Detection of PD-L1-expressing myeloid cell clusters in the hyaluronan-enriched stroma in tumor tissue and tumor-draining lymph nodes

Expression of the transmembrane protein PD-L1 is frequently up-regulated in cancer. Since PD-L1-expressing cells can induce apoptosis or anergy of T lymphocytes through binding to PD1 receptor, the PD-L1-mediated inhibition of activated PD1 + T cells considered as a major pathway for tumor immune escape. However, the mechanisms that regulate the expression of PD-L1 in the tumor microenvironment not fully understood. Analysis of organotypic tumor tissue slice cultures, obtained from tumor-bearing mice as well as from cancer patients, revealed that tumor-associated hyaluronan (HA) supports the development of the immunosuppressive PD-L1 + macrophages. Using genetically modified tumor cells, we identified both epithelial tumor cells and cancer-associated fibroblasts (CAFs) as the major source of HA in the tumor microenvironment. HA-producing tumor cells and, in particular CAFs of bone marrow origin, directly interact with tumor-recruited Hyal2 + myeloid cells forming the large stromal congregates/clusters that are highly enriched for both HA and PD-L1. Furthermore, similar cell clusters comprising of HA-producing fibroblasts and PD-L1 + macrophages were detected in the tumor-draining lymph nodes. Collectively, our findings indicate that the formation of multiple large HA-enriched stromal clusters that support the development of PD-L1-expressing antigen-presenting cells in the tumor microenvironment and draining lymph nodes could contribute to the immune escape and resistance to immunotherapy in cancer.