Fig. 1: TRAIL-resistant cell layers protect TRAIL-sensitive cells within tumor cell spheroids.

a, d Cell viability in HCT116 and NCI-H460 cells, grown in 2D or as spheroids (day 11), following 6 h of treatment with TRAIL. The loss of viability was measured flow cytometrically by staining with Annexin V-EGFP. Data shown are mean values ± SEM from three independent experiments. Insets serve as illustrations. b Cell viability in response to TRAIL (3.55 nM), measured as in (a), in the presence or absence of pan-caspase inhibitor QVD-OPH (50 µM). Data show mean values ± SEM from three independent experiments. c, e 2D-cultivated HCT116 and NCI-H460 cells were detached and HCT116 and NCI-H460 spheroids were dissociated before treatment as in (a, d). Insets serve as illustrations. f The table shows the EC₅₀ values determined by nonlinear regressions from (a, c, d, and e) (sigmoidal dose response). g, h Transcript amounts of TRAILR1 and TRAILR2 (long and short isoforms) in 2D-cultivated cells and cells grown as spheroids (day 11), as measured by qPCR. Relative RNA amounts were calculated by the ΔΔCt method using GAPDH for normalization. Data shown are mean values ± SEM from three independent experiments. i–l Surface expression of TRAILR1 and TRAILR2. 2D-cultivated cells and cells from spheroids (day 11) were analyzed by flow cytometry. Histograms are representative of at least three independent experiments. Population medians were used to calculate the differences in relative surface expression between 2D- and 3D-cultivated cells. AU arbitrary units. Bar graphs show mean values ± SD of at least three independent experiments. Asterisks indicate statistical significance (*p ≤ 0.05; **p ≤ 0.01; ns not significant; unpaired t-test).