Vascular heterogeneity of tight junction Claudins guides organotropic metastasis

Abstract

Carcinomas are associated with metastasis to specific organs while sparing others. Breast cancer presents with lung metastasis but rarely kidney metastasis. Using this difference as an example, we queried the mechanism(s) behind the proclivity for organ-specific metastasis. We used spontaneous and implant models of metastatic mammary carcinoma coupled with inflammatory tissue fibrosis, single-cell sequencing analyses and functional studies to unravel the causal determinants of organ-specific metastasis. Here we show that lung metastasis is facilitated by angiopoietin 2 (Ang2)-mediated suppression of lung-specific endothelial tight junction protein Claudin 5, which is augmented by the inflammatory fibrotic microenvironment and prevented by anti-Ang2 blocking antibodies, while kidney metastasis is prevented by non-Ang2-responsive Claudins 2 and 10. Suppression of Claudins 2 and 10 was sufficient to induce the emergence of kidney metastasis. This study illustrates the influence of organ-specific vascular heterogeneity in determining organotropic metastasis, independent of cancer cell-intrinsic mechanisms.

Extended Data Fig. 1:. Lung and kidney fibrosis induction.

(a) Representative H&E staining, Sirius Red staining, and αSMA immunolabeling of the lung and kidneys of mice with and without fibrosis in the respective organs. Scale bar, 100 μm. (b) Quantification of positive Sirius Red and αSMA⁺ stained area in the indicated organs and groups, n = 5 to 6 mice per group. (c, d) Relative Fn1 and Col1a1 mRNA levels in the respective organs and groups, n = 3 mice per group. KF: kidney fibrosis; LF: lung fibrosis. (e) Representative images for GFP immunolabeling and quantification of percent of the GFP positive area in lung with or without lung fibrosis. Scale bar, 50 μm, n = 6 mice per group. (f) Representative images for GFP immunolabeling of tumor in the mice with or without lung fibrosis. n = 1 mouse per group. Scale bar, 25 μm. (g) Representative liver H&E images in 4T1 tumor orthotopic bearing mice after induction of lung fibrosis. Scale bar, 50 μm. Quantification of metastatic liver nodules in the indicated groups, n = 6 mice per group. (h) Representative images for GFP immunolabeling and quantification of percent of the GFP positive area in liver of mice with or without lung fibrosis. Scale bar, 50 μm, n = 6 mice per group. (i, j) Representative H&E images of heart (i) and intestine (j) in 4T1 tumor orthotopic bearing mice after induction of lung fibrosis. Scale bar, 50 μm. (k) Orthotopic 4T1 tumor volume over time in mice with or without kidney fibrosis, n = 6 mice per group. (l, m) Representative H&E images (l) of the lung of 4T1 orthotopic tumor bearing mice with or without kidney fibrosis and respective quantification (m) of metastatic area. Scale bar, 25 μm, n = 6 mice per group. (n) Representative images for GFP immunolabeling and quantification of percent of the GFP positive area in lung with or without kidney fibrosis. Scale bar, 50 μm, n = 6 mice per group. (o) Representative images for GFP immunolabeling for tumor in the mice with or without kidney fibrosis. Scale bar, 25 μm. (p) Representative liver H&E images in 4T1 tumor orthotopic bearing mice after induction of kidney fibrosis. Scale bar, 50 μm. Quantification of metastatic liver nodules in the indicated groups, n = 6 mice per group. (q) Representative images for GFP immunolabeling and quantification of percent of the GFP positive area in liver of mice with or without kidney fibrosis. Scale bar, 50 μm, n = 6 mice per group. (r) Representative H&E images of heart and intestine of 4T1 tumor orthotopic bearing mice after induction of kidney fibrosis. Scale bar, 50 μm. KF: kidney fibrosis; LF: lung fibrosis. Data are presented as mean values +/− SEM. For k: two-way ANOVA with Sidak’s multiple comparisons test. For all other panels: Unpaired two-tailed t test with Welch’s correction applied for unequal variances (determined by F-test). P values are listed, ns: not significant.

Extended Data Fig. 2. Fibrosis does not promote non-tropic metastasis.

