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. 2025 Jul 30;17(15):2510. doi: 10.3390/cancers17152510

Figure 2.

Figure 2

Comparison of complement-dependent cytotoxicity mediated by the anti-GD2 antibodies dinutuximab beta (DB, intermediate affinity) and naxitamab (NAXI, higher affinity) in a two-dimensional (A,B) and three-dimensional spheroid model (C,D). (A) LAN-1 and (B) CHLA-20 neuroblastoma cells (2500 cells/well) were stained with calcein–AM and incubated with 12.5% active human serum in the presence of a concentration series of DB or NAXI (0.01–1 µg/ml) or absence of antibodies serving as control (complement-independent cytotoxicity, CIC). Cytotoxicity was assessed after 4 h by measuring calcein–AM release. (C,D) For the spheroid assay, 10,000 LAN-1 (C) or CHLA-20 (D) cells stably expressing a near-infrared fluorescence marker were used for seed spheroid formation. Spheroids were treated with 12.5% active human serum and a concentration series of DB or NAXI, with viability determined as the total integrated spheroid fluorescence of the respective time point divided by the total fluorescence at time point 0 h. Statistical differences were analyzed using ANOVA, comparing all groups and using the Holm–Šídák method. Statistical significance is indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus untreated control; $ p < 0.01 versus DB; ns = not significant.