Decoding the tumor microenvironment remodeling orchestrated by CLEC3B+ inflammatory cancer-associated fibroblasts in lung adenocarcinoma immunotherapy: elucidation from pan-cancer spatially single-cell transcriptomics landscape.

INTRODUCTION: The heterogeneity of cancer-associated fibroblasts (CAFs) would possibly influence reaction to immune checkpoint inhibitor (ICI) therapy. However, limited researches investigated inflammatory CAFs (iCAFs) in ICI therapy using pan-cancer single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics RNA sequencing (stRNA-seq) analysis. METHODS: We performed pan-cancer scRNA-seq and stRNA-seq analyses to investigate CLEC3B+ iCAFs, exploring its spatial distribution characteristics. The pan-cancer scRNA-seq and RNA-seq data are incorporated to develop the Inflammatory CAF Signature (ICS), which predicts ICI response based on iCAF marker genes and machine learning techniques. Comprehensive scRNA-seq analysis, with in vivo and in vitro experiments, investigates the mechanisms by which CLEC3B+ iCAFs influence ICI efficacy. RESULTS: The ICS model performs well in predicting ICI response in pan-cancer patients. A higher proportion of total iCAFs is observed in ICI non-responders in pan-cancer landscape and lung adenocarcinoma (LUAD). Cellular communication and stRNA-seq analyses confirm that intercellular interactions and cell-to-cell distances between iCAFs and CD8+ Tex cells impact ICI efficacy. A critical functional heterogeneity within iCAFs modulated by CLEC3B is revealed, while functional experiments confirm that downregulation of CLEC3B on CAFs drives CD8+ T cells toward a dysfunctional state in LUAD. CLEC3B activates JAK2–STAT3 pathway by inhibiting SOCS3 deubiquitylation, enhancing the immunostimulatory effects of CAFs. In a subcutaneously tumor grafted mouse model, combining CLEC3B overexpression with ICI treatment achieves optimal efficacy in LUAD. CONCLUSION: Our study provides ICS model as an outperforming approach for patient selection of ICI, and advances our understanding of CAF biology and suggests potential therapeutic strategies for upregulating CLEC3B in CAFs in cancer immunotherapy.