Fig. 5

Bi-functional MSCs exert specific in vitro cytotoxicity at an early time point against T98G GBM cell line by a cell-to-cell interaction cytotoxicity assay. a Gating strategy applied to quantify the percentage (%) of Annexin V-positive MSC-GBM cell aggregates after 7 h incubation time. Gate 1: forward scatter (FSC) and side scatter (SSC) were used to enrich for intact cells (P1). Gate 2: CFSE staining (P3) and DsRed fluorescence (P2) recorded in the logarithmic scale were used to identify MSC-GBM cell aggregates visualized as CFSE/DsRed double positive (P4). Backgating in P1 population to identify MSC-GBM cell aggregates by morphological parameters. Early cell death in MSC-GBM cell aggregates was evaluated by Annexin V (APC) by FACS gating on CFSE/DsRed-double-positive conglomerates while recording a fixed number of events for all conditions. b Interaction between bi-functional MSCs and GBM cells (smooth columns) produced a significant apoptotic effect on cell aggregates (59 ± 5%), compared to both mTRAIL MSCs (32 ± 7%) and non-expressing TRAIL ones, EV MSCs (9 ± 3%) and GD2 tCAR MSCs (15 ± 4%) (*p < .001). Blocking GD2 antigen–GD2 tCAR binding by anti-idiotype sera incubation (striped columns), significantly reduced the percentage of Annexin V-positive aggregates (40 ± 2%), comparable to mTRAlL MSCs with (36 ± 7%) or without (32 ± 7%) sera incubation. *p < .001, **p < .001, °p < .001, and °°p < .001 by Student’s t test. Data are expressed as mean ± SD