(a) Representative H&E images of the kidneys of 4T1 orthotopic tumor bearing mice +/− lung or kidney fibrosis. Scale bar, 25 μm. (b) Number of mice with microscopic kidney tumors in the indicated groups (c) Representative images for GFP immunolabeling and quantification of percent of the GFP positive area in kidney +/− kidney fibrosis. Scale bar, 50 μm. (d) Number of mice with microscopic kidney tumors in the indicated groups. (e) Representative kidney H&E images in MMTV-PyMT transgenic mouse model with lung or kidney fibrosis. Scale bar, 25 μm. (f) Number of mice with microscopic kidney tumors in the indicated groups. (g) Representative immunofluorescence images of PyMT positive cancer area in tumor, lung, and kidney after kidney fibrosis in MMTV-PyMT transgenic mice. Scale bar, 25 μm. (h–j) Representative images and quantification of positive MTS and Sirius Red staining from the indicated groups. Scale bar, 25 μm, n = 6–7 mice per group. Data are presented as mean values +/− SEM. Unpaired two-tailed t test, with Welch’s correction applied for unequal variances (determined by F-test). P values are listed, ns: not significant.

Extended Data Fig. 3. Intracardiac cancer cell injection does not alter metastatic disease in renal fibrosis & Wound healing does not impact tropic and non-tropic site metastasis.

(a) Representative GFP and CD31 staining of the kidneys from mice with 4T1 orthotopic inoculation and 4T1 i.v. administration with or without kidney fibrosis. Scale bar, 25 μm. (b) Quantification of GFP⁺ cancer cells from lung after 4T1 i.v. administration for 10 days with or without perfusion by flow cytometry. n = 3 mice per group. Gating strategy shown in Supplementary Fig. 1A. (c) Quantification of GFP⁺ cancer cells from kidneys after 4T1 i.v. administration for 10 days with or without perfusion by flow cytometry, n = 3 mice per group. Gating strategy shown in Supplementary Fig. 1B. (d, e) QPCR quantification of GFP DNA (expressed 2^-ΔCt after normalization to Nf1) from the lung (d) and kidney (e) of mice after 4T1 i.v. administration for 10 days with or without perfusion, n = 3 mice per group. (f) Representative H&E images and quantification of lung after intracardiac injection of 4T1 cancer cells together with induction of kidney fibrosis. Control, n = 5 mice; kidney fibrosis, n = 9 mice, Scale bar, 25 μm. (g) Representative H&E images and quantification of metastatic liver nodules after intracardiac injection of 4T1 cancer cells together with induction of kidney fibrosis. Control, n = 5 mice; kidney fibrosis, n = 9 mice. Scale bar, 50 μm. (h) Representative H&E images and number of mice with microscopic kidney tumors in the indicated groups after Intracardiac injection of 4T1 cancer cells together with induction of kidney fibrosis. Scale bar, 25 μm. (i) Representative images for GFP immunolabeling and number of mice with microscopic kidney tumors in the indicated groups after Intracardiac injection of 4T1 cancer cells together with induction of kidney fibrosis. Scale bar, 50 μm. (j) Orthotopic 4T1 tumor volume over time +/− cutaneous wound, n = 6 mice per group. (k) Representative H&E images of the lung of 4T1 orthotopic tumor bearing mice +/− cutaneous wound. Scale bar, 25 μm. (l) Quantification of metastatic area and surface lung nodules in 4T1 orthotopic tumor bearing mice with and without cutaneous wounds, n = 6 mice per group. (m) Tumor volume over time in MMTV-PyMT mice +/− cutaneous wound, n = 5 to 6 mice per group. (n) Representative lung H&E images from in MMTV-PyMT mice +/− cutaneous wound. Scale bar, 25 μm. (o) Quantification of metastatic area and surface lung nodules in MMTV-PyMT mice +/− cutaneous, n = 5 to 6 mice per group. (p) Wound closure rate in 4T1 orthotopic tumor bearing mice (n = 10 mice) and no tumor control (n = 4 mice). (q) Representative lung H&E images from mice with 4T1 i.v. injection and +/− cutaneous wound. Scale bar, 25 μm. (r) Quantification of metastatic area and surface lung nodules in mice with 4T1 i.v. injection +/− cutaneous wound, n = 5 mice per group. Data are presented as mean values +/− SEM or individual values shown. For j, m: two-way ANOVA with Sidak’s multiple comparisons test. For p: mixed effect model with Sidak’s multiple comparison test. For other panels: Unpaired two-tailed t test, with Welch’s correction applied for unequal variances (determined by F-test). P values are listed, ns: not significant.

