Generation of a 3D melanoma model and visualization of doxorubicin uptake by fluorescence imaging.

Melanoma is the most dangerous type of skin cancer and is responsible for 75% of deaths from skin cancers. For an accurate evaluation of potential treatment efficacy, it is important to use study models as close as possible to the in vivo conditions. A 3D model consisting of B16F10 spheroids was developed using liquid overlay technique on plates coated with 1% agarose, in the presence of 1% methylcellulose and L929-conditioned medium. The model is suitable and can be further used for more complex in vitro drug testing than the classical 2D approach. For exemplification, the behavior of a well-known cytostatic, doxorubicin (DOX), was evaluated in spheroids as compared to classical 2D culture conditions. Fluorescence imaging was used to visualize DOX uptake by B16F10 spheroids at different periods of time. The results showed that a much higher DOX concentration is necessary to produce similar effects compared with the monolayer. The fluorescence images revealed that at least 4 h of stimulation is needed for a sufficient DOX uptake. The 3D model developed in this study was suitable to investigate drug penetration in time. Our findings may explain the decrease of the doxorubicin therapeutical effect, suggesting the need of maintaining the drug concentration at the tumoral place for at least 2 h upon administration. Similar or more advanced studies can lead to a better understanding of drug delivery kinetics and distribution upon administration, conducing toward a better performance in designing suitable delivery systems for obtaining the optimum dose-response effect.