Tmic-26. tams and hypoxia enhance glioblastoma stem-like cell enrichment and may impact radioresistance

Abstract The dismal survival rates of glioblastoma (GBM) patients are due, at least in part, to therapeutic resistance of GBM stem-like cells (GSCs) and their ability to repopulate recurrent tumors. Our goal was to determine if tumor-associated macrophages (TAMs) or hypoxia enhance the enrichment of GSCs in a manner that could impact radioresistance. First, we established GBM-brain cortical organoids (GBM-BCOs) to model human GBM. We co-cultured GFP-labeled GBM neurospheres with BCOs for 14 days and observed a tumor take rate of 100%. Next, we evaluated whether hypoxia enhanced TAM migration into GBM-BCOs by incorporating THP-1 cells into GFP-GBM-BCOs and incubating them for 24 h under normoxic or hypoxic conditions. We found that hypoxia significantly increased THP-1 cell influx into GBM-BCOs. Further, we investigated whether TAMs influenced the enrichment of GSCs in GBM neurospheres and in GBM-BCOs. We found that co-culture of THP-1 cells with either GBM neurospheres or with GBM-BCOs enhanced the CD133+ population after 72 h hypoxia, correlating with our IF studies of human GBM samples that show increased CD133+ and IBA1+ cells in hypoxic peri-necrotic zones compared to non-necrotic regions. Next, we established Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI), an advanced methodology for monitoring cell cycle phases for investigating the role of hypoxia and radiation on GBM. Flow cytometry analysis of patient-derived GBM cells revealed that the fraction of G1 cells decreased and the fraction of G2/M cells increased 24 h after 3 Gy radiation compared to non-irradiated cells in normoxia. In contrast, GBM cells exposed to hypoxia do not show such cell cycle shifts after radiation and have an enriched population of cells in G0 in both radiated and non-irradiated cells. Our results indicate that hypoxia and TAMs play critical roles in GSC enrichment and may impact radioresistance.