MSI-N1014 exerted anti-colorectal cancer (CRC) properties. (A) The insert depicts the experimental design where MSI-N1014 effect on cancer-associated fibroblast (CAF) CRC-cells were analyzed. MSI-N1014 dose-dependently reduced the cell viability of CAF-educated DLD1 and HCT116 cells. Reduced migratory (B), colony-forming (C), and tumor sphere-formation abilities (D) in both DLD1 and HCT116 cells post MSI-N1014 treatment. (E) Western blot analysis revealed reduced levels of mammalian target of rapamycin (mTOR), epidermal growth factor receptor (EGFR), interleukin (IL)-6, leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), and β-catenin in MSI-1014-treated cells compared to their control counterparts. (F) Isobologram analysis showing the synergistic effects of MSN-1014 and 5-fluorouracil (5-FU) were achieved in different concentration combinations in both DLD1 and HCT116 cells. Numbers in red indicate the relative expression ratio. ** p < 0.01, *** p < 0.001.