melody Stallings Mann, Bryan McCauley, Tanya Hoskin, Robert Vierkant, Stacey Winham, Amy Degnim, Mark E. Sherman, Derek C. Radisky. Fibroblast diversity and immune microenvironment in pseudoangiomatous stromal hyperplasia (PASH): Associations with breast cancer risk [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 283.

Abstract 1083: Cell membrane-anchored and tumor-targeted IL12 T-cell therapy destroys cancer-associated fibroblasts and disrupts extracellular matrix in heterogenous osteosarcoma xenograft models

Abstract Background: The extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs) play major roles in tumor progression, metastasis, and the poor response of many solid tumors to immunotherapy. CAF-targeted CAR-T cell therapy cannot infiltrate ECM-rich tumors such as osteosarcoma. Method: In this study, we used RNA sequencing to assess whether the recently invented membrane-anchored and tumor-targeted IL12-armed (attIL12) T cells, which bind cell-surface vimentin (CSV) on tumor cells, could destroy CAFs to disrupt the ECM. We established an in vitro model of the interaction between osteosarcoma CAFs and attIL12-T cells to uncover the underlying mechanism by which attIL12-T cells penetrate stroma-enriched osteosarcoma tumors. Results: RNA sequencing demonstrated that attIL12-T cell treatment altered ECM-related gene expression. Immunohistochemistry staining revealed disruption or elimination of high-density CAFs and ECM in osteosarcoma xenograft tumors following attIL12-T cell treatment, and CAF/ECM density was inversely correlated with T-cell infiltration. Other IL12-armed T cells, such as wild-type IL12- or tumor-targeted IL12-T cells, did not disrupt the ECM because this effect depended on the engagement between CSV on the tumor cell and its ligand on the attIL12-T cells. Mechanistic studies found that attIL12-T cell treatment elevated IFNγ production upon interacting with CSV+ tumor cells, suppressing TGFβ secretion and in turn upregulating FAS-mediated CAF apoptosis. CAF destruction reshaped the tumor stroma to favor T-cell infiltration and tumor inhibition. Conclusions: This study unveiled a novel therapy—attIL12-T cells—for targeting CAFs/ECM. These findings are highly relevant to humans because CAFs are abundant in human osteosarcoma. What is already known on this topic: As the major source of ECM factors in tumors, the FAP+αSMA+ subtype of CAFs within tumors induce production of ECM factors, preventing antitumor T cells from accessing the core regions of tumors. What this study adds: Our group demonstrated that attIL12-T cell transfer (US Patent: 11421010) induced T-cell infiltration into and eliminated large osteosarcoma PDX tumors. Comparison of gene expression in sensitive or resistant PDX models to attIL12-T cell therapy elucidated the ECM-targeting mechanism of attIL12-T cells. Our in vitro system further determined that the CSV-attIL12 interaction mediated IFNγ upregulation, which triggered FAS induction in CAFs to induce CAF apoptosis. In sum, attIL12-T cell treatment shifts tumor stroma from a TGFβ-dependent CAF-promoting phenotype to an IFNγ-induced immune-inflamed phenotype. How this study might affect research, practice or policy: attIL12-T cell transfer is a novel anti-ECM strategy that renders ECM-rich tumors more susceptible to T cell-mediated tumor killing. Citation Format: Jiemiao Hu, Alexander Lazar, Wei-Lien Wang, Wendong Zhang, Zhiliang Jia, Dristhi Ragoonanan, Jian Wang, Xueqing Xia, Kris Mahadeo, Richard Gorlick, Shulin Li. Cell membrane-anchored and tumor-targeted IL12 T-cell therapy destroys cancer-associated fibroblasts and disrupts extracellular matrix in heterogenous osteosarcoma xenograft models [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 1083.

