Modl-30. utilizing serum-free co-culture models for in-vitro modeling of tumor-associated macrophages in glioblastoma

Abstract BACKGROUND The GBM tumor microenvironment consists of various cancerous and non-cancerous cells that influence GBM growth, invasion, angiogenesis, and resistance to chemoradiation. In-vitro models of GBM often lack multiple cell types. Some addressed this by using multicellular models with macrophages and astrocytes; however, these models employ serum-containing media, which causes GBM brain-tumor initiating cells to lose their stem-like properties. Moreover, these models use IL-4/13 polarized macrophages (M2), despite tumor-associated macrophages (TAMs) not adhering to the M2 phenotype. Thus, there is a need to develop physiologically accurate in-vitro models of GBM. METHODS We transitioned THP-1 monocytes from serum-containing RPMI 1640 to serum-free TheraPEAKTM X-VIVOTM-15 media. Monocytes were stimulated towards a macrophage-like state with PMA and polarized by co-culturing with GBM patient-derived xenograft (PDX) lines using transwell inserts. Macrophage polarization was characterized through cytokine arrays, invasion, angiogenesis, and phagocytosis assays, comparing them to resting (M0), pro-inflammatory (M1), and M2 macrophages. RESULTS THP-1 monocytes grown in X VIVO-15 media exhibited similar proliferation rates to those in RPMI 1640 for up to 48 hours. However, at 72 hours, there was a slight decrease in proliferation rates in X VIVO-15 media (p< 0.0001). Macrophages polarized by the GBM PDX line, JX39P-RT, did not exhibit the cytokine expression profile of any other group. M2 macrophages displayed higher phagocytic capacity (25%) of JX39P-RT compared to M0, M1, and TAMs (< 15%, p< 0.01). M1 macrophages showed significantly higher invasion rates than M0, M2, and JX39P-polarized TAMs. CONCLUSIONS We have demonstrated a serum-free method by which we can polarize macrophages towards a GBM-supportive phenotype that differs molecularly and functionally from M0, M1, and M2 macrophages. This high-fidelity method of modeling TAMs in GBM will aid in the development and testing of innate immunomodulatory therapeutics that may one day enter the clinic in hopes of improving GBM outcomes