Abstract 4063: Adoptive transfer of NF-kB p50 knockout immature myeloid cells shows a trend towards slower glioblastoma tumor growth in an orthotopic mouse model
Authors: Beck Patrick J. , Barberi Theresa , Friedman Alan D. 
Published: April 2023

Abstract
Background: Glioblastoma (GBM) is a universally fatal brain tumor. Tumor-associated macrophages (TAMs) comprise up to 40% of GBM tumors and predominantly demonstrate an M2 phenotype, favoring tumor growth while suppressing host immune response. Previously, we showed that in p50-/- mice lacking the repressive NF-κB subunit, GBM TAMs are biased toward the pro-inflammatory M1 phenotype, limiting GBM tumor growth by increasing activated tumor T cells. The present study aims to determine whether adoptively transferred immature myeloid cells lacking p50 (p50-IMC) localize to mouse GBM tumors and slow tumor growth.
Methods: Lineage-negative bone marrow (BM) cells were isolated from C57BL/6 (B6) p50-/- mice, expanded, and treated with Macrophage-Colony Stimulating Factor (M-CSF) for 24h to produce p50-IMC. BM-derived myeloid cells from both wild-type (WT) and p50-/- mice were subjected to analysis of M1 (TNFα, IL-12b) and M2 (Fizz1, Mannose Receptor) markers via quantitative PCR after exposure to M-CSF for 24h or after M2-polarization via five days in M-CSF and then two days in IL-4. B6-derived GL261-Luc GBM cells were inoculated intracranially into wild-type B6 mice, and four days post-implantation, mice were given 5-fluorouracil to deplete endogenous leukocytes. Mice then received either no additional treatment or three doses of ten million p50-IMCs 9-, 11-, and 14-days post-implantation, and tumor growth was monitored via bioluminescence, measured following luciferin injection using the Spectrum in vivo imaging system. The number of p50-IMCs localizing to the GBM-containing brain was determined by CFSE-dye-labeling of IMC followed by flow cytometry analysis 24h after injection.
Results: Both TNFα and IL-12b were increased in p50-/- cells after 5 days M-CSF, and increased IL-12b expression was maintained even after M2-polarization with IL-4. Of the 10 million injected p50-IMCs, 0.07% of those cells were found in the brain. Despite such a small number of p50-IMCs localizing to the tissue of interest, inhibition of GBM growth was observed. Mice receiving the p50-IMC treatments (n = 4) demonstrated a 2.3- and 2.4-fold reduction in tumor growth at 15 (p = 0.13) and 18 (p = 0.14) days post-implantation, respectively, compared to control mice (n = 5). At 21 days post-implantation, tumor size was effectively equivalent between the two groups (p=0.76). p50-IMC treatment did not correlate with improved survival.
Conclusion: Loss of p50 skews myeloid cells toward a proinflammatory phenotype, even under M2-polarizing conditions. Adoptive transfer of p50-IMC transiently impairs GBM growth in an orthotopic mouse model. Although statistical significance was not attained, this may be due to the small number of animals used in this experiment. Further investigation is needed to evaluate the full potential of p50-IMC as an immunotherapy option for GBM patients.
Citation Format: Patrick J. Beck, Theresa Barberi, Alan D. Friedman. Adoptive transfer of NF-kB p50 knockout immature myeloid cells shows a trend towards slower glioblastoma tumor growth in an orthotopic mouse model. [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 4063.