Figure 4. EGFR-CAR NK cells enhance eradication of GBM cells and prolong survival in an in vivo xenograft model.
A, Assessment of EGFR-CAR expression 48 hours post retroviral transduction by flow cytometry. Anti-mouse Fab’ antibody was used to stain human anti-EGFR scFv on NK cells. B, Left: unsorted EGFR-CAR NK cells cytotoxicity function against target LN229 (EGFR positive). Right: an eosinophilic leukemia cell line EOL-1 was used as a negative control. P values are corrected by multiple t tests using the Holm-Sidak method. Values are presented as mean ± SEM. *, P < 0.05; **, P < 0.01. The experiment was repeated three times with NK cells isolated from different donors with similar results. C, Degranulation of CD107a as well as secretion of IFN-γ and TNF-α from untransduced (UT), unsorted EV-transduced (EV), or EGFR-CAR transduced NK cells co-cultured with the GBM cell line LN229 (EGFR positive) or U87vIII (EGFRvIII positive). EOL-1 was performed as a negative control for both. The experiment was repeated seven times with NK cells isolated from different donors with similar results. P values correct for ordinary one-way ANOVA using Holm-Sidak multiple comparisons test. Values are presented as mean ± SEM. *, P < 0.05; **, P < 0.01. D, Experimental timeline for in vivo study. On day 0, a xenograft GBM mouse model was established by intracranial injection of 1 × 105 U87vIII-luc cells into NSG mice. On days 3, mice were intratumorally injected with 1 × 106 EV-transduced NK cells, 1 × 106 EGFR-CAR NK cells, or saline. All transduced cells were unsorted. n=5 animals for each group. E, BLI to check brain tumor growth on day 8. F, Quantification of BLI in E. P values correct for ordinary one-way ANOVA using Holm-Sidak multiple comparisons test. Values are presented as mean ± SD. *, P < 0.05. G, Survival of U87vIII-luc-bearing mice treated with EV-transduced NK cells, EGFR-CAR NK cells, or saline alone. Log-rank test was used to compare animal survival curves. **, P < 0.01.