Deciphering the Immunomodulatory Function of GSN+ Inflammatory Cancer‐Associated Fibroblasts in Renal Cell Carcinoma Immunotherapy: Insights From Pan‐Cancer Single‐Cell Landscape and Spatial Transcriptomics Analysis

ABSTRACT The heterogeneity of cancer‐associated fibroblasts (CAFs) could affect the response to immune checkpoint inhibitor (ICI) therapy. However, limited studies have investigated the role of inflammatory CAFs (iCAFs) in ICI therapy using pan‐cancer single‐cell RNA sequencing (scRNA‐seq) and spatial transcriptomics sequencing (ST‐seq) analysis. We performed pan‐cancer scRNA‐seq and ST‐seq analyses to identify the subtype of GSN + iCAFs, exploring its spatial distribution characteristics in the context of ICI therapy. The pan‐cancer scRNA‐seq and bulk RNA‐seq data are incorporated to develop the Caf.Sig model, which predicts ICI response based on CAF gene signatures and machine learning approaches. Comprehensive scRNA‐seq analysis, along with in vivo and in vitro experiments, investigates the mechanisms by which GSN + iCAFs influence ICI efficacy. The Caf.Sig model demonstrates well performances in predicting ICI therapy response in pan‐cancer patients. A higher proportion of GSN + iCAFs is observed in ICI non‐responders compared to responders in the pan‐cancer landscape and clear cell renal cell carcinoma (ccRCC). Using real‐world immunotherapy data, the Caf.Sig model accurately predicts ICI response in pan‐cancer, potentially linked to interactions between GSN + iCAFs and CD8 + Tex cells. ST‐seq analysis confirms that interactions and cellular distances between GSN + iCAFs and CD8 + exhausted T (Tex) cells impact ICI efficacy. In a co‐culture system of primary CAFs, primary tumour cells and CD8 + T cells, downregulation of GSN on CAFs drives CD8 + T cells towards a dysfunctional state in ccRCC. In a subcutaneously tumour‐grafted mouse model, combining GSN overexpression with ICI treatment achieves optimal efficacy in ccRCC. Our study provides the Caf.Sig model as an outperforming approach for patient selection of ICI therapy, and advances our understanding of CAF biology and suggests potential therapeutic strategies for upregulating GSN in CAFs in cancer immunotherapy.