Extended Data Fig. 4. Global gene expression profiling in fibrosis and serum cytokine analysis.

(a, b). Volcano plot and gene ontology (GO) enrichment ratio and false discovery rate (FDR) values associated with overlapped up-regulated and down-regulated genes. (c, d) GO enrichment ratio and FDR values in the lungs of mice with and without lung fibrosis (c) or with and without kidney fibrosis (d). (e, f) Overlapping up-regulated (e) or down regulated (f) genes in the lungs of healthy mice vs. mice with the lung fibrosis, and in the lungs of healthy mice vs mice with kidney fibrosis, with overlapped genes involved in vascular (f) and immune remodeling (e) pathways. (g) Cytokines profile of serum samples from healthy mice, mice with cutaneous wound, mice with lung fibrosis and kidney fibrosis, and heat map of the averaged relative cytokine levels in the indicated groups after normalization to healthy group (set to 1). Healthy, n = 2 mice; wound, n = 2 mice; lung fibrosis (LF), n = 3 mice; kidney fibrosis (KF), n = 3 mice. Cytokines showing minimal changes between healthy and wound (wound/healthy (W/H) ratio between 0.9 and 1.1) were then ranked based on levels in kidney fibrosis.

Extended Data Fig. 5. Immune cells infiltration in lung and kidney after fibrosis induction.

(a) Percentages of CD45⁺, CD3⁺, CD4⁺FoxP3⁻ effector T (Teff) cells, CD8⁺, γδ⁺, CD4⁺FoxP3⁺ regulatory T (Treg) cells, NK1.1⁺ cells, CD11c⁺, CD19⁺, CD11b⁺, CD11b⁺Ly6C⁺Ly6G⁻and CD11b⁺Ly6G⁺ Ly6C⁻ cells in lungs of mice with lung fibrosis or kidney fibrosis. Healthy lung, n = 6 mice; lung fibrosis (LF), n = 5 mice; healthy kidney, n = 6 mice; kidney fibrosis (KF), n = 5 mice. (b) Percentages of CD45⁺, CD3⁺, CD4⁺FoxP3⁻ effector T (Teff) cells, CD8⁺, γδ⁺, CD4⁺FoxP3⁺ regulatory T (Treg) cells, NK1.1⁺ cells, CD11c⁺, CD19⁺, CD11b⁺, CD11b⁺Ly6C⁺Ly6G⁻and CD11b⁺Ly6G⁺Ly6C⁻ cells in kidneys of mice with lung fibrosis or kidney fibrosis. Healthy lung, n = 5 mice; lung fibrosis (LF), n = 6 mice; healthy kidney, n = 6 mice; kidney fibrosis (KF), n = 4 mice. (c) Colocalization of FITC-dextran (green) and CD31 (red) in the liver of mice with and without after rAng2 treatment. Scale bar, 20 μm. Data are presented as mean values +/− SEM. Unpaired two-tailed t test, with Welch’s correction applied for unequal variances (determined by F-test). P values are listed, ns: not significant.

Extended Data Fig. 6. Recombinant Ang 2 enhances metastasis to the lung but not kidney.

(a) Representative lung H&E of mice treated with PBS (control) or recombinant Ang2 (rAng2). Scale bar, 25 μm. (b, c) Representative lung H&E of mice after induction of lung (b) or kidney fibrosis (c). Scale bar, 25 μm. (d) Quantification of edema area per whole lung area, n = 5 to 6 mice per group. (e) Quantification of pleural thickening of lung, n = 5 to 6 mice per group. (f) Representative kidney H&E images of mice treated with PBS (control) or rAng2. Scale bar, 25 μm. (g, h) Representative kidney H&E images of mice after induction of lung (g) or kidney fibrosis (h). Scale bar, 25 μm. (i) Representative lung H&E of mice treated with PBS (control) or recombinant Ang2 (rAng2) with 4T1 cells i.v. injection. Scale bar, 25 μm. (j) Representative kidney H&E of mice treated with PBS (control) or recombinant Ang2 (rAng2) with 4T1 cells i.v. injection. Scale bar, 25 μm. Data are presented as mean values +/− SEM. Unpaired two-tailed t test, with Welch’s correction applied for unequal variances (determined by F-test). P values are listed.

