Abstract A010: A cell culture model of quiescent melanoma cell state that governs cancer evolution, therapy resistance, and recurrence/metastasis

Abstract A crucial adaptability trait of melanoma cells that persist under all selection pressures, including therapies, is their ability to efficiently switch from proliferation to quiescence. With the goal of improving therapeutic strategies for preventing melanoma recurrence, herein we describe an approach to modeling this trait in cell culture. Body imposes a variety of selection pressures and bottlenecks ranging from different components of immunity to metabolic challenges to enforce quiescence and influence cancer evolution. The rationale for the choice of model as cell culture is that it is ideal for modeling the realistic depth of quiescence and for evaluating therapeutic strategies that may inhibit highly abnormal but adaptable cancer cells. As it is not possible to replicate all such bottlenecks in cell culture, our approach is to apply as severe a bottleneck as possible, thus eliminating all proliferative (non-adaptable) cells, leaving behind rare cells surviving in deep quiescence. We hypothesize that if the quiescent cell state being modeled can survive a severe bottleneck, it can also survive other bottlenecks of similar or lower severity. A lack of glutamine proved to be a strong and prolonged selection pressure for the highly metastatic human melanoma cell line A375SM, killing more than 99% of cells and selecting rare cells based on their ability to survive in deep quiescence. After 4 weeks, cells gradually exited quiescence and proliferated indefinitely. Furthermore, we modeled even deeper quiescence lasting longer than 4 weeks by increasing the severity of the selection pressure using dialyzed serum in medium. The cells selected in this manner were much more resistant to paclitaxel than was their parental cell line. We obtained similar results with the highly metastatic mouse melanoma cell line B16-BL6. We conclude that our approach is suitable for modeling abnormal deep quiescence in melanoma that drives the disease, including recurrence and metastasis. The main strength of the approach is that, besides modeling genetic alterations, it incorporates suitable selection pressures and the time element that are important in cancer evolution. Although there is a limitation of lack of body-like microenvironments in cell culture, we believe that abnormal quiescent cancer cell state being modeled here is capable of overcoming restraints imposed by the body. We will also discuss our published and new observations with rare cells (0. 01% in population) surviving in quiescence for several weeks in aggressive triple-negative breast cancer cell lines and their functional characteristics. Our results obtained with adaptable cells from SUM149 TNBC cell line, modeled as above for melanoma cells, suggest that they are stem-like quiescent cells. We will also discuss how we choose and evaluate therapies that would likely inhibit quiescent and slow-growing cancer cells prior to recurrence. This approach may significantly improve how we investigate deep intrinsic resistance and strategies to overcome it. Citation Format: Balraj Singh, Vanessa N. Sarli, Nikil Erry, Anthony Lucci. A cell culture model of quiescent melanoma cell state that governs cancer evolution, therapy resistance, and recurrence/metastasis abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Cancer Evolution: The Dynamics of Progression and Persistence; 2025 Dec 4-6; Albuquerque, NM. Philadelphia (PA): AACR; Cancer Res 2025;85 (23Suppl): Abstract nr A010.