Figure 1 Circulating canine osteosarcoma clusters extravasate by angiopellosis. (A) Illustration of the project workflow. Canine osteosarcoma cell lines were fluorescently labeled and infused into the circulation of Tg(fli1a:egpf) with fluorescent blood vessels. Tumor cells which extravasated as clusters were isolated and expanded, and these sub-lines were then molecularly profiled. (B) Representative image of infused D17 tumor cells aggregating as clusters in zebrafish circulation. Tumor cell clusters begin to extravasate through the endothelial cells of the blood vessels during metastasis. The tumor cluster is eventually completely removed from the inside of the vessels and is lodged in an extravascular cavity while maintaining multicellularity and not disassociating into single cells. SB = 20µM.

Extravasated circulating tumor cells display differential expression of genes and pathways. (A) Overlaps in the upregulated and downregulated genes in extravasated circulating tumor cells. (B) Heat map of pathways commonly downregulated in extravasated circulating tumor cells. (C) Enrichment plots for KRAS signaling, IFN gamma response, and extracellular matrix (ECM) organization in HMPOS and D17 extravasated cells.

Extravasated circulating tumor cells downregulate extracellular matrix remodeling. (A) Gene regulatory networks for the extracellular matrix remodeling pathway in HMPOS and (B). D17. Blue denotes downregulation and red denotes upregulation (FDR < 0.05). Nodes are scaled proportionally to log2FC, with larger nodes depicting greater log2FC between extravasated and parental cell lines. (C) Scatter plot of sum rank of network connectivity parameters by log2FC for HMPOS and (D) D17. (E) Bar graphs of top connected genes in the pathway, colored by log2FC for HMPOS and (F) D17.

Versican-mediated extracellular matrix organization is enriched in extravasated circulating tumor cells. (A) Versican is commonly upregulated in both cell lines and interacts with a core subset of the extracellular matrix organization pathway. Yellow nodes indicate proteins that share at least one interaction with versican. (B) Ranking of top nodes based on neighborhood connectivity, (C) betweenness centrality, and (D) number of edges in the extracellular matrix remodeling pathway. Prognostic value of VCAN for (E) metastasis-free survival and (F) overall survival in osteosarcoma patients.

GRAPHICAL ABSTRACT A graphical abstract of the proposed findings this study identified.