A FOXF1/FOXF2-associated CAF-like transitional fibroblast state is defined and links it to stromal signaling, immune–stromal communication, and cancer-type-specific clinical relevance.
Abstract
Fibroblast heterogeneity shapes tumor progression, yet the transitional states linking normal-associated fibroblasts to cancer-associated fibroblasts (CAFs) remain poorly defined. Here, we integrated single-cell transcriptomic profiles of more than 90,000 stromal cells from 281 samples across nine cancer types to construct a pan-cancer atlas of fibroblast diversity. We identified a distinct CAF-like population positioned between normal-activated fibroblasts and established CAF subsets along the inferred fibroblast activation trajectory. Integration with single-nucleus chromatin accessibility data nominated FOXF1 and FOXF2 as candidate regulators of this CAF-like state. Functionally, CAF-like fibroblasts were characterized by non-canonical WNT activity, WNT5A-associated stromal communication, and a candidate GZMA–F2R/PAR immune–stromal signaling axis supported by spatial transcriptomics. Clinically, the CAF-like signature showed context-dependent prognostic relevance, with high expression associated with poorer survival in the tumor compartment of TCGA stomach adenocarcinoma. Together, this study defines a FOXF1/FOXF2-associated CAF-like transitional fibroblast state and links it to stromal signaling, immune–stromal communication, and cancer-type-specific clinical relevance.
Cancer-associated fibroblasts (CAFs) exhibit substantial heterogeneity in colorectal cancer (CRC), yet the upstream tumor-derived signals associated with their state remodeling during disease progression remain incompletely defined. Through integrative analysis of multi-cohort single-cell transcriptomics, proteomics, and bulk RNA sequencing data, we identified tumor-derived midkine (MDK) as a stage-associated candidate signal linked to CAF remodeling in CRC.
MDK cell-type specificity and stage-associated expression, together with CAF composition and stage-dependent remodeling, were evaluated in two large colorectal cancer single-cell RNA-sequencing cohorts, with the principal findings cross-validated between cohorts and further integrated with bulk transcriptomic, proteomic, and immunohistochemical datasets. Existing malignant-cell-to-CAF CellChat results were re-ranked using a joint breadth–strength framework to identify broadly activated and high-probability signaling pathways. CAF trajectories were reconstructed independently in both cohorts using Monocle 2 and rooted according to the same biological criterion, namely enrichment of the normal-like Fib-MFAP5 population. BayesPrism was used to deconvolve CAF subtype abundance from TCGA colorectal cancer bulk transcriptomes and to evaluate its association with overall survival.
MDK was consistently enriched in malignant epithelial cells across both single-cell cohorts. Within the same-platform bulk RNA cohort, median log2-normalized MDK expression increased from 7.93 in adenoma to 8.19 in carcinoma in situ and 9.02 in adenocarcinoma, supporting a significant ordered increase during colorectal cancer progression (Jonckheere–Terpstra
$$P=0.008$$
; 10,000-permutation
$$P=0.007$$
). Fib-WNT5A abundance likewise showed a stage-associated increase and was positioned significantly later than the normal-like Fib-MFAP5 population in patient-level pseudotime analyses of both the discovery and validation cohorts. Fib-WNT5A exhibited strong TGF-β and extracellular matrix programs, and integration of subtype-associated ECM signatures with bulk proteomics linked this population to collagen-enriched matrix remodeling. Joint breadth–strength analysis identified MK signaling as the top-ranked broadly activated malignant-cell-to-CAF pathway. CellChat further predicted COL1A1/COL1A2–CD44 interactions between Fib-WNT5A and multiple immune populations, supporting an association between Fib-WNT5A expansion and an ECM-rich, immune-restrictive microenvironment.
Patient-level pseudo-bulk analyses were used to evaluate BayesPrism recovery of major CAF states. In TCGA CRC, higher Fib-WNT5A abundance was associated with poorer overall survival in the prespecified median-based univariable analysis (HR = 1.64, 95% CI 1.09–2.49; log-rank
P
= 0.018), whereas higher Fib-MFAP5 abundance was associated with better survival (HR = 0.54, 95% CI 0.35–0.83; log-rank
P
= 0.0045).
