Cancer-associated fibroblasts in bladder cancer: Immunosuppressive mechanisms and therapeutic targeting.

Cancer-associated fibroblasts (CAFs) play a fundamental role in the development of cancers and their response to therapy. In recent years, CAFs have returned to the spotlight as researchers work to unpick the mechanisms by which they impact tumour evolution and therapy responses. However, study of CAFs has largely been restricted to a select number of common cancers, whereas research into CAF biology in bladder cancer has been relatively neglected. In this review, we explore the basics of CAF biology including the numerous potential cellular origins of CAFs, alongside mechanisms of CAF activation and their diverse functionality. We find CAFs play an important role in the progression of bladder cancer with significant implications on tumour cell signaling, epithelial to mesenchymal transition and the capacity to modify components of the immune system. In addition, we highlight some of the landmark papers describing CAF heterogeneity and find trends in the literature to suggest that the iCAF and myCAF subtypes defined in bladder cancer share common characteristics with CAF subtypes described in other settings such as breast and pancreatic cancer. Moreover, based on findings in other common cancers we identify key therapeutic challenges associated with CAFs, such as the lack of specific CAF markers, the paucity of research into bladder-specific CAFs and their relationship with therapies such as radiotherapy. Of relevance, we describe a variety of strategies used to target CAFs in several common cancers, paying particular attention to TGFβ signaling as a prominent regulator of CAF activation. In doing so, we find parallels with bladder cancer that suggest CAF targeting may advance therapeutic options in this setting and improve the current poor survival outcomes in bladder cancer which sadly remain largely unchanged over recent decades.

Colorectal cancer (CRC) progression is heavily influenced by the tumor microenvironment (TME), where cancer-associated fibroblasts (CAFs) are key players. However, the heterogeneity, plasticity, and functional roles of CAFs in CRC remain poorly understood. We integrated single-cell RNA sequencing (scRNA-seq) data from four public CRC datasets and spatial transcriptomics data. Using computational approaches such as Harmony, Monocle2, and CellChat algorithms, we analyzed cellular landscapes, CAF subtype identification, developmental trajectories, transcription factor networks, and cell-cell communications to reveal CAF heterogeneity and their crosstalk with other cell subtypes in CRC. We identified eight distinct CAF subtypes with unique gene expression profiles and developmental plasticity. The CTHRC1+ CAF subtype was significantly associated with T cell exclusion and upregulated expression of immune checkpoint genes. We uncovered a specific communication axis between CTHRC1+ CAFs and MMP7+ malignant epithelial (Malig-Epi) cells mediated by the thrombospondin (THBS)2-SDC4 ligand-receptor signaling. High infiltration of both cell types synergistically correlates with worse prognosis and unfavorable response to immunotherapy. Our study delineates CAF heterogeneity in CRC and highlights the CTHRC1+ CAF subtype as a critical organizer of an immunosuppressive niche. The THBS2-SDC4 signaling pathway between CTHRC1+ CAFs and MMP7+ epithelial cells acts as a potential therapeutic target to disrupt protumorigenic crosstalk and improve clinical outcomes for CRC patients.

Cancer-associated Fibroblasts in Bladder Cancer: Origin, Biology, and Therapeutic Opportunities