Abstract 1577: Cancer associated fibroblast - tumor cell crosstalk enhances epithelial to mesenchymal transition and promotes a classical to basal switch in human pancreatic ductal adenocarcinoma

Abstract Introduction: Cancer associated fibroblasts (CAF) drive a complex tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) through incompletely understood interactions. Two major CAF subtypes, myofibroblastic (myCAF) and inflammatory (iCAF), dominate the tumor landscape and have been defined by gene expression profiles. There is significant plasticity between subtypes and alterations in CAF behavior likely drive changes in tumorigenicity and immune evasion. Additionally, PDAC tumor cells usually present with a predominant classical or basal phenotype that is associated with clinical outcomes. We hypothesized that CAF phenotype impacts tumor cell gene expression and alters the clinically-relevant tumor subtyping. Methods: We interrogated cellular crosstalk in the PDAC TME using a novel three-dimensional, patient-matched coculture system of patient-derived organoids (PDO) and CAFs, established from 12 patients undergoing surgical resection. Molecular characterization included bulk RNA sequencing, proteome profiling (107 factor), flow cytometry, multiplex RNA Scope, imaging mass cytometry (IMC), and RT-qPCR. Results: PDO gene expression analysis from bulk RNAseq demonstrated CAF coculture induced phenotypic shifts in the Moffitt subtype from a classical to a basal phenotype. Further, CAF coculture enhanced epithelial to mesenchymal transition (EMT) across all patients, even those with a baseline basal phenotype. We hypothesized this could be due to both cell-cell crosstalk and secreted factors. We therefore treated PDO with CAF-conditioned media (CM) and demonstrated that CM is sufficient to drive the observed classical-to-basal shift by RT-qPCR. Secretome analysis of CM derived from 3 CAF lines identified HGF, FGF19, and IL-8 as factors contributing to this transcriptional change. Next, we used spatial - omics (transcriptomics and proteomics) to examine the validity of these findings in source patient tissue. Using RNA Scope, we discovered distinct tumor neighborhoods expressing basal mRNAs KRT17 and KRT6a surrounded by CAFs compared to those expressing classical genes TFF1 and GATA6. We then used IMC to further explore CAF subtypes in patient tumors and found FAP+ myCAF regions corresponded to basal tumor regions, while CXCL12+ iCAFs were found in classical regions. Conclusions: PDAC tumor biology is likely driven by interactions between cancer cells and CAFs. Potential mediators of these interactions include both cell-cell contact and secreted factors. We introduce a novel approach combining in vitro coculture with tissue-based assays, demonstrating that modern culture techniques preserve cell type heterogeneity and plasticity. Together, these data demonstrate that a CAF-rich TME drives tumor cell plasticity not only towards EMT but also a more basal disease phenotype. Citation Format: Samantha Guinn, Brayan Perez, Jignasha Patel, Jae Lee, Daniel Zabransky, Won Ho, Elana J. Fertig, Emma Kartalia, Joseph Tandurella, Richard Burkhart, Jacquelyn W. Zimmerman, Elizabeth Jaffee. Cancer associated fibroblast - tumor cell crosstalk enhances epithelial to mesenchymal transition and promotes a classical to basal switch in human pancreatic ductal adenocarcinoma [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 1577.

Abstract 2342: BARTsc: A transcription factor analysis suite for single-cell omics data

