The Dynamic Landscape of the Coagulome of Metastatic Malignant Melanoma

Abstract

The local expression of coagulation-related genes defines the tumor coagulome. The tumor coagulome plays a pivotal role in cancer-associated thrombosis (CAT) and hemostatic complications, such as venous thromboembolism (VTE), which are frequent in patients with advanced/metastatic cancer. Genomic analyses of human tumors, such as skin cutaneous melanoma (SKCM), have unveiled the complexity of the metastatic trajectories. However, no study to date has focused on the metastatic coagulome along these trajectories. Using bulk-tumor and single-cell analyses of primary SKCM, metastastic samples and circulating tumor cells (CTCs), we explored the coagulome of SKCM along metastatic progression. We identified consistent changes in the coagulome of SKCM metastases compared to primary tumors and observed metastatic site specificity. Compared to other metastatic sites, lung metastases of SKCM had a specific coagulome with a higher expression of F3, encoding Tissue Factor. Single-cell analyses were used to chart the inter- and intra-tumor heterogeneity and characterize the metastatic coagulome of SKCM. We found that a subpopulation of CTCs from SKCM expressed high levels of platelet genes, suggesting the contribution of CTC-platelet interactions to the CTC coagulome. These findings highlight the dynamic properties of the metastatic coagulome and its link to cancer progression.

Keywords: cancer-associated thrombosis; metastasis; skin cutaneous malignant melanoma; tumor coagulome.

Figure 1

Comparison of the coagulome of primary vs. metastastic SKCM. (A) Hierarchical clustering based on the normalized expression levels of 138 CRGs. Primary SKCM tumors (SKCM_P) and metastases (SKCM_M), highlighted in yellow, and 19 other types of primary tumors from TCGA were analyzed. (B) The top 20 CRGs with higher variance in primary vs. metastatic SKCM (Fisher F test). (C) GO term analysis (GO Biological Process complete) of CRGs with at least a 2-fold higher expression in SKCM_M vs. SKCM_P (n = 16, p < 0.05, Student t test, FDR). (D) Violin plots showing the expression of GP1BA, the most differentially expressed gene, between SKCM_P and SKCM_M. (E) Plots showing the relationship between the expression of CRGs (RSEM) and their correlation with the copy number (CNA) or methylation levels (Pearson R). Points shown in red have an absolute Pearson correlation coefficient greater than 0.5. Points shown in black have an absolute Pearson r less than 0.5.

Figure 2

Lung metastases of SKCM express higher F3 levels. (A) F3 expression analysis in different metastatic locations of SKCM from TCGA, p < 0.05 with Kruskal–Wallis test. Lung metastases are shown in turquoise, other metastatic sites in grey. (B) Distribution of F3 expression in SKCM lung metastases (n = 8, pink) compared to other locations (n = 63, blue). (C) GSEA showing a significant enrichment in the hallmark “Coagulation” in lung metastases of SKCM compared to other metastatic sites. (D) F3 expression analysis in metastastic samples from four types of tumors from the BCSGC cohort (n = 438). Note that F3 levels are presented for metastastic locations with >2 samples. (E) F3 expression in multiple matched samples from 4 patients with SKCM (PEACE study).

Figure 3

Single-cell RNA seq analysis of the coagulome of metastatic SKCM. (A) UMAP analysis of n = 4645 cells from 19 SKCM metastases (GSE72056), based on the expression levels of n = 138 CRGs. Note that cluster #1 includes all immune cells (NK cells, B, T cells and macrophages), while cancer cells, CAFs and endothelial cells from different patients appear in clusters #2 and #3. (B) Cell type-specific expression of the “core coagulome” genes in SKCM metastases. (C) A Venn diagram showing the overlap of F3, PLAT, and SERPINE1 mRNA expression in cancer cells from GSE72056. The % of overlap is indicated in each case. The GO terms (MSigDB Hallmark) were identified using the 40 most correlated genes with each of the three genes of interest (Pearson r).

Figure 4

The coagulome of circulating tumor cells (CTCs) from SKCM. (A) GO term enrichment analysis of CTCs (GSE255299) compared to cancer cells from GSE72056, using the most significantly differentially expressed genes, between the two groups. (B) Violin plots showing the most differentially expressed platelet-related genes between CTCs and cancer cells. The overall “Platelet score” is obtained from the combination of the corresponding 11 platelet genes.

Figure 5

A subset of CTCs with a platelet coagulome. (A) UMAP analysis of CTCs (GSE255299) according to their expression of CRGs. The colors indicate the platelet score (orange = low, grey = high). (B) Individual patient analysis (n = 7 patients) of CTCs from GSE255299. Note that CTCs with a positive platelet score are in pink and platelet-negative CTCs are in blue. (C) GO term enrichment analysis of genes most correlated with the platelet score (Spearman) in CTCs. (D) Expression analysis of four stemness markers according to the platelet score in CTCs of SKCM.