Microphpthalmia-associated transcription factor (MITF) drives phenotypic heterogeneity in a melanoma spheroid model. A high expression of MITF in a spheroid is associated with a homogenous distribution of cell cycles throughout the spheroid. In contrast, a heterogenous population of cell cycles is observed in low MITF-expressing spheroids. This disparity is brought about by the modulation of extracellular matrix (ECM) components with high MITF expression causing a ‘looser’ matrix and low MITF expression causing a ‘denser’ matrix. Lysosomal enzymes (namely cathepsins) and autophagic activity are in charge of degrading and remodelling ECM components, often positively correlated with tumours' aggressiveness and metastatic potential. The main driver of lysosomal biogenesis and autophagic activity is transcription factor EB (TFEB), a member of the MITF/TFE family of transcription factors. Therefore, it is hypothesised that TFEB exists in the same direction as MITF; high MITF expression would possess a higher autophagic flux, vice versa. The aim of studying the autophagic and lysosomal activity in an MITF-rheostat model is to use it as a prognostic tool to decide on a targeted or combination therapy for patients. Through immunofluorescence and imaging studies, the previous statement was true when cell lines were induced to overexpress MITF. However, this was not the case with knocking down MITF in basally high MITF-expressing spheroids. Autophagy is most active in the quiescent zone of spheroids where cells start to arrest due to hypoxia, drawing interest in this zone. Autophagy was induced in spheroids due to starvation, and most cell lines exhibited an anti-tumoural response. Interestingly, the inhibition of autophagy with chloroquine rescued the population of G2 proliferating cells at the periphery of the spheroid. This suggests that autophagy is crucial in driving phenotypic heterogeneity by killing G2 cells at the periphery. This provides preliminary data that autophagic activity and lysosomal function is essential in determining a personalized treatment option for patients.