Extended Data Fig. 7. tSNE plots of Claudin genes in scRNA-seq data sets.

(a) tSNE plots of different Cldn genes in flow cytometry sorted CD31⁺ endothelial cells from mouse lung tissue. (b) tSNE plots of different Cldn genes in endothelial cells from mouse lung tissue. (c) tSNE plots of Claudin family genes in the endothelial cells from human lung scRNA-seq data. (d) tSNE plots of Claudin family genes in the endothelial cells from mouse kidney scRNA-seq data. (e) tSNE plots of Claudin family genes in the endothelial cells from human kidney scRNA-seq data. Colors coded for the relative gene expressions.

Extended Data Fig. 8. Claudins expression in distinct cell types.

(a) Different Claudin expressions in different endothelial cell types of mouse lung,. (b, c) tSNE plots of Claudin 5 in different cell types of in mouse lung (b) and highly expressed in lung endothelial cells (c). (d) Different Claudins expression in different mouse lung cell types. (e) Expression of diverse Claudins in different mouse kidney cell types. (f, g) Diverse Claudins expression in different mouse (f) and human brain cell types (g). (h) Colocalization of Claudin 2 and CD31 in WT kidney cortex and medulla (individual images in Supplementary Fig. 5a, b). (i) Colocalization of Claudin 10 and CD31 in WT kidney cortex and medulla (individual images in Supplementary Fig. 5c, d). Scale bar, 50 μm (upper), 25 μm (lower). (j) Representative colocalization of Claudin 5 and CD31 in the lung of mice in the indicated groups. Scale bar, 20 μm. (k) Quantification of percentage of Claudin 5 and CD31 double positive area per visual field in the indicated groups, n = 5–9 mice per group. These images and graphs also shown in Supplementary Fig. 6. Data are presented as mean values +/− SEM. Unpaired two-tailed t test, with Welch’s correction applied for unequal variances (determined by F-test). P values are listed, ns: not significant.

Extended Data Fig. 9. Data Figure 9. Ang2 and Claudins signaling and Claudin2/10 suppression.

(a) Relative mRNA expression of Cldn5 in parental NIH-3T3 cells and NIH-3T3 cells treated with Adeno-CMV-luc (Ad-Ctrl) or Ad-CMV-CLDN5v2 (Ad-Claudin 5), n = 3 biologically independent experiments (with each 2 technical replicates). (b) Representative western blot and quantification of his-tagged Claudin 5 (His), Claudin 5, and β-actin in parental NIH-3T3 cells and NIH-3T3 cells treated with Adeno-CMV-luc (Ad-Ctrl) or Ad-CMV-CLDN5v2 (Ad-Claudin 5), n = 3 biologically independent experiments. Uncropped blots are shown in the Image Source Data File. (c) Relative mRNA expression of Cldn5 in NIH-3T3 cells with the indicated treatment. Dox on/off indicates doxycycline exposure, n = 4 biologically independent experiments (with each 2 technical replicates). (d) Orthotopic 4T1 tumor volume over time in the indicated groups, n = 5 mice per group. (e) CLDN5 mRNA levels in metastatic lungs of patients with breast cancer (n = 9) and healthy lungs (n = 6) (GSE193103)62. (f, g) Relative mRNA expression of Cldn2 (f) and Cldn10 (g) in kidney endothelial cells treated with the indicated guide RNAs, n = 3 biologically independent experiments (with each 2 technical replicates). (h) Relative mRNA expression of Cldn2 and Cldn10 in sorted E-cadherin⁺ epithelial cells and CD31⁺ endothelial cells, n = 3 mice per group. Gating strategy is shown in Supplementary Fig. 9. (i, j) Relative mRNA expression levels of Cldn2 and Cldn10 in sorted CD31⁺ endothelial cells (i) and E-cadherin⁺ epithelial cells (j) after treatment with the indicated guide RNAs, n = 3 mice per group. (k) Experimental design for the evaluation of the downregulation of Claudin 2 and Claudin 10 in the kidney of mice with 4T1 orthotopic tumors. (l) Orthotopic 4T1 tumor volume over time in the indicated groups. On day 14, LentiCRISPRv2 control vector, n = 5; LentiCRISPRv2 Cldn2 and Cldn10, n = 5; Day 18, LentiCRISPRv2 control vector, n = 5; LentiCRISPRv2 Cldn2 and Cldn10, n = 6; Day 22, LentiCRISPRv2 control vector, n = 8; LentiCRISPRv2 Cldn2 and Cldn10, n = 6 mice. (m) Representative H&E images of the lung of mice in the indicated groups. Scale bar, 25 μm. (n) Quantification of lung metastatic area. LentiCRISPRv2 control vector, n = 15; LentiCRISPRv2 Cldn2 and Cldn10, n = 20 mice. (o) Representative images for GFP immunolabeling and quantification of percent of the GFP positive area in lung. Scale bar, 50 μm, n = 9 mice per group. Data are presented as mean values +/− SEM, Min-to-Max in E. For a: one-way ANOVA test with Tukey’s multiple comparisons test. For d: two-way ANOVA with Sidak’s multiple comparisons test. For b, c, f, g: one-way ANOVA test with Dunnett’s multiple comparisons test. For e: two-tailed Mann-Whiney test. For i, j, n, o: Unpaired two-tailed t test, with Welch’s correction applied for unequal variances (determined by F-test).

