Tumor necrosis facilitates perihilar cholangiocarcinoma metastasis by ANGPTL6-augmented vessel permeability and tumor dissemination

Figures

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  Fig. 1. Clinic-pathological correlation and prognostic significance of tumor necrosis in pCCA. (A) Representative H&E staining slides of pCCA tumors, where the necrotic region is circled in yellow. (B) Correlation of tumor necrosis with pCCA progression, including tumor T stage, tumor emboli, LNM, and DM (n = 306). (C) Kaplan–Meier overall survival analysis in patients with pCCA (n = 306) dichotomized by the presence or absence of pathological tumor necrosis. (D) Correlation of tumor necrosis with tumor Ki67 proliferation index (n = 219) and CTC dissemination (n = 98) in patients with pCCA. (E) Forest plot illustrating the overall survival hazard ratio of clinic-pathological factors by multivariate Cox regression analysis. (F) Net decision curve analysis demonstrating the comparison of clinical benefits for predicting overall survival in patients with pCCA using different clinic-pathological models. CTC, circulating tumor cell; DM, distant metastasis; LNM, lymph node metastasis; pCCA, perihilar cholangiocarcinoma. Statistical tests: (B) Chi-square test; (C) Log-rank test; (D) Two-sided t-test; (E) Cox proportional-hazards analysis.

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  Fig. 2. Pathological tumor necrosis is associated with dilated vessels and cancer emboli in pCCA. (A) Representative H&E staining slides of pCCA-derived PDX models with different degrees of tumor necrosis, where the necrotic region is circled in yellow. (B) Correlation of tumor necrosis with CTCs in pCCA PDX models (n = 27). Representative immunofluorescence images of single CTCs and CTC clusters are shown on the left. (C) IHC staining of CD34+ vascular vessels (left) or LYVE-1+ lymphatic vessels (right) in pCCA tumor sections. (D) IHC staining of CD34+ vascular vessels (left) or LYVE-1+ lymphatic vessels (right) in pCCA-derived PDX tumor sections. (E) Immunofluorescence co-staining of CD34 and CK19 for detecting the vascular cancer emboli in pCCA tumor sections. (F) Immunofluorescence co-staining of LYVE-1 and CK19 for detecting the lymphatic cancer emboli in pCCA tumor sections. CTC, circulating tumor cell; pCCA, perihilar cholangiocarcinoma; PDX, patient-derived xenograft; IHC, immunohistochemistry. Statistical tests: (B, C, D) Two-sided t-test; (E, F) Chi-square test.

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  Fig. 3. High-resolution spatial proteomics identifies molecular heterogeneity in pCCA cells within the peri-necrotic microenvironment. (A) Representative pathological images of the pCCA tumor H&E-stained slides before and after LCM. (B) PCA plots of all profiled proteome pCCA samples labeled by tissue origins. (C) Volcano plots illustrating the differentially expressed proteins in pCCA cells extracted from peri-necrotic regions compared with other pCCA and normal samples. (D) Venn diagram showing the specifically upregulated proteins in pCCA cells extracted from peri-necrotic regions. (E) Visualization of top 30 upregulated gene ontology biological processes (left) and KEGG pathways (right) enriched in pCCA samples extracted from peri-necrotic regions. Each dot represents an individual biological process, whose size and color indicate the enriched number of genes and adjusted p value, and the connecting line denotes the number of overlapped genes between two processes. ANGPTL6, angiopoietin-like 6; BD: bile duct; CCA, cholangiocarcinoma; KEGG, Kyoto Encyclopedia of Genes and Genomes; LCM, laser capture microdissection; NS: non-specific; PCA, principal component analysis; pCCA, perihilar…

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  Fig. 4. Tumor-derived ANGPTL6 is enriched within peri-necrotic regions and associated with tumor progression in pCCA. (A) IHC staining of ANGPTL6 in pCCA tumor sections (n = 96; top) or pCCA-derived PDX tumor sections (n = 27; bottom). (B) IHC staining of ANGPTL6 encountered with H&E staining in pCCA tumor sections to evaluate the spatial expression pattern of ANGPTL6, where the ANGPTL6-expressing pCCA cells are labeled in red and the necrotic region is circled in yellow. (C) Correlation of ANGPTL6 expression with CTCs (n = 52), lymph node metastasis (n = 96), and distant metastasis (n = 96) in patients with pCCA. (D) Kaplan–Meier overall survival analysis in patients with pCCA (n = 96) dichotomized by ANGPTL6 expression. (E) Correlation of IHC-stained ANGPTL6 expression with the abundance of total and dilated vascular vessels (left; n = 96) or lymphatic vessels in pCCA (right; n = 96). (F) Correlation of IHC-stained ANGPTL6 expression with vascular tumor emboli (left; n = 53) or lymphatic tumor emboli in pCCA (right; n = 53). ANGPTL6, angiopoietin-like 6; CTC, circulating tumor cell; DM, distant metastasis; IHC, immunohistochemistry; LNM, lymph node metastasis; pCCA, perihilar…

