Fig. 2

CAR-T extravasation across the BBB. A Schematic of single-chain variable fragment (scFV) structure of EGFRvIII-CAR. B Schematic illustrating set up of transwell BBB and U87vIII co-culture system to establish blood–brain tumor barrier (BBTB) model. Phase contrast images in the luminal chamber showing CAR-T cell interacting with the iBEC monolayer in the transwell inserts. Scale bar = 50 µm (C) Assessment of BBB integrity using sodium fluorescein (NaFl) permeability (Pe), 24 h following addition of CAR-T cells (CAR-263 and CAR-269) and Mock T cells to iBEC transwell cultures in the presence or absence of U87vIII cells in the companion plates. Pe is expressed as the mean + SD. D Quantification of CAR-T/T cells extravasation in the abluminal chamber of the companion plate after 24 h based on CD45 expression assessed by flow cytometry. E Quantification of CAR-T/T cell activation, in both top (luminal) and bottom (abluminal) compartments based on CD25 expression, as a percentage of CAR-transduced GFP expressing cells, assessed by flow cytometry. Data presented as mean + SD and statistical significance is marked by asterisks assessed by one-way analysis of variance (ANOVA) where *P ≤ 0.05, ** ≤ 0.01 (n = 5)