Abstract In Eukaryotic cells, the expression of a small set of transcription factors (TFs) is usually sufficient to set up a cell-type-specific gene expression program. The underlying complicated epigenetic mechanisms that explain how cells change their states in response to intra- and extra-cellular signals in different scenarios remain to be fully understood. Importantly, epigenetic regulation of gene expression has been considered a central regulatory mechanism of cell fate determination and cellular plasticity. Insights into it will also profoundly impact our understanding of misregulation of gene expression in diseases such as cancer. However, the functional TFs of many cell types remain unclear, which greatly hinders the study of epigenetic regulatory mechanisms. The rapid development of single-cell technology is a promising opportunity that enables us to infer functional TFs for different cell types. Some computational methods have been published in this area. However, existing methods have various pitfalls. For instance, some methods use transcription factor binding motifs (TFBMs) as the reference of TF binding, overlooking widespread non-specific binding. Some other methods hold strict criteria for training data and thus can only predict a small number of TFs. To overcome these issues, we propose to leverage publicly available high-quality ChIP-seq data to predict TFs functional in single-cell omics data. Here, we present BARTsc, a comprehensive suite for a series of TF analysis functionalities, including inference of signature TFs of given cell types/clusters, cross-cell-type/cluster TF activity comparative analysis, and TF clustering based on their cross cell-type distributions. The input of BARTsc can be either scRNA-seq, scATAC-seq, or scMultiome data. We demonstrate that BARTsc outperforms several existing tools in both sensitivity and specificity in identifying key cell type regulators in single-cell datasets of well-studied systems. Applying BARTsc to a scRNA-seq dataset for cancer-associated fibroblasts (CAFs), we identified distinct regulatory activities of some TFs in different subtypes of CAFs, but the TF genes are universally expressed in these subtypes. In addition, by time course BARTsc analysis, we identified a list of TFs that may either promote or suppress CAF differentiation, offering a new approach to find potential candidate therapeutic targets. Citation Format: Hongpan Zhang, Jingyi Wang, Zhenjia Wang, Chongzhi Zang. BARTsc: A transcription factor analysis suite for single-cell omics data [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 2342.

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 6831: Single-cell-based epigenetic and transcriptomic analysis in the KPC pancreatic cancer mouse model revealed dynamiccharacteristic subtypes of cancer-associated fibroblasts

Abstract Pancreatic cancer features robust fibrosis, with 40-80% fibrotic tissue and a dense stroma, highlighting the crucial need to grasp cellular interactions in the cancer microenvironment. In this study, we used single-cell RNA sequencing (scRNA-seq) to analyze pancreatic tissues from 60-day-old normal mouse (n=1) (GSE125588) and KPC (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre) cancer model with tumor diameter of 1-2 cm (n=2) (GSE129455). By examining fibroblast clusters, we identified distinct subtypes of cancer-associated fibroblasts (CAFs), and revealed four clusters: normal fibroblasts cluster, inflammatory CAFs (iCAFs) cluster marked by increased expression of IL6 and other cytokines, myofibroblast CAFs (myCAFs) cluster solely in the KPC model, and antigen-presenting CAFs (apCAFs) cluster expressing Mesothelin. We found that expression pattern of iCAFs in the KPC appeared similar to normal cells than myCAFs, suggesting a potential role in inhibiting cancer. it is reported that mesothelial cells might transition to apCAFs under the influence of IL6 and TGFβ due to tissue damage and inflammatory effects. This transition was visualized in our apCAF cluster, suggesting a potential pathway for the transformation of mesothelial cells into apCAFs. Next, in order to analyze epigenetic profiles of fibroblasts, we conducted single-cell Assay for Transposase-Accessible Chromatin with high-throughput sequencing (scATAC-seq) using 90-day-old KPC mice (n=2) and a wild-type mouse (n=1).scATAC-seq showed that there are mainly two clusters of fibroblasts, one cluster corresponded to myCAF, enriched with PDPN, while the other represented iCAF cluster, enriched with COL1A2. These data suggest that in terms of the epigenetic state, myCAF contain several different CAFs and have the potential to differentiate into either myCAFs or other type of based on certain stimulation. Ultimately, CAF differentiated into three distinct subtypes as revealed by scRNA-seq. This highlights that despite similar epigenetic states, various transcription factors characterize the functionality of these fibroblast subtypes. Further exploration of the underlying mechanism that lead to different CAF subtypes might help in controlling the microenvironment of pancreatic cancer. Citation Format: Keita Sasaki, Keiko Shinjo, Xingxing Wang, Kohei Kumegawa, Reo Raruyama, Shinji Mii, Yoshiteru Murofushi, Miho Suzuki, Atsushi Enomoto, Yutaka Kondo. Single-cell-based epigenetic and transcriptomic analysis in the KPC pancreatic cancer mouse model revealed dynamiccharacteristic subtypes of cancer-associated fibroblasts [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 6831.

