Fig. 4

GD2 tCAR on MSCs surface increases binding to GD2-expressing GBM cells in vitro by a cell-to-cell interaction assay. a Gating strategy for the analysis of the absolute number of MSC-GBM cell aggregates after 1.5 h incubation time. Gate 1: forward scatter area (FSC) and side scatter area (SSC) were used to enrich for intact cells (P1). Gate 2: CFSE staining (P3) and DsRed (or Deep Red) fluorescence (P2) recorded in the logarithmic scale were used to identify MSC-GBM cell aggregates appeared to be CFSE/DsRed (or CFSE/Deep Red) double positive (P4). Backgating in P1 population to identify MSC-GBM cell aggregates by morphological parameters. To quantify the absolute number of aggregates, for all tested conditions, we considered the number of cellular events acquired into the CFSE/DsRed (or CFSE/Deep Red) double-positive gate (P4) over 60 s. b–e Number of MSC-GBM cell aggregates reported as fold of all conditions versus EV MSCs, for all GBM cell lines and primary C3c GBM cells. GD2 tCAR MSCs and bi-functional MSC bound GD2-positive cells, T98G (b), U87MG (c), and C3c (e) greater than EV MSCs and mTRAIL MSCs (*^,°p < .05). For A172 negative line (d), no differences in aggregates formation were found between the MSC types. *^,°p < .05 by Student’s t test. Data are expressed as mean ± SD