Ly6G+ cells in irradiated tumour tissue promote glioblastoma cell dedifferentiation and tumour recurrence

Abstract

Abstract Background Most of glioblastomas (GBMs) frequently recur at sites of radiotherapy, but it is unclear if changes in the tumor microenvironment by radiotherapy affect GBM recurrence. Methods GBM cells were injected into the nude mice brain. At 3 weeks post injection, the whole brains of mice were irradiated 5 times at 2 Gy per day. Rat anti-Ly6G antibodies were used for Ly6G+ inflammatory cell depletion to figure out the roles of Ly6G+ cells in irradiated tumor tissue. To detect cell senescence, non-irradiated or irradiated GBM cells and xenograft tumor sections were stained by using a Senescence β-Galactosidase Staining Kit. Bioinformatics analysis, tubule formation and cell invasion assays were used to unveil GBM cell dedifferentiation and changes in cellular properties. Results Radiation-induced senescent glioma cells alter tumor microenvironment, such as increasing glioma stem cells, angiogenesis, and tumor-associated Ly6G+ neutrophil cells via NFκB signaling-mediated activation of the senescence-associated secretory phenotype. Inhibiting NFκB signaling in irradiated glioma cells using the IκBα super-repressor prevents changes in the tumor microenvironment and glioma stem cells. tumor-associated Ly6G+ cells promote the dedifferentiation of glioma cells into glioma stem cells through the NOS2-NO-ID4 regulatory axis. Depleting tumor-associated Ly6G+ cells using Ly6G-neutralizing antibodies reduces glioma stem cells and prolongs survival in mice with recurrent gliomas after radiotherapy. In recurrent glioblastoma patients, neutrophil markers and tumor-associated neutrophils gene sets correlate with gene sets of NOS2-NO-ID4 regulatory axis and stem cell factors. Conclusions Our results suggest the need to develop new radiation sensitizers targeting neutrophils driven by senescence-associated secretory phenotype and cancer cell dedifferentiation. Legal entity responsible for the study The authors. Funding National Research Foundation of Korea (NRF) [Grant numbers: 2015R1A5A1009024 and 2017R1E1A1A01074205], the School of Life Sciences and Biotechnology for BK21 Plus, Korea University and Institute of Animal Molecular Biotechnology, Korea University. Disclosure All authors have declared no conflicts of interest.