Lattmann, E.; Jovic, A.; Kim, J.; Pham, T.; Corona, C.; Lian, Z.; Saini, K.; Ray, M.; Lu, V.; Tastanova, A.; Boutet, S. C.; Levesque, M. P.

Melanomas are the deadliest skin cancers, in part due to cellular plasticity and heterogeneity. Intratumoral heterogeneity drives varied mutable phenotypes, specifically \"melanocytic\" and \"mesenchymal\" cell states, which result in differential functional properties and drug responses. Definitive and rigorous classification of these phenotypic states has been challenging with conventional biomarker-based methods, and high-parameter molecular methods are cell-destructive, labor-intensive, and time-consuming. To overcome these technical and practical limitations, we utilized label-free artificial intelligence-based morphological profiling to classify live melanoma cells into melanocytic and mesenchymal phenotypes based on high resolution imaging of single cells. To predict the phenotypes of single melanoma cells based on morphology alone, we developed the AI-based \'Melanoma Phenotype Classifier\' trained with 19 patient-derived cell lines with known melanocytic or mesenchymal transcriptional profiles. To link phenotypic state with high-dimensional morphological profiles, cells were subjected to genetic and chemical perturbations known to shift phenotypic states. The AI classifier successfully predicted phenotypic shifts which were confirmed by single-cell RNA-Seq (scRNA-Seq). These results demonstrate that correlations between melanoma cell phenotypes and morphological changes are detectable by AI. Additionally, the Melanoma Phenotype Classifier was applied to dissociated tumor biopsy samples and characterization of phenotypic heterogeneity was supported by scRNA-Seq transcriptional profiles. This work establishes a link between cell morphology and melanoma phenotypes, laying the groundwork for the use of a label-free morphology-based method for phenotyping live melanoma cells combined with additional analyses.