Melanoma is a highly aggressive disease that is difficult to treat, a characteristic that may be due to tumour heterogeneity. Heterogeneity is the occurrence of different subpopulations of cancer cells within a tumour, resulting in multiple cellular phenotypes within a single site. These populations can be proliferating or arrested, invading or quiescent. As these cancer cells are exhibiting variable behaviours, they also respond to therapies uniquely. Understanding the molecular signature influencing cancer cell activity within tumours is therefore crucial to design the most effective therapeutic regimen.
To better understand tumour heterogeneity within melanoma tumours, the fluorescence ubiquitination cell cycle indicator (FUCCI) system was employed to observe different phases of the cell cycle in real-time. Interestingly, it was found that tumour xenografts grown from melanoma cell lines divided into two cohorts. One that contained distinct sub-populations of arrested or proliferating cells, and another that displayed a more homogenous dispersion. Furthermore, it was determined these two phenotypes could be separated by MITF expression, with high MITF levels correlating with random distribution. It was also determined that in WM164 cells, which normally gives rise to homogenous tumour xenografts, knockdown of MITF by shRNA converted the phenotype to become clustered. Cell lines that express MITF were grown into tumour spheroids embedded in collagen, and stained for MITF. MITF expression was found predominantly in the periphery of the spheroid, which also had high Slug and Vimentin expression, with a decrease in E-cadherin, indicative of an Epithelial to Mesenchymal Transition. This area also corresponds with the region of highly proliferative cells. Additionally, serum starvation, resulting in cell cycle arrest, resulted in decreased MITF levels, and knockdown of MITF by shRNA gave rise to more cells arrested in G1. These data outline how MITF and the cell cycle are tightly intertwined within tumour architecture.