Transfection of NCI-H23, HT-29, HCT-116 tumor spheroids by select pyridinium cationic amphiphiles formulations and commercial transfection reagent Lipofectamine®, as revealed by 3D images of GFP expression (a) and luciferase quantitation (b). Boundaries of spheroids are indicated by a dotted white line. Bar represents 200 μm. Note the strong cell dependence of transfection efficiency on cell type. P values were determined by two-way ANOVA within one set of formulations (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001).