Characterization of cancer-associated fibroblast populations that promote tertiary lymphoid structure formation in murine melanoma tumors

In recent years, the clinical relevance of tertiary lymphoid structures (TLS) in cancer has become increasingly clear. However, the mechanisms that promote TLS development have remained obscure, largely because of a lack of animal models in which cause and effect studies can be performed. In this study, we used a mouse model in which TLS develop spontaneously in intraperitoneal tumors to address this issue. Extending earlier studies in which we showed that CXCR5-expressing B cells are essential for TLS development, we found that CXCL13, the ligand for CXCR5, is expressed exclusively by a population of cancer-associated fibroblasts (CAF), and that these CAF are essential to promote B cell accumulation in these tumors. In keeping with earlier work showing that the presence of TLS was associated with smaller tumor size, we also found that the lack of B cell accumulation was associated with larger tumor size. Using scRNA-seq, we identified 7 subpopulations of CAF in TLS containing tumors. While many of these subpopulations appear similar to those de ned in other human and murine tumor models, we also discriminate new immunologically relevant subpopulations of CAF. One of these CAF subpopulations was substantially enriched for CXCL13-expressing cells. This population was also enriched for additional genes that could promote B cell recruitment and enable regulation by a variety of cytokines. However, other genes that we have shown or hypothesize to be important in TLS development were enriched for expression in other CAF subpopulations. Overall, this work indicates that TLS development in this model depends on multiple CAF populations operating at distinct points in the developmental process.