Extended Data Fig. 10. Knockdown of Claudin 2/10 redirects metastasis to the kidney.

(a) Representative H&E images of the kidney of mice in the indicated groups. Scale bar, 50 μm (upper), 25 μm (lower). (b) Percentage of mice with kidney lesion in the indicated groups. LentiCRISPRv2 control vector, n = 15; LentiCRISPRv2 Cldn2 and Cldn10, n = 20 mice. (c) Representative images for GFP immunolabeling in kidney. Scale bar, 50 μm (upper), 25 μm (lower). (d) Number of mice with GFP positive kidney. (e) Quantification of percent of the GFP positive area in the indicated groups, n = 12 mice per group. (f) Orthotopic 4T1 tumor volume over time in the indicated groups. LentiCRISPRv2 control vector without fibrosis, n = 14; LentiCRISPRv2 control vector with fibrosis, n = 11; LentiCRISPRv2 Cldn2 and Cldn10 without fibrosis, n = 12 mice; LentiCRISPRv2 Cldn2 and Cldn10 with fibrosis, n = 10 mice. (g) Number of mice with GFP positive kidney in the indicated groups. LentiCRISPRv2 control vector without fibrosis, n = 14; LentiCRISPRv2 control vector with fibrosis, n = 11; LentiCRISPRv2 Cldn2 and Cldn10 without fibrosis, n = 12 mice; LentiCRISPRv2 Cldn2 and Cldn10 with fibrosis, n = 10 mice. (h) Representative GFP and CD31 staining of the kidneys in mice with Claudin 2 and Claudin 10 down regulation together with or without kidney fibrosis. (i) Representative GFP and CD31 staining in the metastatic kidney with Claudin 2 and Claudin 10 downregulation. For i-j, individual images are shown in Supplementary Fig. 10A-C. Scale bar, 25 μm. (j) Representative images for CD31 and quantification of percent of the CD31 positive area in tumor when downregulate Claudin 2/10 in the kidney. Scale bar, 50 μm. LentiCRISPRv2 control vector, n = 5; LentiCRISPRv2 Cldn2 and Cldn10, n = 7 mice. (k) Representative images for CD31 and quantification of percent of the CD31 positive area in tumor when downregulate Claudin 2 and Claudin 10 with or without kidney fibrosis. Scale bar, 50 μm. LentiCRISPRv2 control vector without fibrosis, n = 5; LentiCRISPRv2 control vector with fibrosis, n = 4; LentiCRISPRv2 Cldn2 and Cldn10 without fibrosis, n = 4 mice; LentiCRISPRv2 Cldn2 and Cldn10 with fibrosis, n = 6 mice. Data are presented as mean values +/− SEM. For b: two-sided Chi-square. For f: two-way ANOVA with Sidak’s multiple comparisons test. For j: Unpaired two-tailed t test, with Welch’s correction applied for unequal variances (determined by F-test). For k: one-way ANOVA with Tukey’s multiple comparisons test. P values are listed, ns: not significant.

