Fig 2: Vcam1 depletion abolished the metastatic growth of iCCA cells in vivo but promote the dissemination of individual tumor cells.A Top four rows: gross GFP images of the livers and lungs from mice orthotopically transplanted with Vcam1Ctrl or Vcam1KO1 cells; bottom row: microscopic GFP images of the lungs from the Vcam1Ctrl or Vcam1KO1 groups showing the higher number of DTCs in the latter. B Quantification of the numbers of DTCs in the lungs in (A). C Top two rows: gross GFP images of the lungs from TVI mice injected with Vcam1Ctrl or Vcam1KO1 cells; bottom row: representative H&E images of the lungs from the Vcam1Ctrl and Vcam1KO1 groups. D Quantification of the numbers of the lung metastases in (C). E The indicated IHC on serial sections of the Vcam1Ctrl and Vcam1KO1 lung metastases. Note the complete loss of E-cadherin in the Vcam1KO tumor (d). Statistics in B & D: Student t-test. P value, ****< 0.0001; **< 0.01.

Fig 3: iCCA-pMF interaction induces Vcam1 expression in the tumor cells.A Cytokine array assays using the CM from the indicated cultures on Day 4 (a–e). (f): Quantitative RT-PCR of Vcam1 mRNA levels in the indicated cultures. Student t-test, P value * <0.05. B Vcam1 detection in the indicated 2.5D cocultures on Day 4 via flow cytometry. The percentage of Vcam1+ tumor cells in each condition was indicated. T:pMF coculture had the most Vcam1+ tumor cells. C Histogram of Vcam1 level in the tumor cells from the indicated cocultures on Day 4 and 42. There was a decrease in Vcam1 levels in Day 42 cultures. D Vcam1 and aSMA IHC on the serial sections of the indicated allograft tumors. All scale bars are 100 µm. Arrows in (a): Vcam1high cells on the tumor border. Dotted lines in (f, g): tumor-pMF interface; arrows in (h, i): red arrows, Vcam1+ tumor cells next to iMFs; black arrows: Vcam1- cells next to iMFs. E VCAM1 and aSMA IHC on serial sections of an iCCA patient tumor. Arrows: VCAM1+ tumor cells at the tumor border next to aSMA+ pMFs. F ZsG/Dapi fluorescence and Vcam1 IHC on the serial sections of the live (a, b) and lung (c, d) from iCCA allografts showing decreased Vcam1 levels in the DTCs. Asterisks in (c, d): Vcam1high tumor cells within the lung metastases; arrows in (c, d): Vcam1low DTCs.

Fig 4: Vcam1 is involved in epithelial-to-mesenchymal transition in mouse and human iCCA tumors.A The mouse and human iCCA RNAseq datasets used in the analysis in (B). B Bubble plot representing gene set enrichment analysis (GESA) of predicted Vcam1 regulatory pathways in both mouse PPTR samples and human TCGA patient samples. Color intensity of the bubbles indicates the statistical significance of enrichment, and bubble size indicates the odds ratio. The bar plot on the left of the bubble plot shows the combined P values of the mouse and human datasets with the Stouffer method. Only the pathways with a combined P value <0.05 are shown. C Quantitative RT-PCR of the Vcam1Ctrl PPTR cells and two Vcam1KO single-cell clones. Student t test. **P < 0.01. D Flow cytometry of the Vcam1Ctrl and Vcam1KO cells. E GSEA analysis of the RNA-seq profiles of Vcam1KO (Vcam1KO1 and Vcam1KO2 combined) vs. Vcam1Ctrl cells. Pathways in red are upregulated in Vcam1KO cells and pathways in black are downregulated. F The GSEA plot of HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION pathway in Vcam1KO vs. Vcam1Ctrl cells. G The volcano plot of the gene expression fold change in Vcam1KO vs. Vcam1Ctrl cells.

Fig 5: Partial Vcam1 neutralization increases the number of small lung metastases in a TVI model.A Transwell migration assay and quantification of the PPTR cells treated with IgG or Vcam1Ab showing increased cell migration with Vcam1 blocking. Student t-test, P value <0.0001. Cells derived from two PPTR tumors were tested. B Flow cytometry of CellTracker proliferation assay of the PPTR tumor cells treated with IgG or Vcam1Ab found no changes in cell proliferation with Vcam1 blocking. C Gross ZsG images of the lungs collected from the indicated TVI models three weeks post injection. Vcam1Ab treatment led to more but smaller lung metastases. D Representative images of the whole-section GFP scans of the lungs from mice treated with IgG or Vcam1Ab. E Quantification of the area of individual lung metastases from the TVI model shows smaller lung metastases in the Vcam1Ab-treated mice. Student t-test, P value, ****<0.0001. F Histogram of the size distribution of the lung metastases from the TVI model shows the larger numbers of small lung metastases in the Vcam1Ab-treated mice. G The indicated staining of the lung metastases from the IgG or Vcam1Ab groups. Arrows: Vcam1+ tumor cells; asterisk: Vcam1- DTCs. All images share the same 100 µm scale bar.