Three-Dimensional Cell Spheroid Culture and Proliferation Activity Study of Uveal Melanoma Cell Line C918

Abstract Objective: This study aims to investigate the morphological and histological characteristics of three-dimensional cell spheroids derived from the uveal melanoma (UM) cell line C918 and assess the impact of luteolin on their proliferation. Methods:C918 cells were cultured in ultra-low adsorption 96-well plates, and morphological changes in C918 three-dimensional cell spheroids were observed over varying time intervals. Histological features of C918 multicellular spheroids cultured in ultra-low adsorption 6-well plates were examined using both HE staining and immunohistochemical staining. The CCK8 reagent was employed to measure the optical density at a 450nm wavelength after 72-hour treatments with varying luteolin concentrations in both two-dimensional and three-dimensional cultured C918 cells. The IC50 values were compared between the two culture conditions. Results: Over time in culture, the volume of C918 three-dimensional cell spheroids gradually increased, and an ischemic and hypoxic region became evident within the spheroids on days 4 to 6 of culture. Histological staining demonstrated positive expression of proliferation marker antibodies (Ki67) and melanoma marker antibodies (MelanA, HMB45, S-100) in the multicellular spheroids from three-dimensional culture. CCK-8 experiments revealed that the IC50 values for luteolin in C918 cells were 183.50μmol/L in three-dimensional culture and 16.19μmol/L in two-dimensional culture after 72 hours. Three-dimensional cultured C918 cells, treated with varying luteolin concentrations for 72 hours, were observed under a microscope. The maximum cross-sectional area showed no statistically significant differences between the groups, but it was reduced in comparison to the control group. Conclusion: Three-dimensional cultured C918 cell spheroids exhibit histological characteristics similar to real tumors and are less responsive to luteolin than their two-dimensional counterparts. They offer a valuable model for anti-tumor drug screening.