A THBS2+ cancer-associated fibroblast subpopulation orchestrates therapeutic resistance in castration-resistant prostate cancer

The progression from hormone-sensitive prostate cancer (HSPC) to lethal castration-resistant prostate cancer (CRPC) involves a poorly understood remodeling of the tumor microenvironment (TME). This study aimed to comprehensively characterize the stromal alterations driving CRPC, with a focus on heterogeneous cancer-associated fibroblast (CAF) subpopulations. Integrated analysis of multiple single-cell RNA sequencing (scRNA-seq) datasets from human HSPC and CRPC samples was performed to map TME dynamics. Fibroblast subclustering identified distinct CAF subsets. Findings for a key subpopulation were validated using independent scRNA-seq cohorts, bulk transcriptomic data, immunohistochemistry (IHC), and multiplex immunofluorescence (mIF) on clinical specimens. Functional roles of the marker gene THBS2 were investigated in vitro using shRNA-mediated knockdown in CAFs and conditioned media assays with prostate cancer cells treated with enzalutamide. scRNA-seq analysis revealed a significant expansion of CAFs in CRPC. Subclustering identified five CAF subsets, with THBS2 + CAFs (CAF-C0) being specifically enriched in CRPC. At the transcriptomic level, THBS2 expression was predominantly localized to CAFs and was consistently validated across independent scRNA-seq datasets. Clinically, high THBS2 expression correlated with ADT resistance, advanced tumor stage, metastasis, and shorter biochemical recurrence-free survival. IHC and mIF confirmed stromal THBS2 protein upregulation in prostate cancer tissues and its association with aggressive features. Functionally, THBS2 knockdown in CAFs significantly diminished THBS2 secretion levels and sensitized prostate cancer cells to enzalutamide treatment in conditioned media assays. This study identifies a specific THBS2 + CAF subpopulation that is expanded in CRPC, associated with therapy resistance and poor prognosis, and functionally contributes to reduced sensitivity to endocrine therapy in vitro. THBS2 + CAFs represent a critical stromal component in CRPC progression and a promising therapeutic target and biomarker.