Antitumoral efficacy of NDI-NIs in advanced 3D models. Advanced 3D spheroids were established from human colorectal cancer cells (HCT116) by growing the cells within drops of a methylcellulose scaffold matrix (day 0). After 2 days of culture (day 2), the spheres were treated with the indicated compound at final concentrations of 1, 2, and 5 μM. (A) Time course analysis of tumor spheroids growth, starting from 24 h after treatment (day 3), monitored by Incucyte^® S3 Live-Cell Analysis System (Essen BioScience, Ann Arbor, MI), 10× magnification. The results were expressed as the percentage of the spheroids area upon treatment relative to their own area before the compound administration. The histogram represents the mean values ± S.E.M. of at least four spheres. (B) Representative images of tumor spheroids growth described in panel (A). (C) Histological and immunohistochemical analysis of the spheroids generated from HCT116 cell lines and grown as in panel (A). Upper panels, representative images of immunostained sections. Lower panels, quantification of Ki-67, cleaved-caspase and γH2AX levels, expressed as percentage of positive cells. Thirty fields for condition were analyzed. The histograms represent the mean values ± S.D. of three independent experiments. *P< .05, **P< .01, ***P < .001, and ****P< .0001.