Proteome and secretome profiling of type 3 dendritic cells

Dendritic cells (DCs) are professional antigen presenting cells endowed with the capacity to initiate strong antitumor immune responses. This function is critical for effective DC-based immunotherapies, but is often hampered by tumor-derived immunosuppressive factors, as is observed for CD14+CD163+ tumor-induced DC3s (ti-DC3s). ti-DC3s are increased in cancer patients where they display an immunosuppressive phenotype, accompanied by weak antigen-specific CD8 T cell activating capacities. While tumor-derived IL-6, M-CSF, and PGE2 have been identified as factors inducing the transition from DC2s to ti-DC3s, a comprehensive unbiased profiling of the resulting changes in secretome and proteome has not been reported. Here, using LC-MS/MS-based techniques, we characterize the proteomic changes in cDC2s during their transition into CD14+ ti-DC3s in vitro, using conditioned-medium from the melanoma cell line BLM. This revealed 157 differentially expressed proteins, including upregulated IDO1 and legumain, which we confirmed to be functionally active. Next, we demonstrate the feasibility of THRONCAT-mediated identification of the newly synthesized secretome in primary human DCs. We detected 17 differentially secreted proteins between DC2 and ti-DC3s, including six cathepsins and the tumor-associated TGFβI. Cathepsin activity was validated in peripheral blood and tumor tissue of melanoma patients, which demonstrated the highest cathepsin activity in ti-DC3s, surpassing DC2s and tumor-associated macrophages. Together, our findings represent the first characterization of the proteome and secretome of primary human melanoma-induced DC3s reported to date, revealing several protein-driven pro-tumor-mechanisms active in ti-DC3s with reduced immunostimulatory abilities.