Background Immunotherapy (IT) has evolved as an essential pillar against cancer due to unprecedented successes in several malignancies. However, only 10% of glioblastoma (GBM) patients respond to IT, presumably due to the paucity of tumor-infiltrating lymphocytes. Radiotherapy (RT) can promote T cells infiltration to generate anti-tumor immune responses, but can also exacerbate potent immune inhibitory mechanism to facilitate immune evasion. Among which, metabolic reprogramming of irradiated GBM represents an emerging mechanism of immune resistance. Notably, increased lipogenesis by the fatty acid synthase (FASN) is a hallmark of GBM that was shown to mediate radioresistance and immunosuppression in other cancer types. Therefore, we hypothesize that de novo lipid biosynthesis mediated by FASN represents an innate immune evasion mechanism in irradiated GBM.
Methods We first defined metabolic changes 24hrs after RT (10 gray - Gy) by seahorse assay and metabolomics in the syngeneic murine GBM model, GL261. To confirm alterations in the lipogenesis pathway, we measured the expression FASN by western blot and the cell lipid content by BODIPY staining and flow cytometry. Finally, GL261 cells were engineered to express an inducible shRNA silencing FASN (GL261shFASN) or its non-silencing control (GL261shNS) and orthotopically implanted on day 0. On day 6, knockdown of FASN was induced by feeding the mice with doxycycline. On day 11, mice received 10Gy irradiation selectively to the tumor. Evaluation of the immune contexture was determined by in situ immunofluorescence on day 19 (n=3/group). Remaining mice were followed for survival (n=7/group).
Results Mitochondrial respiration and glycolysis were significantly enhanced in RT-GL261 cells in vitro. Metabolomic profiling of RT-GL261 cells showed a strong increase in pathways related to nucleotide, amino acids and lipid metabolism. Consistent with this last observation, we found upregulation of FASN and lipids accumulation in RT-GL261 cells as compared to non-RT GL261 cells. In vivo, GL261shFASN tumors presented increased infiltration of CD11c+ and CD8+ T cells as compared to GL261shNS tumors; an observation that was amplified in RT-GL261shFASN tumors. Consistent with a recruitment of CD11c+ and CD8+ T cells, 43% of mice bearing GL261shFASN tumor survived for at least 60 days without tumor regrowth vs. 35 days in GL261shNS tumor bearing mice.
Conclusions Altogether our data suggest that RT is inducing a metabolic reprogramming of GBM by promoting FASN-mediated lipid synthesis to foster immunosuppression. While much work remains to be done, our data propose FASN as a novel therapeutic target to overcome immunosuppression and sensitize irradiated GBM to ITs.
Ethics Approval All mice experiments were approved by the Institutional Animal Care and Use Committee, protocol number 2019-0042.
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