We have demonstrated that targeting the endoplasmic reticulum with fenretinide (synthetic retinoid) or velcade (26S proteosome inhibitor) induces cell cycle arrest and apoptosis of metastatic melanoma cells in vitro and in vivo. This study aims to investigate the effect of ER stress-inducing agents on the dynamics of cell division and cell death of individual melanoma cells within the complex tumor microenvironment.

We have developed a novel model to visualize the cell cycle dynamics in melanoma cells in real-time in vitro and in vivo. Cells transfected with fluorescence ubiquitination cell cycle indicator (FUCCI) plasmids appear red in G1, yellow in S and green in S/G2/M phase with a fluorescence gap during cytokinesis. FUCCI-melanoma cells were grown as 3D spheroids and implanted into a collagen matrix to mimic tumor architecture and microenvironment, or as xenografts in NOD/SCID mice. Using confocal and intravital multiphoton microscopy, respectively, we observed in 3D spheroids in vitro and in xenografts in vivo, that melanomas are composed of differentially cycling tumor cells in a subcompartment-specific distribution, which may result in differential sensitivity to apoptosis. Flow cytometry and confocal microscopy indicated that treatment of FUCCI-melanoma cells with fenretinide or velcade induced G1 or G2 accumulation, respectively, in 2D culture over the course of 24 h. Interestingly, while spheroids treated with low concentrations of velcade accumulated in G2 phase after 24 h, by 72 h the majority of cells appeared to be in G1 phase, suggesting cell cycle synchronization and indicating that cells which do not undergo apoptosis can recover and re-enter the cell cycle. Furthermore, combined treatment caused synergistic cytotoxicity in 2D and in 3D culture. Subsequent experiments utilizing our established in vivo FUCCI-model will optimize the velcade/fenretinide combination treatment for metastatic melanoma.