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

BackgroundOsteosarcoma (OS), with a peak incidence during the adolescent growth spurt, is correlated with poor prognosis for its high malignancy. The tumor microenvironment (TME) is highly complicated, with frequent interactions between tumor and stromal cells. The cancer-associated fibroblasts (CAFs) in the TME have been considered to actively involve in the progression, metastasis, and drug resistance of OS. This study aimed to characterize cellular heterogeneity and molecular characterization in CAFs subtypes and explore the potential targeting therapeutic strategies to improve the prognosis of OS patients. MethodsThe single-cell atlas of human OS tumor lesions were constructed from the GEO database. Then significant marker genes and potential biological functions for each CAFs subtype were identified and explored using the Seurat R package. Next, by performing the survival analyses and constructing the risk scores for CAFs subtypes, we aimed to identify and characterize the prognostic values of specific marker genes and different CAFs subtypes. Furthermore, we explored the therapeutic targets and innovative drugs targeting different CAFs subtypes based on the GDSC database. Finally, prognoses related CAFs subtypes were further validated through immunohistochemistry (IHC) on clinical OS specimens. ResultsOverall, nine main cell clusters and five subtypes of CAFs were identified. The differentially expressed marker genes for each CAFs clusters were then identified. Moreover, through Gene Ontology (GO) enrichment analysis, we defined the CAFs_2 (upregulated CXCL14 and C3), which was closely related to leukocyte migration and chemotaxis, as inflammatory CAFs (iCAFs). Likewise, we defined the CAFs_4 (upregulated CD74, HLA-DRA and HLA-DRB1), which was closely related to antigen process and presentation, as antigen-presenting CAFs (apCAFs). Furthermore, Kaplan-Meier analyses showed that CAFs_2 and CAFs_4 were correlated with poor clinical prognosis of OS patients. Meanwhile, therapeutic drugs targeting CAFs_2 and CAFs_4, such as 17-AAG/Docetaxel/Bleomycin and PHA-793887/NG-25/KIN001-102, were also explored, respectively. Finally, IHC assay confirmed the abundant CAFs_2 and CAFs_4 subtypes infiltration in the OS microenvironment compared with adjacent tissues. ConclusionOur study revealed the diversity, complexity, and heterogeneity of CAFs in OS, and complemented the single-cell atlas in OS TME.

Cancer-associated fibroblasts (CAFs) play a crucial role in the progression of pancreatic ductal adenocarcinoma (PDAC). Here, integrated single-cell RNA sequencing analysis is utilized to comprehensively map CAFs in the human PDAC tumor microenvironment (TME). Normal fibroblasts (NFs) and nine distinct CAF subtypes are identified including newly identified CAF subtypes, CDCP1+FTL+ CAFs, transitional CAFs (tCAFs), interferon simulated genes (ISG)+ myofibroblastic CAFs (myCAFs), and proliferative CAFs (pCAFs). CDCP1+FTL+ CAFs, pCAFs, and ISG+ myCAFs are associated with unfavorable clinical outcomes. CDCP1+FTL+ CAFs exhibit enhanced glycolysis and iron metabolism, resisting ferroptosis. The antigen-presenting CAFs (apCAFs) show high heterogeneity, consisting of multiple subtypes expressing distinct immune cell signatures. The CAF subtypes display differentiation plasticity, transitioning from early normal-like CAFs (nCAFs) to inflammatory CAFs (iCAFs) and myCAFs, ultimately leading to more invasive pCAFs. AP-1 family members FOS and JUN regulate the malignant phenotype conversion of NFs to nCAFs, while transforming growth factor-β (TGFβ) and interferon-γ (IFNγ) signals trigger the interconversion between classic myCAFs and iCAFs, respectively. A close interaction between CAFs and myeloid cells (especially neutrophils) is further observed in PDAC-TME, mainly mediated by CXCR4-CXCL12 chemotaxis. This work depicts a detailed CAF map and its dynamic interconvertible shift, providing important insights for combined targeted CAFs therapy.

