Morphological and functional responses of three-dimensional-cultured colorectal cancer spheres to anticancer drugs

BACKGROUND Three-dimensional (3D) culture systems closely recapitulate the tumor microenvironment, allowing the assessment of drug effects on cell-cell interactions and metabolic heterogeneity. Colorectal adenocarcinoma (CRAC) cells exhibit epithelial and mesenchymal features, reflecting tumor heterogeneity, which may influence drug sensitivity. However, the morphological alterations and functional states of the residual CRAC spheres after chemotherapy remain unclear. Understanding these morphological and metabolic adaptations may help identify cancer stem cell (CSC)-like and epithelial-mesenchymal transition (EMT)-related phenotypes that contribute to therapeutic resistance and recurrence. AIM To elucidate the morphological and functional responses of 3D-cultured CRAC spheres to anticancer drugs and to characterize the residual CSC/EMT phenotypes. METHODS Five CRAC cell lines were cultured as 3D spheres, treated with 5-fluorouracil, irinotecan, or oxaliplatin, and analyzed morphologically and molecularly. RESULTS All drugs significantly suppressed sphere growth, but the morphological and metabolic responses varied. Scanning electron microscopy revealed cell surface flattening or detachment in epithelial-type spheres, whereas mesenchymal-type SW480 spheres exhibited a bowl-shaped structure with depressed centers. ATP levels decreased in all lines, except for 5-fluorouracil-treated SW480 spheres, which retained ATP but showed reduced water-soluble tetrazolium 8 activity, suggesting metabolic dormancy. Caspase 3/7 activity markedly increased in irinotecan-treated HCT-15 cells. Quantitative reverse transcriptase PCR revealed the upregulation of CD44 , ALDH1A1 , and PROM1 in HCT-15 cells, and increased CD44 /ALDH1A1 with reduced PROM1 in SW480 cells. CDH1 , VIM , and ZEB1 levels increased in HCT-15 cells, whereas SW480 cells upregulated CDH1 and VIM but downregulated ZEB1 , indicating partial EMT and mesenchymal-epithelial reverting transitions. CONCLUSION Anticancer drugs induce distinct morphological and molecular adaptations, and residual CRAC spheres acquire hybrid epithelial-mesenchymal phenotypes associated with chemoresistance.