Abstract 4274: Unraveling collagen signatures in cancer-associated fibroblasts: A biomarker-driven approach

Abstract Background: The tumor microenvironment (TME) plays a crucial role in driving tumor development. Among the constituents of the tumor stroma, cancer-associated fibroblasts (CAFs) are a significant component. CAFs are actively involved in tumor progression by modulating the architecture of the TME through increased deposition of various collagens resulting in tumor fibrosis. Several studies have shown that CAFs have heterogeneity within, and between, individual tissues. TGF-β is thought to be the main driver of tumor fibrosis, however, the field lacks a characterization of the specific collagen deposition of CAFs from different tissues. In this study, we investigated the fibrotic activity of CAFs from various tissues by measuring the production of three specific collagen peptides in vitro by use of non-invasive clinically validated biomarkers. Methods: Primary human CAFs from pancreas (pCAF), colon (cCAF), breast (bCAF) and lung (lCAF) were cultured over a 12-day period in ficoll-based media under both unstimulated conditions and TGF-β stimulation. Additionally, cells were subjected to treatment with the ALK5/TGF-β1 receptor kinase inhibitor (ALK5i). The assessment of collagen I (PRO-C1), collagen III (PRO-C3), and collagen VI (PRO-C6) formation was conducted in the cell supernatant at day 3, day 6, day 9, and day 12 using competitive enzyme-linked immunosorbent assay (ELISA). Results: For lCAFs, cCAFs, and pCAFs, the addition of TGF-β led to a notable elevation in the levels of PRO-C1, PRO-C3, and PRO-C6 within the supernatant. Conversely, in the case of bCAFs, stimulation with TGF-β did not elicit remarkable alterations in the levels of PRO-C1 and PRO-C3 compared to unstimulated cells. Notably, bCAFs exhibit elevated intrinsic activity (high PRO-C3) even in the absence of TGF-β stimulation whereas lCAFs and cCAFs exhibited relatively low level of PRO-C3. Additionally, across all different CAFs, the introduction of ALK5 inhibitor (ALK5i) to the culture medium resulted in a considerable reduction in PRO-C1, PRO-C3, and PRO-C6 levels, returning them to the baseline of the unstimulated control or even lower. Distinct collagen formation patterns were observed among the various CAFs. CAFs derived from breast cancer exhibited the highest release of PRO-C1 and PRO-C3, while CAFs isolated from colon cancer displayed the highest levels of PRO-C6. Conclusion: These findings underscore the heterogeneity in collagen production among different CAFs, providing valuable insights into the extracellular matrix dynamics within distinct tumor microenvironments. Moreover, non-invasive biomarkers demonstrate the capability to differentiate between different CAF subtypes. Lastly, our model proves to be a valuable tool for anti-fibrotic drug screening, as evidenced by the consistent suppression of TGF-β1 signaling and reduced levels of PRO-C1, PRO-C3, and PRO-C6 upon treatment with ALK5i across all cases. Citation Format: Annika Hettich, Neel I. Nissen, Morten A. Karsdal, Nicholas Willumsen. Unraveling collagen signatures in cancer-associated fibroblasts: A biomarker-driven approach [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 4274.

Abstract 1597: CD144+cancer-associated fibroblasts drive the malignancy of pancreatic cancer via stimulating inflammatory paracrine

