486 Targeted Inhibition of Extracellular Matrix Protein, Laminin 411 in Glioblastoma In Vivo Upregulates Innate Immune System and Increases Animal Survival

INTRODUCTION: Glioblastomas (GBM) escape the immune system and are heterogenous making it difficult to target the tumor directly for treatment. However, tumor microenvironment is independent of glioma heterogeneity and might be a universal target. GBMs overexpressing laminin-411 are associated with poor patient survival. We synthesized a blood brain barrier (BBB) crossing drug delivery system that can selectively inhibit laminin-411 synthesis in vivo, upregulating innate immune system and improving survival. METHODS: GBM cell lines CT-2A and GL261 with CRISPR/Cas9 depletion of laminin-411 or control wild-type were intracranially implanted in C57BL/6J mice, n = 8 per group. Depletion was validated by western blot. We used flow cytometry (FC) to verify molecular changes in tumor microenvironment after laminin-411 depletion and examined mouse survival. Additionally, wild-type GL261-implanted mice (n=8) were I.V. treated with nanoimmuno polymeric drug crossing BBB and specifically blocking laminin-411 synthesis using morpholino antisense. Mouse survival was determined, and tumors were assessed by RNA-seq, immune markers (FC), and immunohistochemistry. RESULTS: Animals with laminin-411 knockout GBMs showed significant tumor upregulation of innate immune system markers, CD3, CD8, as well as NK T cells, NK, IFNg+ NK cells, and M1 macrophages by FC analyses vs. control tumors. These mice had increased median survival, 47 vs. 34 days in CT-2A (p = 0.003) and over 50 vs. 26 days in GL261 (p = 0.001). Similarly, in vivo treatment of wild-type GBM GL261-bearing mice with nanoimmuno polymeric drug showed 32% greater survival (33 vs. 25 days, p = 0.0001) vs. PBS. Immunohistochemistry-validated RNA-seq showed increase in IFN-g, TNF-a, and M1 macrophage iNOS, and decrease of proliferation markers Ki-67 and c-Myc after treatment. CONCLUSIONS: For the first time, laminin-411 was studied as a targetable regulator of GBM immunity. Our nanoimmuno drug inhibiting laminin-411 can cross BBB and inhibit tumor growth.