Myeloid Cells in the Glioblastoma Microenvironment Maintain a Glycolysis-Dominant Metabolic Profile and Develop an Enhanced Immunosuppressive Phenotype under Hypoxia

Introduction: The glioblastoma (GBM) tumor microenvironment (TME) is marked by severe hypoxia. While most cells switch to glycolysis under hypoxia and favor mitochondrial respiration in normoxia, cancer cells prefer glycolysis even in oxygen-rich conditions (Warburg effect). It remains unclear how pro-tumoral myeloid cells adapt metabolically to the hypoxic conditions of the GBM TME. Here, we aimed to characterize metabolic and immunologic changes of myeloid cells under hypoxia.

Methods: Hematopoietic stem cells isolated from C57BL/6J mice bone marrow were differentiated into myeloid-derived suppressor cells (MDSCs) using GM-CSF and IL-6. T cells from mice splenocytes were sorted and activated using CD3/CD28 agonists. Tumor-associated macrophages (TAMs) and tumor-infiltrating lymphocytes (TILs) were also isolated from the brains of CT2A glioma-bearing mice. A subset of these cells was exposed to hypoxia (5% O2) at 37°C for 24 and 48 hours. With Seahorse XF analyzer, glycolysis and mitochondrial respiration were measured by extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively. Immunophenotyping was performed via flow cytometry.

Results: In normoxia, MDSCs exhibited a strong glycolytic preference, with significantly higher basal ECAR compared to T cells (P < 0.0001). Under hypoxia, T cells showed stable OCR and an increase in ECAR (P=0.0005), though this remained below the basal level of normoxic MDSCs. Hypoxia did not affect MDSC ECAR but reduced OCR after 24 (P=0.002) and 48 hours (P=0.001). Similar trends were observed with TAMs, which displayed a glycolysis-dominant profile and higher basal ECAR (P < 0.0001) than TILs. Extended hypoxia from 24 to 48 hours upregulated arginase 1 expression in MDSCs (P=0.029), suggesting a more immunosuppressive phenotype.

Conclusion : Myeloid cells demonstrate high glycolytic activity and reduced oxidative phosphorylation in response to hypoxia. Unlike T cells, myeloid cells maintain glycolysis even in normoxia—a metabolic profile resembling cancer cells. Exposure of myeloid cells to prolonged hypoxia is correlated with a resultant immunosuppressive phenotype.