Summarizing, the results generated by this study demonstrated the generation of pretreated cell lines namely HCT116, LS174T, DLD1, and SW620. By ODC chronic treatment consisting of FA, 5-FU, SN38, and OX at low doses. In 2D and 3Dcc models, all ODC lines-Pretreated were less affected by ODC treatment than ODC-naive lines. Whereas in the 3D model, only ODC-pretreated LS174T and SW620 were less affected by ODC treatment than ODC-naive cells. Finally, in 3D culture, the optimized drug combination composed of regorafenib, vemurafenib, vatalanib, and AZD4547 at low doses, showed considerable efficacy only in metastatic origin DLD1 cells, and an irrelevant effect on the HCT116 cells.