SUMMARY It is currently accepted that activated cancer-associated fibroblasts (CAF) participate in T cell exclusion from tumor nests, but it remains unclear how they promote barrier phenotypes, and whether specific subsets are involved. Here, using single-cell RNA sequencing coupled with multiplex imaging on a large cohort of lung tumors, we identify four main CAF populations, of which only two are associated with T cell exclusion: (i) MYH11 + αSMA + CAF, which are present in early-stage tumors and form a single-cell layer lining cancer aggregates, and (ii) FAP + αSMA + CAF, which appear in more advanced tumors and organize in patches within the stroma or in multiple layers around tumor nests. Both CAF populations show a contractility phenotype together with dense and aligned matrix fiber deposition compared to the T cell-permissive CAF. Yet they express distinct matrix genes, including COL4A1/COL9A1 (MYH11 + αSMA + CAF) and COL11A1/COL12A1 (FAP + αSMA + CAF). Hereby, we uncovered unique molecular programs of CAF driving T cell marginalization, whose targeting should increase immunotherapy efficacy in patients bearing T cell-excluded tumors. SIGNIFICANCE The cellular and molecular programs driving T cell marginalization in solid tumors remain unclear. Here, we describe two CAF populations associated with T cell exclusion in human lung tumors. We demonstrate the importance of pairing molecular and spatial analysis of the tumor microenvironment, a prerequisite to develop new strategies targeting T cell-excluding CAF.

Spatial positioning and matrix programs of cancer-associated fibroblasts promote T cell exclusion in human lung tumors