Abstract
This study investigates the formation of cadmium-induced CSCs within individual HepG2 tumor spheroids, which are fabricated using 3D bioprinting of HepG2 cancer cell-laden hydrogel composed of alginate and gelatin. The quantification of cadmium-induced single CSCs in single spheroids was performed through multicolor single-cell 3D imaging of CSC biomarkers, without the need to isolate spheroids from the hydrogel. Single HepG2 CSCs exhibiting the aldehyde dehydrogenase 1 (ALDH1)+/CD133+ phenotype were detected. The percentage of CSCs in the spheroids increased proportionally with the concentration of cadmium exposure. Additionally, the number of CSCs expressing Nanog and clusterin also increased in response to higher cadmium concentrations. Flow cytometric quantification of cadmium-induced CSCs isolated from single spheroids in the hydrogel yielded results similar to those obtained through single-cell imaging of CSCs within spheroids. This similarity underscores the reliability of this multicolor single-cell imaging technique for detecting cadmium-induced CSCs in spheroids. Overall, this study demonstrates that intact single-cell imaging of 3D bioprinted tumor spheroids is a powerful tool for illustrating the risk of developing drug-resistant CSCs upon exposure to carcinogens such as cadmium.