Background
Radiation therapy (RT) induces nucleic acid sensing followed by cancer cell-intrinsic interferon (IFN-I) responses in various cancers. However, combining RT with immunotherapy in glioblastoma has failed to improve patient survival, suggesting robust immunosuppression. Our previous findings showed that RT induces a metabolic shift towards fatty acid synthase (FASN)-mediated lipid synthesis, supporting glioblastoma survival. Since fatty acid metabolism hampers IFN-I responses in viral immunity, we hypothesized that FASN might similarly inhibit IFN-I in irradiated glioblastoma, acting as an immune evasion mechanism.
Methods
In vitro, FASN was targeted using a doxycycline-inducible shRNA, CRISPR/Cas9, and FASN inhibitors (FASNi) in murine glioblastoma cells (GL261, CT2A and SB28). Nucleic acid sensors (cGAS, RIG-I, and MDA5) were blocked using CRISPR/Cas9. Glioblastoma cells derivatives were irradiated with 6Gy and collected 24hrs post RT to assess IFN-I by RNAseq, LEGENDplex assay, and ELISA. In vivo, CT2AshNS and CT2AshFASN cells were intracranially injected and selective irradiation (10Gy) was administered on day 10. Anti-IFNAR antibody was injected on days 6, 10 and 14. To evaluate the translation potential, mice bearing glioblastoma tumors received systemic FASNi (daily, i.p., days 9–18) and clinically relevant radiation (5 fractions of 6Gy). The immune landscape was evaluated by flow cytometry, immunofluorescence, and multiplex imaging with CellScape ChipCytometry (Canopy Biosciences) followed by analysis using Phenoplex (Visiopharm) (n=3–5/group).
Results
RNAseq gene expression analysis identified IFN-I signaling as the most significantly enriched pathway in irradiated FASN knockout cells. FASN blockade using orthogonal methods enhanced the secretion of IFN-beta and CXCL10 in irradiated glioblastoma cells; an effect that was significantly reduced when cGAS, RIG-I, or MDA5 were blocked. In vivo experiments showed increased CD11c+ and CD8+ T cell infiltration within irradiated CT2AshFASN tumors compared to CT2AshNS. Interestingly, this observation was lost when the IFN-I pathway was blocked, thus suggesting that targeting FASN selectively in cancer cells promotes immune infiltration. Similar results were obtained in mice treated with systemic FASNi in combination with fractionated RT. Flow cytometry and multiplex imaging analysis revealed a significant increase in activated CD8 T cells infiltrating RT+FASNi-treated tumors.
Conclusions
Our study highlights the role of FASN in shaping irradiated glioblastoma immunogenicity. Specifically, FASN inhibition restores nucleic acid sensing and cancer cell-intrinsic IFN-I, thus inducing immune cell infiltration. This work identifies fatty acid metabolism as a potent driver of immune evasion in irradiated glioblastoma and FASN as a novel target to convert a cold tumor into an immune inflamed glioblastoma in the context of RT.
Ethics Approval
Ethics Approval: Mice experiments were approved by the Institutional Animal Care and Use Committee #2019-0042.