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Abstract

Immunotherapeutic strategies for malignant melanoma have made significant progress but are still challenged by the development of resistance in a subset of patients. One of the factors contributing to this resistance is the accumulation of myeloid-derived suppressor cells (MDSC) in the melanoma microenvironment. MDSC are a heterogeneous population of myeloid cells that can inhibit anti-tumor T cell responses, thereby promoting immunosuppression and facilitating tumor progression. TLR4 and RAGE signaling are considered as important regulators of MDSC accumulation and acquisition of their immunosuppressive functions. Damage-associated molecular patterns (DAMPs) such as S100A8/9, high mobility group box 1 (HMGB1), and heat shock proteins (HSPs) that act as ligands for TLR4 or RAGE were reported to drive MDSC accumulation and to be significantly expressed in the tumor microenvironment (TME) of solid tumors. However, a precise role of TLR4 and RAGE signaling in the acquisition of immunosuppressive properties by MDSC requires further elucidation. The present study aims to evaluate the impact of endogenous TLR4 ligands and TLR4 signaling on MDSC-mediated immune suppression in malignant melanoma. MDSC were purified from the peripheral blood of late-stage melanoma patients and were generated in vitro from healthy donor-derived monocytes. Normal monocytes were treated with recombinant (r) HSP90a for 24h or with rS100A9 and rHMGB1 in the presence of GM-CSF for 72 h. The immunosuppressive capacity of MDSC and stimulated monocytes were assessed in T cell inhibition assays. In addition, TLR4 inhibitor (Resatorvid) and RAGE inhibitor (FPS-ZM1) were tested in these assays. Expression of immunosuppression related markers and pathways involved in the MDSC stimulation were assessed by flow cytometry, Western Blot, and gene expression profiling. The levels of S100A8/9 and HMGB1 were measured in the plasma of melanoma patients by ELISA. The Cancer Genome Atlas (TCGA) data analysis was performed to evaluate the association between the expression of immunosuppressive markers and S100A9 and HMGB1 in the TME of melanoma. Stimulation of monocytes with HSP90a, S100A9 and HMGB1 resulted in the acquisition of suppressive activity against T cells via increased production of reactive oxygen species (ROS) and nitric oxide (NO) as well as upregulated expression of programmed death ligand-1 (PD-L1) and indoleamine 2,3-dioxygenase (IDO). Increased plasma levels of S100A8/9 was found to be correlated with the expression of immunosuppressive markers on MDSC in the peripheral blood of melanoma patients. Importantly, the blockade of TLR4 signaling and, to a lesser extent RAGE signaling, resulted in a substantial attenuation of T cell inhibition. The supernatant of in vitro generated MDSC contained a significantly higher levels of S100A8/9 and HMGB1 than in that from patient derived MDSC Furthermore, elevated plasma levels of S100A8/9 were found to be associated with a poor progression free survival (PFS) in melanoma patients. In conclusion, this study highlights the crucial role of TLR4 and, to a lesser extent RAGE signaling, as well as TLR4 ligands S100A9 and HMGB1 in the acquisition of immunosuppressive properties by MDSC in malignant melanoma. These findings suggest that targeting TLR4 signaling pathway may represent a promising therapeutic strategy to overcome MDSC-mediated immune suppression and enhance the efficacy of melanoma immunotherapy.