Figure 1.. Fibrosis enhances tropic metastasis with no impact on non-tropic sites

(A-B) Representative MTS of lung (A) and kidney (B) fibrosis. Scale bar, 100 μm. (C) Orthotopic 4T1 tumor volume over time +/− lung fibrosis, n=6 mice per group. (D) Representative lung H&E of 4T1 orthotopic tumor bearing mice +/− lung fibrosis. Scale bar, 25 μm. (E) Quantification of metastatic area and surface lung nodules in 4T1 orthotopic tumor bearing mice +/− lung fibrosis, n=6 mice per group. (F) MMTV-PyMT tumor volume over time +/− lung fibrosis, n=5 to 7 mice per group. (G) Representative lung H&E of MMTV-PyMT tumor bearing mice +/− lung fibrosis. Scale bar, 25 μm. (H) Quantification of metastatic area and surface lung nodules in MMTV-PyMT tumor bearing mice +/− lung fibrosis, n=5 to 7 mice per group. (I) Orthotopic 4T1 tumor volume over time +/− kidney fibrosis, n=6 mice per group. (J) Representative lung H&E of 4T1 orthotopic tumor bearing mice +/− kidney fibrosis. Scale bar, 25 μm. (K) Quantification of metastatic area and surface lung nodules in 4T1 orthotopic tumor bearing mice +/− kidney fibrosis, n=6 mice per group. (L) MMTV-PyMT tumor volume over time +/− kidney fibrosis, n=6 mice per group. (M) Representative lung H&E of MMTV-PyMT tumor bearing mice +/− kidney fibrosis. Scale bar, 25 μm. (N) Quantification of metastatic area and surface lung nodules in MMTV-PyMT tumor bearing mice +/− kidney fibrosis, n=5 to 6 mice per group. (O) Representative lung H&E of the lung of mice following 4T1 i.v. injection +/− lung fibrosis and +/− kidney fibrosis. Scale bar, 25 μm. (P) Quantification of lung colonization following 4T1 i.v. injection +/− lung fibrosis or kidney fibrosis, n=6 mice per group. Data are presented as mean values +/− SEM. ctrl: control, LF: lung fibrosis, KF: kidney fibrosis. For B, E, H, K, N, P: unpaired two-tailed t test with Welch’s correction applied for unequal variances (determined by F-test); for C, I: two-way ANOVA with Sidak’s multiple comparisons test; for F, L: multiple unpaired t tests of tumor measurements. P values are listed, ns: not significant.

Figure 2.. 4T1 cells form tumors when injected under the renal capsule

(A) Experimental design for the evaluation of tumor formation in mice injected with 4T1 cells under the renal capsule +/− kidney fibrosis induced immediately post injection. (B) Representative kidney H&E of mice with 4T1 cells injected under the renal capsule and +/− kidney fibrosis. Scale bar, 100 μm. (C-D) Kidney weight (C) and quantification of primary tumor area (D) in kidneys of the indicated groups, n=7 mice per group. (E-F) Representative kidney H&E of mice with 4T1 cells injected under the renal capsule together without (E) or with kidney fibrosis (F). Scale bar, 20 μm. (G-H) Representative images for GFP immunolabeling (G) and quantification of percent of the GFP positive area (H) in the indicated groups, n=4 mice per group. Scale bar, 50 μm. (I) Representative lung H&E images of mice with 4T1 cells injected under the renal capsule +/− kidney fibrosis. Scale bar, 25 μm. (J-K) Quantification of metastatic area/whole lung (J) and surface lung nodules (K) in the indicated groups, n=7 mice per group. (L-M) Representative H&E images of kidney in mice with 4T1 i.v. administration and with lung (L) or kidney fibrosis (M). Scale bar, 25 μm. (N) Number of mice with microscopic kidney tumors in the indicated groups, n=6 mice per group. Data are presented as mean values +/− SEM. For D, H, J, K: unpaired two-tailed t test, with Welch’s correction applied for unequal variances (determined by F-test). For C: one-way ANOVA with Tukey multiple comparisons test. P values are listed, ns: not significant.