In pancreatic ductal adenocarcinoma (PDAC), cancer associated fibroblasts (CAFs) play critical and complex roles in the tumor microenvironment. CAFs are also a major cell type in the desmoplastic stroma in PDAC and may account for half of the entire tumor tissue. Multiple subtypes of CAFs have been suggested, but the tissue origin(s) of CAF subtypes are unknown and genetic tools to robustly target them in vivo are lacking. Here we aimed to examine three potential tissue sources of CAFs: the pancreatic epithelium (through epithelium-to-mesenchyme transition), the bone marrow (through circulation), and the pancreatic mesenchyme or tissue resident fibroblasts (TRFs) in the normal pancreas (through proliferation). We utilized a genetically engineered mouse model of PDAC, where Kras and p53 mutations were engineered in the pancreatic epithelium using an Flp-Frt system. To determine whether the pancreatic epithelium gives rise to CAFs, we permanently labeled the pancreatic epithelium with a GFP reporter and traced their cell descendants by GFP expression. Despite robust GFP labeling of the epithelium, GFP expression was rarely identified in CAFs, suggesting little contribution of epithelium to the CAF pool. To determine whether the bone marrow gives rise to CAFs, we transplanted donor bone marrow carrying a ubiquitously expressed GFP reporter allele to GFP-negative recipient mice. We found that only a small proportion of pancreatic CAFs were tagged with GFP, suggesting their bone marrow origin. Lastly, to determine whether pancreatic TRFs give rise to CAFs, we used an inducible CreER-LoxP system to allow for permanent Tomato labeling in TRFs progenitors, the splanchnic mesenchyme, during mid-gestation. Lineage tracing in PDAC showed that the vast majority of CAFs were labeled with Tomato expression, suggesting their splanchnic origin. Furthermore, certain splanchnic gene expression signatures were persistent in subsets of CAFs in both the PDAC mouse model and human patient samples. In summary, we found that bone marrow contributes to a small proportion of CAFs in PDAC, and the pancreatic epithelium contributes even less. Meanwhile, pancreatic TRFs are derived from the splanchnic mesenchyme during fetal development and they expand to contribute to the vast majority of CAFs in PDAC. Moreover, the persistence of splanchnic signature defines subtypes of CAFs. This study provides approaches to robustly target CAFs in vivo and novel insights into CAF heterogeneity in PDAC. Citation Format: Lu Han, Yongxia Xu, Sean Sweeney, Ulyss Roesner, Melodie Parrish, Khushbu Patel, Xuezhong Yu, Michael Ostrowski, Gustavo Leone. The splanchnic mesenchyme during fetal development is the major source of pancreatic cancer associated fibroblasts [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PR001.

In pancreatic ductal adenocarcinoma (PDAC), cancer associated fibroblasts (CAFs) play critical and complex roles in the tumor microenvironment. CAFs are also a major cell type in the desmoplastic stroma in PDAC and may account for half of the entire tumor tissue. Multiple subtypes of CAFs have been suggested, but the tissue origin(s) of CAF subtypes are unknown and genetic tools to robustly target them in vivo are lacking. Here we aimed to examine three potential tissue sources of CAFs: the pancreatic epithelium (through epithelium-to-mesenchyme transition), the bone marrow (through circulation), and the pancreatic mesenchyme or tissue resident fibroblasts (TRFs) in the normal pancreas (through proliferation). We utilized a genetically engineered mouse model of PDAC, where Kras and p53 mutations were engineered in the pancreatic epithelium using an Flp-Frt system. To determine whether the pancreatic epithelium gives rise to CAFs, we permanently labeled the pancreatic epithelium with a GFP reporter and traced their cell descendants by GFP expression. Despite robust GFP labeling of the epithelium, GFP expression was rarely identified in CAFs, suggesting little contribution of epithelium to the CAF pool. To determine whether the bone marrow gives rise to CAFs, we transplanted donor bone marrow carrying a ubiquitously expressed GFP reporter allele to GFP-negative recipient mice. We found that only a small proportion of pancreatic CAFs were tagged with GFP, suggesting their bone marrow origin. Lastly, to determine whether pancreatic TRFs give rise to CAFs, we used an inducible CreER-LoxP system to allow for permanent Tomato labeling in TRFs progenitors, the splanchnic mesenchyme, during mid-gestation. Lineage tracing in PDAC showed that the vast majority of CAFs were labeled with Tomato expression, suggesting their splanchnic origin. Furthermore, certain splanchnic gene expression signatures were persistent in subsets of CAFs in both the PDAC mouse model and human patient samples. In summary, we found that bone marrow contributes to a small proportion of CAFs in PDAC, and the pancreatic epithelium contributes even less. Meanwhile, pancreatic TRFs are derived from the splanchnic mesenchyme during fetal development and they expand to contribute to the vast majority of CAFs in PDAC. Moreover, the persistence of splanchnic signature defines subtypes of CAFs. This study provides approaches to robustly target CAFs in vivo and novel insights into CAF heterogeneity in PDAC. Citation Format: Lu Han, Yongxia Wu, Melodie Parrish, Khushbu Patel, Xuezhong Yu, Michael Ostrowski, Gustavo Leone. The splanchnic mesenchyme during fetal development is the major source of pancreatic cancer associated fibroblasts [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-013.

