Figure 9.

(A) Phase-contrast images of DU145, DU145GemR, and PC3GemR at 10× objective treated with a combination of 50 μM cyclo-RGDfK(TPP) peptide and OP or without OP at concentrations of 50, 100, or 200 μg/mL for 6 days. A total of 10,000 cells were plated per well in a 96-well plate for 6 days. (B) MCTS volume was measured by using V = (4/3) πr³ where π = 3.1415 and r = average radius (microns). Radius was measured from the scale bar. Each bar in the graph represents mean MCTS volume ± standard error (error bars) for all MCTS within the representative images. Results were compared by a one-way ANOVA at 95% confidence using Fisher’s LSD test. Data are representation of one of two independent experiments showing similar results. (C) Viability of DU145, DU145GemR, and PC3GemR attached cells and MCTS using WST-1 assay. Cells were plated at a density of 10,000 cells/well in a 96-well plate and incubated at 37°C in a CO₂ incubator for 3 hours for the cells to attach. The attached cells were treated with OP at the indicated doses with a combination of cyclo-RGDfK(TPP) peptide at 50 μM for 6 days. On day 7, 10 μL of WST reagent was added to each well. Cells were incubated for 2 hours at 37°C. Cell viability was expressed as a percent of control ± SEM of two independent experiments. Statistical analysis was carried out by using GraphPad Prism, and the results were compared by one-way ANOVA at 95% confidence using Fisher’s LSD test. (D) Phase-contrast images of PC3 cells at 10× objective treated with a combination of 50 μM cyclo-RGDfK(TPP) peptide and OP or without OP at 50, 100, or 200 μg/mL for 6 days. A total of 10,000 cells were plated per well in a 96-well plate for 6 days. (E) Spheroid volume and cell viability assays on PC3 MCTS. (F) Phase-contrast images of PC3 MCTS at 4× objective in the presence of 50 μM cyclo-RGDfK(TPP) peptide with 100 μg/mL OP or without OP with 10,000 cells per well in 96-well plate for 1–6 days of incubation.

Abbreviations: MCTS, multicellular tumor spheroid; cyclo-RGDfK(TPP), cyclic Arg-Gly-Asp-D-Phe-Lys peptide modified with 4-carboxybutyl-triphenylphosphonium bromide; OP, oseltamivir phosphate; ANOVA, analysis of variance; SEM, standard error of mean; LSD, least significant difference; WST, cell proliferation reagent.