Glioblastoma (GBM) is the most common and lethal malignant brain tumor that invariably recurs after standard therapy, with a median survival of only ~16 months. A major determinant of high GBM lethality is the overall immunosuppressive tumor microenvironment and the presence of treatment resistant hypoxic niches. In a recent study, we engineered murine GBM cell line GL261 with a lentiviral HRE driven fluorescent reporter, HRE-UnaG-PEST to study the relationship between tumor hypoxia and immunosuppression. By 3-4 weeks post-transplant in mice, we observed an abundance of UnaG+ GBM cells that were organized as pseudopalisading patterns around necrotic cores, similar to histological features observed in GBM patient specimens. We also observed an accumulation of TAMs and cytotoxic T cells inside hypoxic cores surrounded by UnaG+ GBM cells, which coincided with a gradual increase in vascular disarray and development of hypoxic niches. Single cell RNA sequencing (scRNA-seq) revealed that UnaG+ GBM cells were negatively enriched for neuroinflammation, Type II IFN signaling, TNFα and NF-κB signaling pathways, and IFNα and IFNγ responses. Furthermore, the gene ontology term “Negative Regulation of Immune System Process” (including genes such as Mif, Lgals3, Sdc4, Cd44, Dusp1) was predominantly upregulated in UnaG+ GBM cells. Hypoxia transcriptional program in GL261 GBM thus favors immunosuppression. To longitudinally study the immunosuppressive transcriptional programs of hypoxic GBM cells, we combined lentiviral HRE-CreERT2 with a knock-in of CAG-loxP-STOP-loxP-tdTomato (LSL-tdTom) reporter into the Rosa26 locus as a lineage tracing reporter for hypoxia-exposed GBM cells. A subclone of GL261-HRE-CreERT2; LSL-tdTom cells that displayed low baseline expression and faithful induction of tdTomato upon exposure to 1% O2 and 4-hydroxytamoxifen was identified and intracranially transplanted in mice. After a single tamoxifen injection day 21 post-transplant, mice were traced for 2 days or 7 days before analysis. At both timepoints, we observed abundant tdTom+ GBM cells spread throughout the tumor. Few tdTom+ GBM cells showed GLUT1 expression, more so in 7-day tracing than 2-day tracing. Hence, as the tumor progressed, most tdTom+ GL261 cells no longer experienced hypoxia, likely due to new angiogenesis or migration away from hypoxic areas. No tdTom+ GL261 cells were observed in parallel control experiments where tamoxifen was not injected. To investigate if hypoxia-induced immunosuppressive adaptations are preserved along tumor progression, further scRNA-seq and FACS studies will be performed to characterize the transcriptional profile of tdTom+ GBM cells. To test if hypoxic exposure is a critical conditioning event that promotes an immunosuppressive environment in GBM, we plan to genetically eliminate hypoxic GBM cells by inducing expression of Diptheria Toxin and evaluate effect on functional state of immune cells.

Citation Format: Anirudh Sattiraju, Sangjo Kang, Hongyan Zou, Roland Friedel. Tumor hypoxia conditions glioblastoma cells for immunosuppression [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 1260.