Messenger rna challenge rapidly predicts brain tumor responsiveness to checkpoint inhibitors

Abstract BACKGROUND Pathogen recognition receptor (PRR) activation is requisite for igniting the immune response and making the tumor microenvironment ‘hot’ during oncogenesis. We theorized that these PRRs would remain present in ICI responsive tumors and could be challenged using pan-PRR mimic to prospectively predict brain cancer immunogenicity and response to immune checkpoint inhibitors (ICIs). METHODS By layering mRNA into multilamellar nanoparticles (ML RNA-NPs), multiple pathogen recognition receptors can be simultaneously activated in individualized cancer cells serving as a broad screen to test tumor immunogenicity. We challenged murine brain tumor lines known to respond (GL261 and SMA-560) or resist ICI treatment (KR158b and CT2A) with ML RNA-NPs and analyzed downstream production of proinflammatory cytokines as predictors of ICI response. RESULTS We demonstrated that ML-RNA-NPs can activate multiple PRRs simultaneously. Following ML RNA-NP challenge, ICI responsive tumors GL261 and SMA-560 (compared to more ICI resistant lines KR158b and CT2A) elicited increased production in pro-inflammatory cytokines such as IFN-β and IL-6 and in CCL4. To further develop this work into a commercially viable tool to predict ICI response, we developed 3D modeling of glial tumors for ML RNA-NP challenge and immunogenicity prediction. We enrolled canines with primary gliomas and began growing their tumoroids in 3D using liquid-like solid (LLS) technology. Using this pipeline, we successfully perfused 3D tumoroids with ML RNA-NP demonstrating ability to set up real-time patient derived explants to predict immunogenicity. We demonstrated that mRNA perfusion of 3D tumoroids elicited similar cytokine response observed with 2D culture of cell-lines. CONCLUSION Our results indicate that cytokine signatures following ML RNA-NP challenge differ significantly from ICI responsive versus non-responsive brain tumors. These findings allow for creation of a diagnostic assay to quickly assess brain tumor immunogenicity, allowing for informed treatment with ICIs.