Single-cell and spatial transcriptomics reveals the key role of MCAM+ tip-like endothelial cells in osteosarcoma metastasis

Abstract

Osteosarcoma, the most common primary malignant bone tumor in children and adolescents, is highly aggressive and prone to metastasis. Endothelial cells (ECs) are involved in angiogenesis and play a key role in promoting the metastasis of tumor. However, research on tip-like ECs within osteosarcoma was extremely rare. In this study, a single-cell atlas of ECs was constructed using single-cell transcriptomic data. It was found that tip-like ECs were abundant in the primary tumors and metastatic foci. Gene sets score analysis indicated their enrichment in pathways associated with angiogenesis and metastasis. What's more, MCAM was highly expressed in tip-like ECs and was likely to promote the metastasis of osteosarcoma. MCAM was also found to be highly expressed in the ECs of metastatic lymph nodes when compared to normal lymph node samples. Meanwhile, spatial transcriptomics data confirmed the presence of MCAM-positive ECs in metastatic lymph node, closely localized to osteoblasts. In vitro assays, including qRT-PCR, tube formation, and immunofluorescence, validated the role of the MCAM gene in promoting angiogenesis. In conclusion, tip-like ECs may promote tumor metastasis by enhancing angiogenesis. MCAM was a functional gene for tip-like ECs and could serve as a target for the treatment of osteosarcoma.

Fig. 1. Single-cell atlas of osteosarcoma.

A The overall flowchart of the research (Created in BioRender.com). B The tSNE plot of the multicellular ecosystem of 19 tissue samples. C tSNE plot of 7 cell clusters in the multicellular ecosystem. D The marker genes of each cell cluster. E Composition of cell clusters in different samples.

Fig. 2. Classification of EC subtypes.

A ECs were categorized into seven distinct subtypes. B The marker genes of each EC subtype. C Gene expression heatmap in EC subtypes. D Composition of EC subtypes among different groups. E GSVA analysis showed that the tip-like ECs subtype was significantly enriched in the NOTCH SIGNALING pathway, WNT BETA CATENIN SIGNALING, and APICAL JUNCTION pathway etc. F Lung metastasis scores were the highest in tip-like ECs by AUCell. G Angiogenesis scores were the highest in tip-like ECs by AUCell.(Proli: proliferative).

Fig. 3. Cellular communication and differential gene analysis between osteosarcoma groups.

A Heatmap of cellular interactions between tip-like ECs and other clusters. B Cellular ligand-receptor bubble maps of osteoblasts and tip-like ECs. C Differential gene volcano plots of tip-like ECs in the PT group versus the PC group. D GSEA plot of differentially expressed genes in tip-like ECs in PT versus the PC group. E Differential gene volcano plot of tip-like ECs in Met-Lung group versus the PC group. F GSEA plot of differentially expressed genes in tip-like ECs in Met_Lung versus the PC group (PT primary tumor, PC paracancerous, Met-Lung lung metastasis).

Fig. 4. Landscape of EC subtypes in pre- and post-chemotherapy.

A tSNE plots of ECs in different groups. B Composition of EC subtypes across different groups. C Differences in pathway activities scored per cell by GSVA between pre-chemotherapy and post-chemotherapy tip-like ECs. D Differential gene volcano plot of tip-like ECs between pre- and post-chemotherapy. E GSEA plot of differentially expressed genes in tip-like ECs between pre- and post-chemotherapy. F Scores of tip-like ECs in chemotherapy efficacy groups.

Fig. 5. Pseudotemporal trajectory analysis of EC subtypes.

A–C Pseudotemporal analysis of EC subtypes. D CytoTRACE plot of EC subtypes. E Heatmap depicts the expression of the top 20 genes in EC subtypes across two branches, classified into three gene clusters.

Fig. 6. Validation of the effect of MCAM on osteosarcoma progression.

A Violin plot of MCAM in EC subtypes. B Violin plot of the expression of MCAM in different sample groups. C Prognostic analysis of patients with high and low MCAM expression. D Representative immunofluorescence images of MCAM in osteosarcoma tissue and peritumoral tissue (n = 2). E PCR detection of siRNA-MCAM knockdown efficiency. F Representative images of tube formation assay. G Number of connections of cell-forming tubes. H Total length of cell-forming tubes. (NC negative control, PC paracancerous, PT primary tumor, Scale bar = 200 μm,***p < 0.001, ****p < 0.0001).

Fig. 7. Single-cell and spatial transcriptomic characterization of ECs in metastatic lymph node samples.

A Violin plot of MCAM expression in MLN and NLN groups. B UMAP plot of ST data from MLN. C AddModuleScore analysis of ECs gene set for ST cell clusters. D AddModuleScore analysis of osteoblasts gene set for ST cell clusters. E Spatial location of MCAM expression. F Spatial location of cell clusters in MLN (ST spatial transcriptomics, MLN metastatic lymph node, NLN normal lymph node).