RESUMEN
Background: Uveal melanoma is the most common non-cutaneous melanoma and is an intraocular malignancy affecting nearly 7,000 individuals per year worldwide. Of these, approximately 50% will progress to metastatic disease for which there are currently no effective therapies. Despite advances in molecular profiling and metastatic stratification of uveal melanoma tumors, little is known regarding their underlying biology of metastasis. Our group has identified a disseminated neoplastic cell population characterized by co-expression of immune and melanoma proteins, circulating hybrid cells (hybrids), in patients with uveal melanoma. Compared to circulating tumor cells, which lack expression of immune proteins, hybrids are detected at an increased prevalence in peripheral blood and can be used as a non-invasive biomarker to predict metastatic progression. Methods: To ascertain mechanisms underlying enhanced hybrid cell dissemination we identified hybrid cells within primary uveal melanoma tumors using single cell RNA sequencing and evaluated their gene expression and predicted ligand-receptor interactions in relation to other melanoma and immune cells within the primary tumor. We then verified expression of upregulated hybrid pathways within patient-matched tumor and peripheral blood hybrids using cyclic immunofluorescence and quantified their protein expression relative to other non-hybrid tumor and disseminated tumor cells. Results: Among the top upregulated genes and pathways in hybrid cells were those involved in enhanced cell motility and cytoskeletal rearrangement, immune evasion, and altered cellular metabolism. In patient-matched tumor and peripheral blood, we verified gene expression by examining concordant protein expression for each pathway category: TMSB10 (cell motility), CD74 (immune evasion) and GPX1 (metabolism). Both TMSB10 and GPX1 were expressed on significantly higher numbers of disseminated hybrid cells compared to circulating tumor cells, and CD74 and GPX1 were expressed on more disseminated hybrids than tumor-resident hybrids. Lastly, we identified that hybrid cells express ligand-receptor signaling pathways implicated in promoting metastasis including GAS6-AXL, CXCL12-CXCR4, LGALS9-P4HB and IGF1-IGFR1. Conclusion: These findings highlight the importance of TMSB10, GPX1 and CD74 for successful hybrid cell dissemination and survival in circulation. Our results contribute to the understanding of uveal melanoma tumor progression and interactions between tumor cells and immune cells in the tumor microenvironment that may promote metastasis.
RESUMEN
Uveal melanoma (UM) is the most common non-cutaneous melanoma and is an intraocular malignancy that affects nearly 7,000 individuals per year worldwide. Of these, nearly 50% will progress to metastatic disease for which there are currently no effective therapies. Despite advances in the molecular profiling and metastatic stratification of class 1 and 2 UM tumors, little is known regarding the underlying biology of UM metastasis. Our group has identified a disseminated tumor cell population characterized by co-expression of immune and melanoma proteins, (circulating hybrid cells (CHCs), in patients with UM. Compared to circulating tumor cells, CHCs are detected at an increased prevalence in peripheral blood and can be used as a non-invasive biomarker to predict metastatic progression. To identify mechanisms underlying enhanced hybrid cell dissemination we sought to identify hybrid cells within a primary UM single cell RNA-seq dataset. Using rigorous doublet discrimination approaches, we identified UM hybrids and evaluated their gene expression, predicted ligand-receptor status, and cell-cell communication state in relation to other melanoma and immune cells within the primary tumor. We identified several genes and pathways upregulated in hybrid cells, including those involved in enhancing cell motility and cytoskeleton rearrangement, evading immune detection, and altering cellular metabolism. In addition, we identified that hybrid cells express ligand-receptor signaling pathways implicated in promoting cancer metastasis including IGF1-IGFR1, GAS6-AXL, LGALS9-P4HB, APP-CD74 and CXCL12-CXCR4. These results contribute to our understanding of tumor progression and interactions between tumor cells and immune cells in the UM microenvironment that may promote metastasis.