In pancreatic ductal adenocarcinoma (PDAC), cancer associated fibroblasts (CAFs) play critical and complex roles in the tumor microenvironment. CAFs are also a major cell type in the desmoplastic stroma in PDAC and may account for half of the entire tumor tissue. Multiple subtypes of CAFs have been suggested, but the tissue origin(s) of CAF subtypes are unknown and genetic tools to robustly target them in vivo are lacking. Here we aimed to examine three potential tissue sources of CAFs: the pancreatic epithelium (through epithelium-to-mesenchyme transition), the bone marrow (through circulation), and the pancreatic tissue resident fibroblasts (TRFs) in the normal pancreas (through proliferation). We utilized a genetically engineered mouse model of PDAC, where Kras and p53 mutations were engineered in the pancreatic epithelium using an Flp-Frt system. To determine whether the pancreatic epithelium gives rise to CAFs, we permanently labeled the pancreatic epithelium with a GFP reporter and traced their cell descendants by GFP expression. Despite robust GFP labeling of the epithelium, GFP expression was rarely identified in CAFs, suggesting little contribution of epithelium to the CAF pool. To determine whether the bone marrow gives rise to CAFs, we transplanted donor bone marrow carrying a ubiquitously expressed GFP reporter allele to GFP-negative recipient mice. We found that only a small portion of pancreatic CAFs were tagged with GFP. Lastly, to determine whether pancreatic TRFs give rise to CAFs, we used an inducible CreER-LoxP system to allow for permanent Tomato labeling in TRFs progenitors, the splanchnic mesenchyme, during mid-gestation. Lineage tracing in PDAC showed that the vast majority of CAFs were labeled with Tomato expression, suggesting their splanchnic origin. Furthermore, certain splanchnic gene expression signatures were persistent in subsets of CAFs in both the PDAC mouse model and human patient samples. In summary, we found that bone marrow contributes to a small portion of CAFs in PDAC, and the pancreatic epithelium contributes even less. Meanwhile, pancreatic TRFs are derived from the splanchnic mesenchyme during fetal development and they expand to contribute to the vast majority of CAFs in PDAC. Moreover, the persistence of splanchnic signature defines subtypes of CAFs. This study provides approaches to robustly target CAFs in vivo, and novel insights into CAF origin and heterogeneity in PDAC. Citation Format: Lu Han, Yongxia Wu, Melodie Parrish, Khushbu Patel, Tony Trimboli, Julia Lefler, Xuezhong Yu, Michael Zimmermann, Angela Mathison, Raul Urrutia, Michael Ostrowski, Gustavo Leone. The splanchnic mesenchyme is the main tissue origin of fibroblasts in the pancreas during homeostasis and tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3645.

