Figure 4.

CRISPRoff repression of MGMT sensitizes GBM xenografts to TMZ in vivo. (A) Schematic of CRISPRoff mRNA and sgRNA delivery using electroporation into GBM cells, followed by intracranial transplantation of monoclonal populations (2 million cells per animal, n = 5 animals per condition) into immunocompromised mice. Animals were treated with TMZ between days 6–10 at 500 µg/kg/day by oral gavage. (B) Bioluminescent imaging (BLI) of orthotopic xenografts of CRISPRoff modified LN18 cells. P value = 2-way ANOVA comparing across all groups at day 55. (C) Representative BLI images for orthotopic xenografts for each CRISPRoff modified LN18 tumor at the specified timepoint post-transplantation. (D) Time to complete tumor regression, as measured by BLI, for orthotopic tumors following 5d TMZ treatment between days 6–10. P value = log-rank test comparing all time-to-event curves across the entire duration of the experiment. (E) H&E and immunohistochemistry against MGMT or cleaved caspase 3 from orthotopic tumors 30 minutes following the final dose of TMZ (day 10). Representative images are shown for n = 3 – 4 tumors per condition. (F) Quantification of IHC against MGMT or cleaved caspase 3. P value = 2 tailed Student’s t-test.