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

Abstract:
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. Cxcl13-cre-tdTomato mice were implanted with B16-OVA melanoma tumors in either intraperitoneal or subcutaneous locations to establish which cells expressed CXCL13. Single cell suspensions from tumors were analyzed by flow cytometry. To address the impact of CXCL13 + CAF on TLS formation, we bred Cxcl13-cre tdTomato mice to R26R-iDTR flox mice, which express the diphtheria toxin receptor under the control of a floxed stop codon. The resulting Cxcl13-cre/iDTR mice were treated with diphtheria toxin beginning on day 7 after IP tumor implantation. Single cell suspensions from tumors were analyzed by flow cytometry. To characterize their functional properties, CAF in IP melanoma tumors from Cxcl13-cre/eGFP mice were collected by cell sorting, analyzed for transcript expression using scRNA-seq, clustered into groups using the Seurat package, and profiled using pathway analysis and differential gene analysis. 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, were expressed exclusively by a population of cancer-associated fibroblasts (CAF), and that these CAF were 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 groups of CAF in TLS containing tumors. One of these CAF groups 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 groups. This work indicates that TLS development in this model depends on multiple CAF populations operating at distinct points in the developmental process. Some of these populations appear similar to, but distinct from, those defined in other human and murine tumor models. However, we also discriminated new immunologically relevant populations of CAF, particularly one expressing CXCL13, which we propose acts as a central organizer of tumor-associated TLS.

Introduction:
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.

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