Abstract: Objective To investigate the mechanism of metformin for inhibiting self- renewal of colorectal cancer stem cells (CSCs). Methods CSCs were sorted from Wnt reporter- transfected colorectal cancer patient-derived organoids (PDOs) by fluorescence-activated cell sorting (FACS) and treated with metformin. The changes in self-renewal of the cells were assessed using sphere formation, colony formation and limiting dilution assays. The mRNA expressions of genes related with stemness and differentiation and Wnt target genes was detected by qRT-PCR. Wnt activity was assessed using flow cytometry in the CSCs. Seahorse analysis was used to evaluate cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) after metformin treatment. Mitochondrial membrane potential levels were detected with TMRE staining, and reactive oxygen species (ROS) levels were detected using MitoSOX staining. Galactose (10 mmol/L), metformin (10 μmol/L), NAC (5 mmol/L), and galactose+metformin were used to modulate ROS levels in the CSCs, and sphere-formation assays and flow cytometry were used to assess the changes in self- renewal capacity and Wnt activity. The effect of lentiviral transfection of yeast NADH dehydrogenase NDI1 on TMRE staining, MitoSOX staining and Wnt activity in the CSCs were analyzed with flow cytometry. Results Metformin significantly decreased the capacities of CSCs to form spheres, colonies and xenografts and reduced Wnt activity in the cells (P<0.01). The mRNA levels of stemness- related genes and Wnt target genes decreased significantly while those of differentiation-related genes increased in metformin- treated CSCs (P<0.05), which also showed significantly decreased OCR, TMRE and ROS levels with enhanced ECAR (P<0.001). Galactose significantly increased sphere-forming capacity, ROS levels and Wnt activity of the cells, and these effects were significantly inhibited by metformin (P<0.05). Transfection of the CSCs with NDI1 significantly attenuated the inhibitory effects of metformin on proportion of CSCs and Wnt signaling pathway activity. Conclusion Metformin reduces mitochondrial oxidative phosphorylation and ROS levels by inhibiting mitochondrial complex I, thereby suppressing Wnt signaling pathway to reduce self-renewal ability of colorectal CSCs.

Key words: metformin, cancer stem cells, self-renewal, colorectal cancer, mitochondrion, Wnt