LAUSR

Tumor Associated Macrophages (TAMs), copiously found in the Tumor Microenvironment (TME) are key players in creating an immunosuppressive environment, increasing angiogenesis and metastasis in prostate cancer (PCa). They are plastic in nature and usually exist as, M1 (anti-tumorigenic) or M2 (pro-tumorigenic) phenotype, with relative abundance of M2. In this study, we aimed to explore the phenotypic effects of TAMs on PCa and investigate the potential of glycyrrhizin, a natural compound, to reprogram M2 TAMs, as a feasible treatment approach against PCa. THP-1 monocytes and RAW 264.7 were used to study macrophage differentiation and glycyrrhizin-mediated repolarization. THP-1 cells were cultured in conditioned media (CM) of LNCaP and BPH-Cd to obtain PCa associated macrophages. Flow cytometric analysis revealed increase in expression of surface markers CD32, CD64, CD206 and CD163 confirming M1/M2 differentiation. qPCR analysis also demonstrated upregulation of CD80, CD86, CD68, TGF-β and IL10. M2 markers such as CD68, CD163, CCL2, CCL5 and TGF-β were expressed in BPH-Cd TAMs. LNCaP TAMs also co-expressed CD68 and CD163. In spheroid assay on BPH-Cd cells, treatment with BPH-Cd TAM resulted in abnormally shaped, enlarged spheroids. Furthermore, the CM enhanced migration of BPH-Cd cells in transwell migration assay and downregulated P21 in western blot analysis, thus confirming its tumor-promoting role. MTT assay performed prior to studying the polarization potential of glycyrrhizin showed an increase in viability of glycyrrhizin-treated THP-1 and RAW 264.7 and selectively promoted viability in M1. The M1-polarizing properties of glycyrrhizin was revealed in the qPCR analysis of glycyrrhizin-treated M0 macrophages. The reduction in the size of the spheroids and decreased migration of BPH-Cd upon treatment with glycyrrhizin-treated M0 CM confirmed the same. Differentiation media (DM) of glycyrrhizin-treated M2 TAMs resulted in smaller, disintegrated BPH-Cd spheroids indicating possible reprogramming effect. Interestingly, glycyrrhizin-treated M2 CM downregulated the gene expression of stemness-associated markers such as NANOG, SNAIL, OCT3/4 and COX2 with concurrent suppression of EMT marker, N-Cadherin in LNCaP cells. Cell cycle analysis on LNCaP revealed a decrease in the percentage of cells in S phase compared to glycyrrhizin-treated M2 CM, suggesting hindered proliferation. A notable observation to help understand the plausible mechanism of M2 TAM suppression was the decrease in Reactive Oxygen Species (ROS) levels in glycyrrhizin-treated M1 and M2 TAMs derived from THP-1 cells. The glycyrrhizin-mediated inhibition of ROS was also confirmed in RAW 264.7. In conclusion, our findings suggest that glycyrrhizin could potentially reprogram TAMs to intercept proliferation, metastasis and stemness characteristics in PCa. Citation Format: Amy Thomas, Gnanasekar Munirathinam. Reprogramming M2-like tumor associated macrophages using a natural compound, glycyrrhizin, as a potential therapeutic strategy against prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2334.