Fig. 4

Induction of AST factors in disseminated breast cancer CTCs. A Schematic overview of the isolation and scRNA-seq analysis of primary tumor cells and CTCs from de novo metastatic breast cancer patients. CTCs were isolated using HDM chips. B tSNE plot representing 1,509 cancer cells (EpCAM⁺) sorted from breast cancer tissues and PBMC of three de novo metastatic breast cancer patients analyzed by scRNA-seq. Turquoise dots, 1,488 cells from breast tissues; orange dots, 21 CTCs from blood samples. CTCs are in region marked with orange boundary. C, Scaled and aggregated transcriptional expression of 4AST factor genes. Cell positions are based on tSNE plot in (B). D Violin plots showing normalized expression levels of IKZF1, IRF8, NFE2, and BTG2 in each group of (B). E Bar chart showing gene ontology (GO) analysis of differentially expressed genes (DEGs) between primary tumor cells and CTCs. Length of each bar, FDR-corrected p-value of each term. F Representative histologic images of AST factor expression in lymphovascular invasion (LVI) from primary human breast cancer patients. G Schematic overview of the isolation and scRNA-seq analysis of LM2-derived primary tumor cells and CTCs from orthotopic model of breast cancer metastasis. PuroR⁺-LM2 cells were injected into mammary fat pads of NSG mice and CTCs were subsequently isolated using HDM chips. H Immunofluorescence analysis of EpCAM⁺ and CD45⁻ CTCs. Yellow, EpCAM; red, CD45. I UMAP embedding of analyzed transcriptomes of 3,006 CTCs (orange) and 5,511 primary tumor cells (turquoise). J Scaled and aggregated transcriptional expression of 4AST factor genes. Cell positions are from the UMAP plot in (I). K GSEA of ECM organization- and focal adhesion-related genes in CTCs compared to primary tumor cells. L Heatmap of GO analysis of 105 and 100 genes differentially expressed in CTCs and primary tumor cells. M–N Feature plots of cells showing the expression levels of TEAD2 (M) and HBA1/2 (N). Cell positions are from the UMAP plot in (l). O Schematic showing the procedure used for the isolation and scRNA-seq analysis of B16F10-derived primary tumor cells, CTCs, and metastatic tumor cells from an orthotopic model of melanoma metastasis. GFP-positive cells were injected into the footpads of C57BL/6N mice and tumor cells were subsequently isolated using FACS. P UMAP embedding of analyzed transcriptomes of 1,139 CTCs (orange), 1,129 primary tumor cells (turquoise) and 1,630 metastatic tumor cells (light grey). Q Feature plots of cells showing the expression levels of 4AST factor genes. R Dot plot showing the average expression level of the 4 factors, Btg2, Ikzf1, Irf8, and Nfe2, in primary tumor, CTCs, and metastasized lung nodules, respectively