Pancreatic cancer (PC) is marked by extensive heterogeneity, posing significant challenges to effective treatment. The tumor microenvironment (TME), particularly cancer-associated fibroblasts (CAFs), plays a critical role in driving PC progression. However, the prognostic and functional contributions of distinct CAF subtypes remain inadequately understood. Here, we introduce a novel 7-gene risk model that not only robustly stratifies PC patients but also unveils the unique role of PHLDA1 as a key mediator in tumor-stroma crosstalk. By integrating single-cell RNA sequencing (scRNA-seq), spatial transcriptomics, and bulk RNA sequencing data, we comprehensively characterized the heterogeneity of CAFs in PC. We identified five CAF subtypes and focused on matrix CAFs (mCAFs), which were strongly associated with poor prognosis. A 7-gene mCAF-associated risk model was constructed using advanced machine learning algorithms, and the biological significance of PHLDA1 was validated through co-culture experiments and pan-cancer analyses. Our multiomics analysis revealed that the novel 7-gene model (comprising USP36, KLF5, MT2A, KDM6B, PHLDA1, REL, and DDIT4) accurately predicts patient survival, immunotherapy response, and TME status. Notably, PHLDA1 was uniquely overexpressed in CAFs and correlated with the activation of key protumorigenic pathways, including EMT, KRAS, and TGF-β, underscoring its central role in modulating the crosstalk between CAFs and malignant ductal cells. Pan-cancer analysis further supported PHLDA1's prognostic and immunomodulatory significance across multiple tumor types. Our study presents a novel 7-gene prognostic model that significantly enhances risk stratification in PC and identifies PHLDA1+ CAFs as promising prognostic biomarkers and therapeutic targets. These findings provide new insights into the TME of PC and open avenues for personalized treatment strategies.

Background: Cancer-associated fibroblasts (CAFs) are a highly diverse cell population that plays a crucial role in shaping the tumor microenvironment (TME). Their spatial interactions within the TME remain to be systematically characterized, and the factors driving their phenotypic heterogeneity are not yet fully understood. Recent advancements in single-cell spatial transcriptomics technologies and cell segmentation approaches enable the precise measurement of thousands of transcripts at subcellular resolution. Combined with innovative bioinformatics, these advancements have unlocked unprecedented opportunities for in-depth characterization of CAFs and their interactions within the TME. Methods: We conducted a comprehensive spatial transcriptomic analysis of CAFs across 8 cancer types, using the CosMx and MERSCOPE platforms to profile nearly 6 million cells as part of our discovery cohorts. We developed a novel computational method to spatially cluster CAF subpopulations and defined four distinct spatial CAF subtypes. We further examined their phenotypic differences, spatial niche organization, and interactions with neighboring cells. Additionally, we investigated the influence of CAF subtypes on immune cell phenotypes and infiltration levels. To validate our findings, we employed multiple spatial platforms, including Visium, Xenium, COMET, CODEX, and IMC, to ensure reproducibility across diverse technologies. Moreover, we performed correlative studies in large-cohorts with rich clinical data to evaluate the clinical significance of these CAF subtypes. Results: Our approach provided an unprecedented high-resolution examination of the spatial phenotypes of CAFs in large, complex tissues, uncovering novel insights. We comprehensively characterized CAFs across various solid tumor types and identified four conserved spatial CAF subtypes. These subtypes are characterized by distinct cell organizational patterns, neighboring cell compositions, interaction networks, and transcriptomic features. Moreover, they demonstrate significant correlations with the abundance, distribution, and phenotypes of tumor-infiltrating immune cells, as well as associations with tumor histopathological features and clinical outcomes. Conclusions: Our study provides a pan-cancer, high-resolution characterization of CAF spatial organization and interactions in solid tumors and identified conserved spatial CAF subtypes linked to distinct organizational patterns. Our findings reveal that the transcriptomic states and phenotypes of CAFs are significantly shaped by their interactions with neighboring cancer and immune cells. Beyond offering novel insights into the spatial heterogeneity of CAFs, this work paves the way for developing strategies to effectively target and modulate CAFs for therapeutic benefit. Citation Format: Yunhe Liu, Ansam Sinjab, Jimin Min, Guangchun Han, Francesca Paradiso, Yuanyuan Zhang, Yang Liu, Ruiping Wang, Guangsheng Pei, Kyung Serk Cho, Enyu Dai, Akshay Basi, Jared K. Burks, Kimal I. Rajapakshe, Yanshuo Chu, Jiahui Jiang, Daiwei Zhang, Xinmiao Yan, Paola A. Guerrero, Alejandra Serrano, Mingyao Li, Tae Hyun Hwang, Andrew Futreal, Jaffer A. Ajani, Luisa M Solis Soto, Amir A. Jazaeri, Humam Kadara, Anirban Maitra, Linghua Wang. Spatial subtypes and cellular interactions of cancer-associated fibroblasts revealed by single-cell spatial omics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 166.