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  Fig. 5. ANGPTL6 is the critical target in the positive feedback loop of necrosis-associated vessel permeability, tumor vessel intravasation, and cancer metastasis. (A) Distance analysis evaluating the abundance of total and dilated CD34+ vascular vessels (left) or LYVE-1+ lymphatic vessels (right) in the periphery (within 200 μm) of pCCA cells with different expression levels of ANGPTL6. (B) Human pCCA cell line EGI-1 was subcutaneously implanted in Balb/C nude mice (n = 12) to evaluate the impact of knocking down ANGPTL6 on pCCA tumor necrosis. (C) Evans Blue in vitro leakage assays to evaluate the time-dependent influence of recombinant human ANGPTL6 (rhANGPTL6) on the permeability of monolayer HUVECs (left) and HLECs (right). (D) Immunofluorescence staining of VE-cad to evaluate the influence of rhANGPTL6 on the integrity of intercellular junctions in monolayer HUVECs (left) and HLECs (right). (E) Tumor transendothelial migration assays to evaluate the migration of pCCA cells through rhANGPTL6-treated monolayer HUVECs (left) and HLECs (right). (F) Western blots investigating the influence of rhANGPTL6 on the VE-cad integrity and PAK1/ERK signaling in HUVECs/HLECs. ANGPTL6,…

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  Fig. 6. ScRNA-seq profiling of molecular heterogeneity of pCCA cells in the tumor necrosis microenvironment. (A) The H&E-stained slides of five pCCA tumors profiled using scRNA-seq, with the necrotic region circled in yellow and clinic-pathological characteristics listed on the right. (B) UMAP dimension-reduction plots of profiled cells labeled by sample, necrosis, unsupervised clustering, and annotated cell types. (C) Heatmap illustrating the expression pattern of a panel of authoritative markers for each cell type. (D) UMAP dimension-reduction plots of malignant pCCA cells labeled by sample and necrosis. (E) Heatmap illustrating pairwise correlations of 50 intratumoral gene programs derived from malignant cells from five profiled pCCA tumors. Clustering identified two coherent gene programs enriched in pCCA cells from the necrosis microenvironment. (F) Enrichment analysis of the top 10 upregulated KEGG pathways (left) and transcriptional factor inferring analysis of the potential activated transcriptional factors (right) in pCCA cells using necrosis-enriched gene programs. KEGG, Kyoto Encyclopedia of Genes and Genomes; pCCA, perihilar cholangiocarcinoma; scRNA-seq, single-cell RNA…

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  Fig. 7. Single-cell RNA sequencing profiling of the heterogeneous tumor microenvironment in pCCA with pathological tumor necrosis. (A) Boxplots showing the proportions of each cell type in the tumor microenvironment from pCCA tumors. (B) Enrichment analysis of the top 10 downregulated KEGG pathways in B cells (left) or T cells (right) from the necrosis microenvironment. (C) Bar chart illustrating the cellular proportion of total T cells, CD4+ T cells, macrophages, and CAFs in pCCA tumors. (D) Volcano plot showing the differentially expressed genes in CD8+ T cells from the necrosis microenvironment, where genes associated with functional exhaustion are highlighted. (E) UMAP dimension-reduction plots of macrophages labeled by sample, necrosis, clustering, and the expression of CCL20. apCAF, antigen-presenting CAF; CAF, cancer-associated fibroblast; KEGG, Kyoto Encyclopedia of Genes and Genomes; pCCA, perihilar cholangiocarcinoma; TME, tumor microenvironment; vCAF, vascular CAF; mCAF: matrix-remodeling CAF. Statistical tests: (A) Two-sided t-test.

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  Fig. 8. CCL20 is associated with the immunosuppressive microenvironment in pCCA with pathological tumor necrosis. (A, B) Intercellular ligand–receptor analysis inferred by CellPhoneDB in pCCA tumors with or without pathological tumor necrosis. (A) The network visualization of the number of significant cellular interactions, where the size of the directional connecting line denotes the number of significant interactions between the two types of immune–stroma cells (above: no necrosis; bottom: with necrosis). (B) The significantly enriched immunosuppressive ligand–receptor interactions (left: no necrosis; right: with necrosis). (C) IHC staining of protein markers denoting the functional status of CD8+ T cells (GZMB, PD-1, CTLA4, TIGIT, and TIM3), Tregs (FOXP3), and macrophages (CD206) in pCCA tumor sections (n = 54). (D) IHC staining of CCL20 in pCCA tumor sections (n = 54). (E) Multiplex immunofluorescence staining in pCCA tumor sections to evaluate the abundance of CCL20, FOXP3+ Tregs, and CD206+ M2-type macrophages. (F) Schematic diagram demonstrating that tumor-derived ANGPTL6 is a pivotal fulcrum within the positive feedback loop in necrosis-promoted pCCA progression: upregulated…