Abstract 5926: High dimensional morphology analysis reveals new insights in melanoma cell heterogeneity
Authors: Lattmann Evelyn , Tastanova Aizhan , Jovic Andreja , Saini Kiran , Pham Tiffine , Corona Christian , Mei Jeanette , Phelan Michael , Boutet Stephane C. , Carelli Ryan , Jacobs Kevin B. , Kim Julie , Ray Manisha , Johnson Chassidy , Li Nianzhen , Salek Mahyar , Masaeli Maddison , Levesque Mitchell P. 
Published: April 2023

Abstract
Melanomas are the deadliest skin cancers, in part due to cellular plasticity and heterogeneity within the tumors. These characteristics have made a deeper understanding of melanomas challenging. Classically, melanoma cells are characterized with a limited set of protein biomarkers. Gene expression signatures and mutational analysis (e.g., BRAF and NRAS genotyping) can provide a more detailed view of heterogeneity but may not translate to readily available biomarkers for functional studies. The Deepcell platform enables multi-dimensional morphology analysis and enrichment of unlabeled single cells using artificial intelligence (AI), advanced imaging, and microfluidics, enabling high resolution profiling of population heterogeneity. Label-free multi-dimensional morphology analysis may have higher resolution than a limited set of protein biomarkers, minimizes perturbation to the transcriptome, and reduces cell handling steps. We used patient-derived cell lines and dissociated biopsy samples to train a Deepcell AI model to identify and sort for melanoma cells based on morphology alone. The model was tested on metastatic melanoma biopsies, with identification and enrichment of melanoma cells verified by various downstream assays, including scRNASeq. In addition to melanoma populations, the AI model classified various cells of the microenvironment, such as stromal cells and immune subtypes, based on morphology only. To further characterize tumor heterogeneity, we imaged &gt;25 patient-derived melanoma cell lines representing melanocytic, mesenchymal, and intermediate phenotypic states on the Deepcell platform. Morphology analysis of these images revealed distinct clusters of cells for each phenotype, indicating that there are morphological differences associated with each state. We developed a random forest (RF) classifier to identify the top differential morphological features between the different cell lines, thereby providing a label-free means of phenotyping melanoma samples. The morphology analysis of the cell lines uncovered significant variability in pigmentation; an RF classifier distinguished between pigmented vs non-pigmented cells with &gt;90% accuracy. Pigmentation is a hallmark of melanoma cells, and it has been associated with the melanocytic phenotype and differential drug response in vitro. However, there is not currently a robust method to profile and study pigmentation in live cells. We further investigated this observation by correlating morphological profiles, molecular, and functional information with the level of cell pigmentation. The Deepcell platform presents a new method for sorting and characterizing cellular heterogeneity using morphology, including pigmentation status. As such, multi-dimensional morphology analysis will bolster the understanding of complex melanoma states and tumor microenvironment, particularly in patient derived biopsies.
Citation Format: Evelyn Lattmann, Aizhan Tastanova, Andreja Jovic, Kiran Saini, Tiffine Pham, Christian Corona, Jeanette Mei, Michael Phelan, Stephane C. Boutet, Ryan Carelli, Kevin B. Jacobs, Julie Kim, Manisha Ray, Chassidy Johnson, Nianzhen Li, Mahyar Salek, Maddison Masaeli, Mitchell P. Levesque. High dimensional morphology analysis reveals new insights in melanoma cell heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5926.