Proteome and secretome profiling of the melanoma-induced transition towards immune incompetent dendritic cells reveals enhanced IDO1, cathepsin, and legumain activity.

Abstract

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, we characterized by tandem LC-MS/MS 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 profiled the newly synthesized secretome in human DCs with THRONCAT metabolic labeling. We detected 17 differentially secreted proteins between DC2 and ti-DC3s, which included six cathepsins and tumor-associated TGFβI. Cathepsin activity was validated in peripheral blood and tumor tissue of melanoma patients. We detected 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 human melanoma-induced DC3s. This revealed several protein-driven pro-tumor-mechanisms active in ti-DC3s that potentially contribute to creating an immune environment favorable for tumor progression.