Abstract B055: Optimized human monocyte-derived macrophage cultures accurately reflect macrophage immunomodulatory function
Authors: Groot Amber E. de , Pienta Kenneth J. 
Published: February 2019

Abstract
Prostate cancer tumor growth and disease progression is highly influenced by noncancerous host cells within the tumor microenvironment. Tumor-associated macrophages (TAMs) are a critical component of this microenvironment, and high TAM infiltrate correlates with poor prognosis in prostate cancer. Macrophages can inhibit or support tumor growth depending on their polarization to an M1 or M2 state. M1 macrophages, as part of the Th1 immune response that fights bacterial or viral infection, promote helper and killer T-cell recruitment and activation and induce cell death of infected cells. In the tumor, M1s and their recruited T cells target the cancer cells for cell death. Conversely, as part of the Th2 immune response that repairs tissue injury, M2 macrophages promote cell growth and proliferation, inhibit helper and killer T-cell function, and induce regulatory T-cell differentiation. Protumor M2s TAMS make up the majority of macrophage infiltrate in prostate cancer tumors and protect the cancer cells from attack by the immune system. Techniques for studying TAMs in vitro are limited, which is confounded by the lack of procedure consensus for in vitro macrophage differentiation and polarization. The Pienta Lab established an optimized protocol for generating homogenous populations of M1 and M2 macrophages from human monocytes isolated from whole blood (doi: 10.2144/000114435). While these cell populations have been well characterized by their marker expression, their functional ability to modulate T-cell activation is critical to determining their validity as an in vitro TAM model. Following our optimized protocol, monocytes isolated from human blood were differentiated and cultured as resting macrophages or polarized into M1 or M2 macrophages. Conditioned media were collected at 24, 48, and 72 hours. The immunomodulatory capacity of the macrophages was evaluated by measuring the secreted levels of Th1 cytokines IL-1B and TNF known to recruit and activate helper and killer T cells and Th2 cytokine IL-10 known to prevent helper and killer T-cell function and promote regulatory T-cell induction. The in vitro-polarized human M1s secreted high levels of Th1 cytokines IL-1B and TNF, while levels from both the resting and M2-polarized macrophages were below the limit of detection. Likewise, conditioned media from M2-polarized macrophages showed high levels of Th2 cytokine IL-10, but none was detected in resting or M1 cultures. Taken together, this indicates that M1 and M2 macrophages isolated and polarized using our optimized protocol accurately reflect anticipated macrophage immunomodulatory function. Further work will confirm these data by RT-qPCR of RNA taken from M1, M2, and resting macrophage cultures, and conditioned media from serum-free cultures will be assessed to determine the secretion of additional immunomodulatory cytokines present at abundant levels in serum (e.g., TGFB1).
Citation Format: Amber E. de Groot, Kenneth J. Pienta. Optimized human monocyte-derived macrophage cultures accurately reflect macrophage immunomodulatory function [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B055.