Fig. 3.

CAR-T mediated cytotoxicity of U87vIII-mKate2 target cells. A Post-BBB anti-EGFRvIII CAR-T effector function of CAR-F263, CAR-F269 and Mock T cells, in transwells contained iBECs, assessed in the abluminal compartment using continuous live-cell imaging (Incucyte) of U87vIII-mKate2 cells (red) proliferation as a measure of CAR-mediated killing. Data shown presented as percentage (%) of red object count per well. B anti-EGFRvIII CAR-T effector function of CAR-F263, CAR-F269 and Mock T cells, in empty inserts, assessed in the abluminal compartment using continuous live-cell imaging (Incucyte) of U87vIII-mKate2 proliferation as a measure of CAR-mediated killing. Data shown presented as percentage (%) or red object count per well. Representative data form three independent experiments. C–D Representative images of U87vIII-mKate2 killing in the albuminal chamber, for A and B respectively, after 48 h. Scale bar = 100 µm. See Additional file 3: Movie S1 for real-time movie showing CAR-F263, CAR-F269 and Mock T cell mediated U87vIII-mKate2 killing in the abluminal chamber. Quantification of CAR-T/T cell extravasation (mean + SD), across the BBB and empty inserts, in the presence and absence of U87vIII cultures in the companion plates assessed by CD45⁺ expression using flow cytometry at (E) 3, (F) 6 and (G) 24 h. Statistical significance marked by asterisks assessed by one-way analysis of variance (ANOVA) where *P ≤ 0.05