Figure 3.. Ang2 mediates fibrosis-induced lung metastasis

(A, C) Volcano plots depicting the number of differentially regulated genes in healthy lung vs. lung fibrosis (A) and healthy lung vs. lung from mice with kidney fibrosis (C). (B, D) Gene ontology (GO) enrichment ratio and false discovery rate (FDR) values for deregulated genes implicated in vascular remodeling in lung with fibrosis (B) or lung of mice with kidney fibrosis (D). The numbers of deregulated genes out of all the genes in the pathways are indicated in parenthesis with a star denoting common pathways in B and D. (E) Heatmap of relative mean levels of the listed cytokines in the serum of mice in the indicated group. Healthy, n=2; wound, n=2; lung fibrosis, n=3; kidney fibrosis, n=3 mice. (F) ELISA quantification of circulating Ang2 levels in the serum of mice in the indicated groups. Healthy, n=5; wound, n=3; 4T1 orthotopic tumor bearing mice (T), n=4; control for lung fibrosis (ctrl LF), n=6; lung fibrosis, n=6; control for kidney fibrosis (ctrl KF), n=6; kidney fibrosis (KF), n=5 mice. (G-H) Representative images and respective quantification of dextran extravasation (green) and CD31 immunolabeling (red) in the lung (G) and kidney (H) after rAng2 or PBS control treatment (individual images in Supplementary Figure 2). Scale bar, 20 μm. Quantification of FITC-Dextran area per CD31 positive area, n=4 to 5 mice per group. (I-J) Representative lung H&E of mice with PBS or rAng2 treatment and with (J) and without (I) i.v. injection of 4T1 tumor cells. Scale bar, 200 μm. Quantification of pleural edema and thickness in the lungs, n=5 to 6 mice per group (I), and quantification of lung colonization and surface lung nodules (J) in the indicated groups, n=5 to 6 mice per group. Data are presented as mean values +/− SEM. Unpaired two-tailed t test, with Welch’s correction applied for unequal variances (determined by F-test). P values are listed, ns: not significant.

Figure 4.. Ang2 neutralization is sufficient to revert lung metastasis induced by kidney fibrosis

(A) Orthotopic 4T1 tumor volume over time +/− kidney fibrosis (KF) and IgG or anti-Ang2 treatment, n=9 mice per group. Arrows indicate a ~30% reduction in tumor volume at experimental endpoint with anti-Ang2 compared to IgG (ctrl groups). (B-C) Quantification of metastatic area (B) and number of microscopic lung nodules (C) in the indicated groups, n=9 mice per group. (D) Representative lung H&E of mice in the indicated groups. Scale bar, 25 μm. (E) Representative images of kidneys with H&E, MTS, and Sirius Red staining from the indicated groups. Scale bar, 25 μm. (F-G) Quantification of positive MTS (F) and Sirius Red stained area (G) in the indicated groups, n=9 mice per group. (H) Relative mRNA expression levels of Cldn2, Cldn3, Cldn4, Cldn5, Cldn7, Cldn10, Cldn17, Cldn19, Jam2, Jam3, Occludin and Tjp1 in healthy kidney, lung, and liver tissues. Expression value is normalized to endogenous Pecam1 (CD31) levels and is presented as 2^-ΔCt, n=3 mice per group. Data are presented as mean values +/− SEM. For A: two-way ANOVA with Tukey’s multiple comparisons test. For B, C, F and G: one-way ANOVA test with Tukey’s multiple comparisons test. P values are listed, ns: not significant.

Figure 5.. Differential Claudin expression levels in lung and kidney

(A-B) t-SNE plots (A) and violin plots (B) of scRNA-seq evaluation of Cldn5 mRNA levels in endothelial cells from the listed organs. (C-D) t-SNE plot of sRNA-seq of murine lung cells (C) with a focus on endothelial cells (EC) showing the relative mRNA levels for Cldn2, Cldn5 and Cldn10 (D). (E) tSNE plots and analysis of Claudin 5 and CD31 colocalization percentage in mouse lung endothelial cells (EC). (F-G) t-SNE plot of sRNA-seq of murine kidney cells (F) with a focus on endothelial cells (EC) showing the relative mRNA levels for Cldn2, Cldn5 and Cldn10 (G). (H) Colocalization of CD31 (green) and Claudin 5 (red) in WT liver, brain, and kidney. Scale bar, 25 μm (individual images in Supplementary Figures 3, 4A). (I) Colocalization of Lyve-1 (green) and Claudin 5 (red) in lung. Scale bar, 25 μm (individual images in Supplementary Figure 4B). Data are presented as mean values +/− SEM.

