Abstract 5128: Role of adherent-to-suspension transition in melanoma metastatic dissemination and circulating tumor cell dynamics

Abstract Background: Melanoma is the most fatal form of skin cancer, presenting significant therapeutic challenges due to its high metastatic potential. Once melanoma spreads to distant sites, prognosis declines sharply, underscoring the need to uncover the mechanisms driving metastasis. This study investigates the role of transcriptional regulators in melanoma metastasis, focusing on how primary tumor cells undergo “Adherent-to-Suspension Transition” (AST) to reprogram their anchorage dependency and disseminate into the bloodstream. Methods: Using fluorescent protein-tagged melanoma cells, we developed an orthotopic melanoma metastasis mouse model and performed single-cell RNA sequencing (scRNA-seq) on melanoma cells isolated from the primary footpad tumor, blood, and lung metastases. Publicly available spatial transcriptomics data were analyzed to identify the cells undergoing AST within the primary tumor. Doxycycline-inducible AST-overexpressing melanoma cell lines were generated for functional validation. Results: We identified three AST factors—IKZF1, IRF8, and NFE2—known hematopoietic transcriptional regulators that reprogram adherent cells into suspension cells. Induction of these AST factors upregulated hemoglobin genes while downregulating extracellular matrix (ECM)-integrin genes, leading to cell-matrix dissociation and anoikis resistance. During metastasis, AST factor expression was induced in circulating tumor cells (CTCs) but reversed in metastatic niches. Notably, AST factor expression was independent of epithelial-mesenchymal-like transition (EMT-like transition). Phylogenetic analysis of scRNA-seq data revealed that AST-positive clones exhibited greater aggressiveness and metastatic potential. To evaluate AST factors as therapeutic targets, we performed an intervention study using lenalidomide, an IKZF1 inhibitor. Treatment significantly reduced the number of CTCs and metastatic burden without affecting the primary tumor. Spatial transcriptomics analysis demonstrated an inverse correlation between AST factor expression and proximity to blood vessels, further implicating AST in early metastatic processes. To explore AST-mediated regulation of anchorage dependence, we subjected B16F10-3ASTTetR cells to repeated cycles of doxycycline treatment. Doxycycline induction caused cell detachment, which was reversed upon withdrawal, highlighting the dynamic nature of AST. RNA sequencing revealed that AST-induced cells were depleted of the Hallmark EMT pathway, suggesting that AST reprograms anchorage dependency through distinct mechanisms. Conclusion: AST represents a novel, EMT-independent mechanism driving melanoma metastasis by reprogramming anchorage dependency. These findings position AST factors as actionable therapeutic targets for preventing metastatic progression in melanoma. Citation Format: Dong Ki Lee, Jongwook Oh, Soyeon Lee, Hyun Woo Park, Heon Yung Gee. Role of adherent-to-suspension transition in melanoma metastatic dissemination and circulating tumor cell dynamics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5128.