Abstract 2751: Secreted fatty acids from head and neck cancer cell lines mediate M2-like macrophage polarization
Authors: Albakri Marwah M. , Huang Stanley C. , Tashkandi Hammad , Sieg Scott F. 
Published: July 2021

Abstract
Head and neck cancers (HNC) can persist despite infiltration of immune cells into the tumor microenvironment. One mechanism that may contribute to failed immune clearance of tumors stems from the conditioning of tumor infiltrating macrophages into a cell type that supports tumor survival rather than a cell with pro-inflammatory characteristics. To understand mechanisms that lead to conditioning of macrophages by tumors, we assessed the effects of HNC cell lines on macrophage maturation. Macrophages were matured by incubating purified human monocytes for 3-4 days in medium alone or in medium supplemented with supernatants from HNC cell lines (FADU and SCC9) or a human leukemia monocytic cell line (THP-1). Macrophages were recovered and examined by flow cytometry for markers of maturation (CD14, CD68), activation (HLA-DR, CD86, and IL-15R), scavenger receptor (CD36), toll-like receptor (TLR4), M2 marker (CD206), immune checkpoint (PD-L1) and intracellular chemokine expression (IP-10). Relative to unconditioned macrophages or cells exposed to THP-1 supernatants, cells incubated with FADU or SCC9 supernatants displayed enhanced survival, downregulation of cell surface HLA-DR, CD86, IL-15R, CD36 and intracellular IP-10 expression. In contrast, the expression of PD-L1, CD14, and CD206 were increased by HNC cell line supernatants. To assess function, matured macrophages were stimulated overnight with the TLR4 agonist, LPS. Induction of macrophage activation was measured by increased expression of HLA-DR and IL-15R. Despite sustained the expression of TLR4 and CD14, macrophages matured in HNC supernatants failed to upregulate HLA-DR and IL-15R expression when stimulated with the canonical TLR4 agonist, LPS. These phenotypic and functional changes were accompanied by a decrease in intracellular phospho-p38 expression and a robust induction of WIP-1 phosphatase. Next, we considered the hypothesis that fatty acids released from tumors might contribute to these phenotypes. To test this, we added Etomoxir, an inhibitor of fatty acid oxidation, into cell cultures during exposure to tumor supernatants. The addition of Etomoxir reversed a number of these phenotypic changes induced by tumor supernatants with the exception of PD-L1 induction. Moreover, depletion of fatty acids from tumor supernatants with lipid removal agent, mitigated the effects of supernatants on macrophage maturation. Additionally, we found that macrophages incubated with either palmitic acid or oleic acid for 3-4 days developed similar phenotypes as cells incubated in tumor supernatants. Together, our data suggest that fatty acids are both necessary in tumor supernatants and sufficient on their own to mediate maturation of macrophages into a cell type with limited pro-inflammatory characteristics (M2-like). Approaches that modify the expression or effects of fatty acids in tumor microenvironment may be useful in HNC.
Citation Format: Marwah M. Albakri, Stanley C. Huang, Hammad Tashkandi, Scott F. Sieg. Secreted fatty acids from head and neck cancer cell lines mediate M2-like macrophage polarization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2751.