Figure 6.. Ang2 downregulates Claudin 5 via nuclear enrichment of FOXO1

(A) Experimental design for the evaluation of Ang2 mediated Claudin 5 downregulation in the lung endothelial cells. (B) Relative Cldn5 mRNA levels in lung endothelial cells treated with the rAng2 (PBS control normalized to 1) for the indicated timepoints, n=3 biologically independent experiments (n=3 PBS and n=3 rAng2 with each 2 technical replicates). (C-D) Relative Claudin 5 protein levels in lung endothelial cells treated with rAng2 for the indicated timepoints, n=5 biologically independent experiments (n=5 PBS and n=5 rAng2). Uncropped blots are shown in the Image Source Data File. (E) Representative immunofluorescence staining of Claudin 5 in lung endothelial cells after treatment with rAng2 for 24h. n=1 biologically independent experiment. Scale bars, 25 μm. (F) FOXO1 protein levels in nuclear and cytoplasmic fractions at the indicated timepoints, with alpha-tubulin and Lamin A/C as markers for cytoplasmic and nuclear fraction, respectively. n=1 biologically independent experiment. Uncropped blots are shown in the Image Source Data File. (G) Relative Jam2, Cldn3 and Cldn4 mRNA levels of in lung endothelial cells after treatment with rAng2 for 24h, n=3 biologically independent experiments (with each 3 technical replicates). (H) Relative Cldn5 mRNA levels of in brain endothelial cells after treatment with the rAng2 for 24h, n=3 biologically independent experiments (with each 2 technical replicates). Data are presented as mean values +/− SEM. Unpaired two-tailed t test, with Welch’s correction applied for unequal variances (determined by F-test). P values are listed, ns: not significant.

Figure 7.. Critical role of Claudin 5 in 4T1 cell extravasation in lung

(A) Experimental design for the evaluation of Cldn5 overexpression (Ad-Claudin 5) in mice with 4T1 i.v. injection +/− rAng2 treatment. (B) Representative colocalization of Claudin 5 and CD31 in the lung of mice in the indicated groups (individual images in Supplementary Figure 7). Scale bars, 50 μm. (C) Quantification of percentage of Claudin 5/CD31 double positive area per visual field in the lung of mice in the indicated groups, n=4 to 5 mice per group. (D) Representative lung H&E of mice in the indicated groups. Scale bar, 25 μm. (E) Quantification of lung colonization area of mice in the indicated groups, n=4 to 5 mice per group. (F) Experimental design for the evaluation of the downregulation of Claudin 5 (AAV-shRNA) in mice with 4T1 orthotopic tumors. Dox: doxycycline in the drinking water (Dox on). (G) Representative colocalization of Claudin 5 and CD31 in the lung of mice in the indicated groups individual images in Supplementary Figure 8). Scale bar, 50 μm. (H) Quantification of percentage of Claudin 5/CD31 double positive area per visual field in the lung of mice in the indicated groups, n=4 to 5 mice per group. (I) Representative lung H&E of mice in the indicated groups. Scale bar, 25 μm. (J) Quantification of lung metastatic area in mice in the indicated groups, n=4 to 5 mice per group. Data are presented as mean values +/− SEM. One-way ANOVA with Tukey ‘s multiple comparisons test. P values are listed.

Figure 8.. Knockdown of Claudin 2/10 in kidney fibrosis enables renal metastasis

(A) Experimental design for the evaluation of the downregulation of Claudin 2 and Claudin 10 in kidney together with induction of kidney fibrosis in mice with 4T1 orthotopic tumors. (B) Representative lung H&E of mice in the indicated groups. Scale bar, 25 μm. (C) Quantification of lung metastatic area. LentiCRISPRv2 control vector without fibrosis, n=14; LentiCRISPRv2 control vector with fibrosis, n=11; LentiCRISPRv2 Cldn2 and Cldn10 without fibrosis, n=12 mice; LentiCRISPRv2 Cldn2 and Cldn10 with fibrosis, n=10 mice. (D) Representative kidney H&E of mice in the indicated groups. Scale bar, 50 μm (upper), 25 μm (lower). (E-F) Representative lung (E) and kidney (F) images for GFP immunolabeling in the indicated groups and associated quantification. E: scale bar, 50 μm, F: scale bar, 25 μm. (G) Percentage of mice with kidney lesion in the indicated groups. (H) Graph abstract. Data are presented as mean values +/− SEM. For C, E: one-way ANOVA with Tukey’s multiple comparisons test. For F: Kruskal–Wallis with Dunn’s multiple comparison test. For G: two-sided Chi-square. P values are listed, ns: not significant.