Collectively, the data support a progression-linked association among tumor-cell-enriched MDK, CAF state remodeling, an ECM-high Fib-WNT5A program, and predicted collagen-mediated immune interactions in CRC. Functional perturbation studies are required to establish the direction and causality of the proposed MDK-CAF axis.
BACKGROUND
Cancer-associated fibroblasts (CAFs) are key components of the bladder cancer microenvironment, but how specific CAF subpopulations drive malignant progression remains unclear. This study aimed to identify CAF driver genes and elucidate the role of caveolin-1 (CAV1) in CAF-mediated bladder cancer progression.
METHODS
Single-cell RNA sequencing and public bladder cancer datasets were integrated to identify fibroblast-associated candidate genes and evaluate the clinical significance of CAV1. Clinical samples were analyzed by immunofluorescence. The biological functions of CAV1 were further evaluated through CAF-bladder cancer cell co-culture systems, gain- and loss-of-function experiments, and comprehensive molecular analyses. The downstream mechanism was explored through LIF stimulation, STAT3 silencing, and validated in a subcutaneous xenograft model.
RESULTS
Single-cell analysis identified CAV1 as a fibroblast-associated gene linked to stemness- and ferroptosis-related signatures. High CAV1 expression predicted poor prognosis and was independently associated with unfavorable survival. CAV1 expression positively correlated with fibroblast infiltration and was enriched in a subset of ACTA2-positive CAFs. CAFs markedly promoted multiple malignant behaviors of bladder cancer cells, whereas silencing CAV1 effectively counteracted these pro-tumorigenic effects. Mechanistically, CAV1 enhanced LIF expression in CAFs, thereby activating JAK2/STAT3 signaling in tumor cells. Pharmacological inhibition of STAT3 abolished the tumor-promoting effects of CAFs both in vitro and in vivo.
CONCLUSIONS
CAF-derived CAV1 promotes bladder cancer progression by activating the LIF-STAT3 signaling axis. Targeting stromal CAV1-mediated STAT3 signaling may represent a promising therapeutic strategy for bladder cancer.
Introduction Although the role of cancer-associated fibroblasts (CAFs) in cancer progression is increasingly recognized, their spatial dynamics and interactions with immune cells remain poorly understood. Methods Here, we present a computational framework that integrates single-cell resolution spatial transcriptomics and standard spatial transcriptomics across multiple tumor types to investigate CAF heterogeneity and its roles in situ. Results Our analysis presents a continuous transition from fibroblast progenitors to COL11A1-expressing CAFs, which we term aggressive CAFs (aCAFs), within a spatial context. We show that aCAFs, whose expression has been associated with poor prognosis, tend to localize at tumor boundaries, where proximity to tumor cells predicts increased expression of aCAF-associated genes. Spatial modeling shows that regions enriched for COL11A1-expressing CAFs were depleted of non-exhausted immune cells, including naive T cells, activated cytotoxic T cells, and activated B cells, suggesting a role in immune exclusion. Spatial correlation analysis further reveals that aCAFs co-localize with lipid-associated macrophages, a pattern linked to extracellular matrix remodeling and altered lipid metabolism. Conclusion Our study provides insights into the interactions of aCAF, tumor cells, and immune cells in the tumor microenvironment. We also provide an open-source implementation of SpatialAttractor, a toolkit for exploring gene co-expression in the spatial context.
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These findings define the cellular organization and molecular programs that shape the cSCC microenvironment and provide insights into the coordinated immune, stromal, and epithelial interactions involved in cSCC progression.
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These findings define functionally distinct and pharmacologically targetable CAF programs in HNSCC and support stromal-directed interventions as a rational component of future combination treatment strategies.
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Integrative single-cell RNA sequencing analysis of publicly available datasets from non-small cell lung cancer and breast cancer is performed to systematically map transcriptional heterogeneity and regulatory networks within the TME, providing a systems-level framework of TME organization.
M. O. Odubote, Chiemeka Elochi Emeribe· bioRxiv· 0 citations
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