Differential tumour cell behaviour caused by environmental conditions, termed dynamic heterogeneity, is a prime source for drug resistance, and understanding its underlying mechanisms is crucial to design effective therapies. Due to their dynamic nature and three-dimensionality, tumours and tumour models are challenging systems to study spatio-temporal phenomena. The FUCCI cell cycle sensor allowed us to assess individual cycling cells within their endogenous multicellular environment. Whole measurements of the three-dimensional structures of samples required single-plane illumination microscopy to achieve sufficient penetration depths, while being minimally invasive. Using these techniques, we have demonstrated dynamic heterogeneity in melanoma xenograft tumours and melanoma spheroids. This heterogeneity was characterised by the presence of clusters of proliferating cells and clusters of G1-arrested cells in the same tumour/spheroid. The location of the quiescent zones suggested oxygen/nutrient deprivation as the cause of cell cycle arrest, and the G1-arrested cells reversed to cycling when cultured under normoxia in 2D culture. Here we demonstrate that this heterogeneity is consistently decreased in vivo and in vitro by MITF, a transcription factor strongly associated with melanoma development, progression and therapy response. While this phenomenon was not associated with a reduced hypoxic core, we show that high MITF expression allows proliferation under hypoxia. Importantly, modulation of MITF expression leads to changes of spheroid architecture, tensile stress and in extracellular matrix (ECM) and cell-ECM adhesion and crosstalk proteins. Currently undergoing atomic force microscopy measurements of spheroids will reveal whether these changes are accompanied by stiffness modulation of cells, ECM or both. In addition, we are in the process of incorporating fluorescent stress beads into spheroids to assess forces that cells undergo at different locations within these structures. Furthermore, inhibition of the Rho/ROCK signalling pathway mimics the morphology and cell cycle effects of high MITF expression. These findings support a novel role of MITF in controlling intratumour melanoma heterogeneity through changes in cell-ECM crosstalk and mechanotransduction.