Uncovering Uveal Melanoma Heterogeneity: The Role of SPP1^high Melanoma Cells in Early Metastasis and their Interactions with CCL3^high Macrophages

Abstract

Purpose : Emerging evidence suggests that a minute subset of cells, conferring a poor prognosis within uveal melanoma (UM), may play a pivotal role in tumor metastasis. This study aims to unravel the intra-tumor heterogeneity of UM through single cell sequencing, shedding light on the mechanisms underlying early UM metastasis.

Methods : We conducted an in-depth bioinformatics analysis of 11 samples obtained through primary enucleation at Beijing Tongren Hospital. Our analysis focused on characterizing the cellular components of tumor cell subtypes and their interactions with immune cells at the single-cell level, comparing the early-metastasis group (M+; metastasis < 2 years from diagnosis) and late-metastasis group (M-; metastasis > 2 years from diagnosis). Gene expression was knocked down using specific shRNAs/siRNAs and functional assays, including CCK8 and transwell assays, were employed to assess cell proliferation and invasive potentials. Cell cycle and apoptosis were analyzed by flow cytometry after PI or PI/Annexin V-APC staining. Standard immunoblotting and quantitative RT-PCR were used to assess the mRNA and protein abundance.

Results : Our analysis revealed that M+ group samples exhibited a higher proportion of melanoma cells and a lower proportion of immune cells compared to M- group. A subcluster of melanoma cells expressing high levels of Secreted phosphoprotein 1 (SPP1) was associated with early metastasis. Immunofluorescence confirmed the presence of SPP1^high melanoma cells in UM tissues. Functional experiments demonstrated that SPP1 promoted the proliferation, migration, and invasion of UM cells. Additionally, a C-C Motif Chemokine Ligand 3 (CCL3) ^high macrophage sub-cluster was significantly enriched in both M+ and M- groups and correlated with poor prognosis. Further intercellular interaction analysis revealed that SPP1^high melanoma cells were the primary cluster interacting with CCL3^high macrophages in the M+ group.

Conclusions : Our study provides insights into key distinctions between early and late metastatic UM at a single-cell resolution. We identified SPP1^high melanoma cells as a predictive marker of early metastasis and elucidated their potential interplay with CCL3^high macrophages.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.

 

Graphical Abstract.