TMOD-25. UTILIZING PRIMARY MACROPHAGES AS AN IMPROVED SERUM-FREE IN VITRO MODEL OF TUMOR-ASSOCIATED MACROPHAGES IN GLIOBLASTOMA

Abstract BACKGROUND Tumor-associated macrophages (TAMs) account for up to 40% of tumor mass in glioblastoma and over 70% of the immune cells contained therein. TAMs influence glioblastoma (GBM) growth, invasion, and resistance to therapy. Unfortunately, in vitro macrophage models typically employ immortalized cell lines, such as THP-1, which require serum for their growth and maintenance, and are often stimulated with IL-4/13 to mimic TAM function in vitro. Unfortunately, THP-1-derived macrophages do not readily adopt the M2 state and may be poor TAM models. METHODS The buffy coat from peripheral blood was isolated from healthy donor blood and plated in RPMI 1640 supplemented with 10% human serum and 50ng/mL of M-CSF. 25 ng/mL of M-CSF was added on day 3. The macrophages were washed, lifted, and plated in serum-free X VIVO-15 supplemented with M-CSF. Afterwards macrophages were left in an M0 (resting) state, or polarized towards an M1(pro-inflammatory) by stimulating with LPS/IFNγ, M2 (anti-inflammatory) by polarizing with IL-4/13, or TAM state by co-culturing with GBM patient-derived xenolines. Macrophage polarization was characterized through cytokine arrays, invasion, angiogenesis, and phagocytosis assays, comparing them to M0, M1, and M2 macrophages. RESULTS M1-polarized THP-1 macrophages displayed an increase in M1-associated markers, such as CD80 and HLA-DR (p<0.0001); however, M2-polarized THP-1 macrophages did not display an increase in the M2-associated surface marker, CD206 (p = 0.6167). Interestingly, M1-polarized peripheral blood mononuclear cell (PBMC)-derived macrophages displayed increases in the M1-associated markers, CD80 and CD369 (p< 0.01). Additionally, M2-polarized PBMC-derived macrophages displayed increases in CD206 (p<0.0001). Interestingly, TAMs derived from either THP-1s or PBMCs did not closely align with either polarization state using cell-surface markers, dPCR, or cytokine arrays. CONCLUSIONS We have demonstrated a serum-free method of generating a TAM model by polarizing primary macrophages towards a GBM-supportive phenotype that differs molecularly and functionally from M0, M1, and M2 macrophages.