Detection of neoplastic-immune hybrid cells with metastatic properties in uveal melanoma

Abstract

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 curative 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 (n = 8) 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 (n = 4) 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.

Keywords: Disseminated tumor cells; Metastasis; Multiplexed cyclic immunofluorescence; Single-cell RNA sequencing; Uveal melanoma.

Fig. 1

Neoplastic-immune hybrids detected in primary UMs identified using cyCIF. Hybrid cells identified by their co-expression of pan-leukocyte immune protein, CD45 (orange), and one or more melanocytic proteins [HTR2B (green), MITF (light pink), MLANA (magenta), Tyrosinase (red), or GP100 (yellow)]. Class 2 hybrid shows expression of HTR2B and decreased expression of tyrosinase compared to class 1 hybrid cells. Hybrid cells shown in image insets were identified in the primary tumor. H&E whole globe images for both patient samples with marked area of analyses

Fig. 2

Neoplastic-immune hybrids identified in primary UMs by scRNA-seq. A) tSNE of all cells sequenced from primary tumor biopsies colored according to major cell type (tumor, macrophage/monocyte, immune, stromal/other) [13] and B) hybrid cells identified across all patient samples overlaid on tSNE in red. C-G) Hybrid analyses of scRNA-seq dataset for patient UMM059. C) Leiden-based clustering shown as a UMAP and annotation by major cell type in D) where identified hybrid cells (cluster 8) are in red. E) Individual UMAPs for gene expression of melanocytic and macrophage/monocyte genes. F, G) Tumor score and macrophage/monocyte score violin plots for each cluster and colored according to major cell type, where hybrid cells (cluster 8, red) have significantly higher tumor scores (* = p-value ≤ 2 × 10^-8. Hybrid cells (cluster 8, red) have significantly higher macrophage/monocyte scores than all other tumor cell clusters (* = p-value ≤ 2 × 10^-16). All other patient data provided in Supplemental Figs. 2, 3, 4 and 5

Fig. 3

Differential gene expression and pathway analysis for all hybrid cells compared to tumor cells and macrophages. A, B) Selected significantly upregulated and downregulated genes (adjusted p-value ≤ 0.05 and log twofold change $\ge$ 0.5 or ≤ -0.5) shared across hybrid cells from all patient samples compared to tumor cells (A) and compared to macrophages (B), values shown for patient UMM059. A complete list of all differentially expressed genes for each patient sample provided in supplemental file 2. C) Reactome pathway enrichment analysis based on all differentially expressed genes shared across patient samples and organized by biological category, values shown for patient UMM059 and all other patient sample pathway enrichment analysis in Supplemental File 3

Fig. 4

Hybrid cells that successfully disseminate display upregulated expression of TMSB10, CD74 and GPX1. Representative images of TMSB10, CD74, and GPX1 staining in A) primary UM tumor FFPE sections and in B) hybrid cells identified in circulation. C) The number of positive hybrid cells and tumor cells identified within primary tumor sections and peripheral blood for each biomarker, TMSB10, CD74 and GPX1, expressed as a percentage of total hybrid cells or tumor cells identified, where each dot represents 10 s-10000 s of cells from each individual patient sample (n = 4, supplemental Fig. 7) (Welch’s t-test, TMSB10 CHCs to CTCs p value = 0.03, CD74 tumor hybrids to CTCs p value = 0.03, GPX1 tumor hybrids to CHCs p value = 0.0007 and GPX1 CHCs to CTCs p value = 0.04, all other comparisons not significant p value > 0.05)

Fig. 5

Predicted ligand-receptor interactions between hybrid cells and cells of the UM tumor microenvironment. A) Heatmap showing the number of significant interactions between hybrid cells and other major cell types within the tumor microenvironment (p-value ≤ 0.05). B) Dot plot representing all significant ligand-receptor interactions between hybrid cells, macrophages/monocytes, tumor cells, and T-cells for patient UMM059. A complete list of significant interactions for all patient samples is included in Supplemental file 4