Abstract 4668: In-vitro modeling of tumor-associated macrophages in glioblastoma

Abstract

Abstract BACKGROUND: The Glioblastoma (GBM) tumor microenvironment (TME) is comprised of a plethora of cancerous and non-cancerous cells that contribute to GBM growth, invasion, and chemo- and radio-resistance. In-vitro models of GBM typically fail to incorporate multiple cell types. Some groups have addressed this problem by employing 3D bioprinting to incorporate macrophages, astrocytes, and other parenchymal cells in an extracellular matrix. However, these models also use serum-containing media, which has been shown to cause GBM brain-tumor initiating cells to lose their stem-like properties, and IL-4/13 polarized macrophages (M2), even though tumor-associated macrophages (TAMs) do not adhere to the traditional M2 phenotype. Taken together, these observations highlight a critical need to develop physiologically accurate in-vitro model systems for GBM. METHODS: THP-1 monocytes were transitioned from serum-containing RPMI 1640 to serum-free CTS™ AIM V™ SFM and TheraPEAKTM X-VIVOTM-15 Serum-free Hematopoietic Cell Medium. Monocytes were stimulated towards a macrophage-like state with PMA and polarized by co-culturing them with GBM patient-derived xenograft (PDX) lines, using transwell inserts. dPCR, a cytokine array, and a phagocytosis assay were used to characterize macrophages polarized by this method relative to classically polarized resting (M0), pro-inflammatory (M1), and anti-inflammatory (M2) macrophages. RESULTS: There was no significant difference in proliferation rates up to 48 hours for THP-1 monocytes grown in serum-free CTS AIM V or X-VIVO-15 media compared to serum-containing RPMI 1640. At 72 hours, both serum-free alternatives had slightly decreased proliferation relative to the serum-containing RPMI 1640; however, the X-VIVO-15 had higher proliferation relative to the CTS AIM V (p<0.0001). THP-1 macrophages polarized towards a tumor-supportive phenotype by the GBM PDX cell line, JX39P-RT, did not recapitulate the cytokine expression profile of any other polarization group. M2 macrophages had significantly higher phagocytic capacity (25%) of JX39P-RT relative to M0, M1, and TAMs (<15%, p<0.01). CONCLUSIONS: We have demonstrated a serum-free method by which we can polarize macrophages towards a GBM-supportive phenotype. This high-fidelity method of modeling TAMs in GBM will aid in the development of innate immunomodulatory therapeutics that may one day enter the clinic in hopes of improving GBM outcomes. Citation Format: Hasan Alrefai, Andee M. Beierle, Taylor L. Schanel, Lauren C. Nassour, Joshua C. Anderson, Patricia H. Hicks, Christopher D. Willey. In-vitro modeling of tumor-associated macrophages in glioblastoma. [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 4668.