Fig. 4: Biological effects of PCMdt-MMAE on CRC cell cycle, survival, and death.

a Changes in cell cycle: BxPC-3 and control HCC1806 (1 × 10⁶ cells per dish) cells were treated at 37 °C with 5 µg/mL of PCMdt-MMAE for various times. They were then collected, stained with propidium iodide, and analyzed using a flow cytometer [32,33,34]. Changes in cell cycle were marked with arrows. b Reduction of cell viability: A panel of seven cancer cell lines expressing variable levels of MET, RON, or both receptors (5000 or 8000 cells per well in a 96-well plate in triplicate) were treated with different amounts of PCMdt-MMAE for 96 h. HCC1806 cells without MET or RON expression served as the control. Cell viability was determined by the MTS assay [32]. c Dose-dependent cell death: Three cancer cell lines expressing MET, RON, or both receptors were treated with different amounts of PCMdt-MMAE for 96 h as described in (b). HCC1806 cells were used as the control. At the end of the study, the dead cells were counted using the Trypan blue exclusion assay to determine the percentage of cell death [32]. d Morphological evidence of cell death. Treatment of cells with PCMdt-MMAE was performed as described in (c). Cellular morphological changes from individual cell lines were observed at 96 h under the Olympus BK-41 inverted microscope and photographed. For all studies described above, the percentages of cell viability and/or cell death and the individual IC₅₀ values from individual groups were calculated using the GraphPad Prism 6 software. Results shown here are from one of three experiments with similar results. ADC antibody–drug conjugate, CRC colorectal cancer, MET mesenchymal-epithelial transition, PCMdt-MMAE monomethyl auristatin E was conjugated to PCMbs–MR to generate the dual-targeting ADC, RON recepteur d’Origine nantais