Tumor-associated macrophages (TAMs) have been implicated in suppressing T-cell activity against cancer cells, representing an important target for the development of new immune therapy combinations. Accumulating evidence indicates that increased numbers of TAMs infiltrating cancer including melanoma are associated with poor prognosis. However, TAMs remain poorly defined in human cancers as they can exhibit a range of different molecular phenotypes and functional properties, and their cellular precursors are also uncertain, necessitating further investigation to enable their therapeutic manipulation. Using multiplex immunohistochemistry up to 7 colors and flow cytometry, here we sought to examine molecular characteristics of TAMs in human melanoma metastases to gain better understanding of the mechanisms driving their immune-suppressive activity in tumors as wells as the pathways recruiting their precursors. In lymph node and dermal melanoma metastases, we found that a population of TAMs expressing the marker CD163 frequently express molecules that are highly suppressive of T-cell function, including the PD-1 ligands PD-L1 and PD-L2, IDO (indoleamine 2,3-dioxygenase), TGF-β, and IL-10. In particular, the predominant source of PD-L1 is not the melanoma cells present but the infiltrating CD163+ TAMs. We also observed that many of the CD163+ TAMs downmodulate MHC class II molecules, represented by HLA-DR, suggesting they have suppressed capacity to present melanoma cell antigens to T cells. CD163+ TAMs in melanoma metastases often express weak levels of CD14 and CCR2, while cells adjacent to them express these markers brightly, suggesting that these TAMs are likely to derive from recently extravasated monocytes. Further analysis confirmed that CCL2, the chemokine ligand for CCR2, is indeed present in many melanoma tissue samples examined. Interestingly, the majority of CD163+CD14+ TAMs in melanoma metastases also express high levels of CD16, suggesting that these TAMs are either more closely related to the nonclassical CD14+CD16+ monocytes or derive from the classical CD14+CD16- monocytes that subsequently gain CD16 expression after entering the tumor tissue. In vitro, we were able to generate cells from primary human monocytes that recapitulate the phenotype of CD163+ TAMs observed in situ and are capable of suppressing T cells, which will provide us a valuable tool to carry out functional studies to reveal how TAMs suppress T-cell function and how to reverse such activities. Collectively, our data demonstrate the complex molecular features of CD163+ TAMs in melanoma metastases, necessitating further investigations to determine the dominant T cell-suppressive mechanisms used by TAMs and to prioritize therapeutic targets.

Citation Format: Saem Park, Anna Brooks, Chun-Jen Chen, Rod Dunbar. Molecular characteristics of tumor-associated macrophages in human melanoma metastases [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B101.