Abstract Purpose: Cancer-associated fibroblasts (CAFs) are highly heterogeneous in pancreatic ductal adenocarcinoma (PDAC). We previously reported that CAFs can acquire an endothelial phenotype under stress. Thus, we hypothesized that there are CAFs highly expressing endothelial markers and substantiated a novel subtype of CAFs promoting tumor progression. Experimental design: We performed single-cell RNA sequencing to identify the existence of CD144+CAFs. The prospective and retrospective analysis were combined to figure out the correlation between CD144+CAFs proportion and clinical outcome. The proliferation and invasion assay of cell lines, organoids and pancreatic cancer xenograft model were used to detect the effect of CD144+CAFs on tumor progression. Cytokine array assay, RNA-sequencing, IP-mass spectrum, Ch-IP and luciferase analysis were conducted to elucidate the underlying mechanism. siRNA delivery nanosystem was designed and administrated to precisely target CD144+CAFs in vivo. Results: CD144+CAFs were present in tumor microenvironment of PDAC and breast cancer and patients with a higher CD144+CAFs proportion have worse prognosis in PDAC. CD144+CAFs can promote the metastasis of PDAC through enhancing epithelial-mesenchymal transition. CD144-β-catenin-STAT3 signaling axis was activated and downstream inflammatory secreted cytokines were transcriptionally upregulated to maintain the functions and phenotype of CD144+CAFs. A novel CAF-targeting siRNA delivery nanosystem, loading FAPα and siCDH5, was designed and administrated to precisely target CD144+CAFs, which substantially inhibited the protumoral roles in vivo. Conclusion: CD144+CAFs can promote the growth and metastasis of PDAC by activating CD144-β-catenin-STAT3 signaling axis and stimulating inflammatory secretion. CAF-targeting siRNA delivery nanosystem can inhibit tumor progression by precisely targeting CD144+CAFs. Citation Format: Xugang Sun, Jun Yu, Tiansuo Zhao, Jihui Hao. CD144+cancer-associated fibroblasts drive the malignancy of pancreatic cancer via stimulating inflammatory paracrine [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 1597.

Abstract 1589: Integrated multiomics analysis revealed the significance of Transgelin in driving pancreatic cancer-associated fibroblast-mediated cancer progression

Abstract Pancreatic ductal adenocarcinoma (PDAC) is primarily characterized by a desmoplastic reaction, mainly consisting of cancer-associated fibroblasts (CAFs). In order to investigate the heterogeneity of CAFs, we initially conducted single-cell Assay for Transposase-Accessible Chromatin with high-throughput sequencing (scATAC-seq) using pancreas tissue from 12-week-old KPC (K-rasLSL.G12D/+; Trp53R172H/+; Pdx-1-Cre) PDAC mouse models. We identified three clusters enriched with myofibroblast CAF (myCAF) gene markers and one cluster enriched with inflammatory CAF (iCAF) gene markers. Next, we performed single-cell RNA sequencing (scRNA-seq) on pancreas tissues from KPC mice. scRNA-seq revealed four distinct fibroblast clusters, each exhibiting characteristic gene expression profiles of normal fibroblasts, iCAF, myCAF and antigen-presenting CAF (apCAF). These findings suggest that CAFs in the myCAF cluster identified in scATAC-seq may have potential to differentiate into either myCAFs or other CAF subtypes based on certain cues. Next, we focus on the expression of myCAF marker genes, particularly two commonly used markers, Tagln and Acta2. Among myCAFs, two subtypes were identified: Tagln-positive and Acta2-positive CAFs, and Tagln-negative and Acta2-positive CAFs. To confirm our findings, we conducted Immunohistochemical analysis (IHC) on KPC pancreas samples. We observed CAFs that were positive for both Talng and αSMA exclusively in close proximity to tumor cells. Fibroblast expressing only αSMA mostly located in normal area. Tagln is known as an actin-binding protein and plays an important role in activated fibroblasts. Orthotopic PDAC mouse models using Tagln homozygous knockout mice showed a significant reduction in tumor weight compared to the wild-type mice (P = 0.0076). Indeed, we found that high TAGLN expression in PDAC samples was associated with poor survival, as demonstrated by the analysis of the TCGA dataset. These data suggest that myCAFs expressing TAGLN contribute to the promotion of PDAC growth. Targeting stromal TAGLN might be a potential therapeutic strategy to counteract PDAC progression. Citation Format: Keiko Shinjo, Xingxing Wang, Kohei Kumegawa, Reo Maruyama, Shinji Mii, Yoshiteru Murofushi, Miho Suzuki, Atsushi Enomoto, Yutaka Kondo. Integrated multiomics analysis revealed the significance of Transgelin in driving pancreatic cancer-associated fibroblast-mediated cancer progression [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 1589.

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.