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

Cancer-associated fibroblasts (CAFs) are a critical component of the glioma microenvi-ronment and play essential roles in tumor progression and resistance to immunotherapy. To comprehensively characterize CAF heterogeneity and their interactions with immune cells, we conducted an integrative multi-omics analysis incorporating single-cell and bulk RNA sequencing, spatial transcriptomics, and multiplex immunofluorescence. This approach identified nine distinct CAF subtypes with phenotypic and functional diversity, including tumor-like CAFs (tCAFs), myofibroblast-like CAFs (myCAFs), vascular CAFs (vCAFs), metabolic CAFs (meCAFs), proliferative CAFs (pCAFs), antigen-presenting CAFs (apCAFs), interferon-responsive CAFs (infCAFs), inflammatory CAFs (iCAFs), and a group of CAFs with unknown identity. Several subtypes were significantly associated with poor clinical outcomes. Notably, apCAFs engaged in extensive crosstalk with M2-polarized macrophages via TGF-β signaling pathways. Spatial transcriptomic pro-filing and immunofluorescence imaging revealed the co-localization of apCAFs and M2 macrophages at the tumor periphery, indicating the formation of an immunosuppressive niche. Moreover, AQP4 was identified as a specific marker of apCAFs, and its expression was significantly correlated with poor prognosis and resistance to immunotherapy. These findings offer a comprehensive atlas of CAF heterogeneity in glioma and highlight the therapeutic promise of targeting apCAF-M2 macrophage interactions or AQP4 to over-come immune resistance and improve clinical outcomes.

BackgroundUse of anti-PD-1 immune checkpoint inhibitors (ICI) is currently the first line therapy for recurrent/metastatic head and neck squamous cell carcinoma (HNSCC), but critical work remains in identifying factors guiding...

Cancer-associated fibroblasts (CAFs) play a pivotal role in Gastric cancer (GC) progression and immune modulation. This study aimed to identify CAF subtypes using single-cell analysis and evaluate their prognostic and therapeutic relevance in GC. CAF gene sets were derived from 13 single-cell datasets and quantified via ssGSEA in bulk transcriptomic cohorts (TCGA and GEO). Consensus clustering defined CAF-based subtypes. Immune infiltration was evaluated using CIBERSORT, xCell, MCPcounter, and ESTIMATE. Immunotherapy response was predicted using TIDE and ImmuCellAI. Chemotherapeutic sensitivity was assessed via PRISM, CTRP, and GDSC databases. Hub genes were identified by WGCNA, and a prognostic model was constructed and validated in external cohorts and at the single-cell level. Two CAF subtypes, FA_H and FA_L, were identified. FA_H was associated with poor prognosis, higher M2 macrophage infiltration, and immunosuppressive pathways, while FA_L correlated with improved survival and stronger predicted response to immune checkpoint inhibitors. Dasatinib was predicted as a potential therapeutic agent specifically for FA_H subtype. A five-gene prognostic model (COL1A2, NDN, SPARC, VCAN, TCEAL7) showed consistent predictive performance across datasets. Functional validation confirmed upregulation of TCEAL7 in CAFs and its role in promoting GC cell invasion. Single-cell-based CAF subtyping defines clinically relevant heterogeneity in GC. The FA_H subtype may serve as both a prognostic biomarker and therapeutic target, particularly for dasatinib-based or immunomodulatory strategies.