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. 2018 Feb 1;128(2):834-845.
doi: 10.1172/JCI94674. Epub 2018 Jan 22.

Endothelial Tie1-mediated Angiogenesis and Vascular Abnormalization Promote Tumor Progression and Metastasis

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Free PMC article

Endothelial Tie1-mediated Angiogenesis and Vascular Abnormalization Promote Tumor Progression and Metastasis

Silvia La Porta et al. J Clin Invest. .
Free PMC article

Abstract

The endothelial tyrosine kinase receptor Tie1 remains poorly characterized, largely owing to its orphan receptor status. Global Tie1 inactivation causes late embryonic lethality, thereby reflecting its importance during development. Tie1 also plays pivotal roles during pathologies such as atherosclerosis and tumorigenesis. In order to study the contribution of Tie1 to tumor progression and metastasis, we conditionally deleted Tie1 in endothelial cells at different stages of tumor growth and metastatic dissemination. Tie1 deletion during primary tumor growth in mice led to a decrease in microvessel density and an increase in mural cell coverage with improved vessel perfusion. Reduced angiogenesis and enhanced vascular normalization resulted in a progressive increase of intratumoral necrosis that caused a growth delay only at later stages of tumor progression. Concomitantly, surgical removal of the primary tumor decreased the number of circulating tumor cells, reduced metastasis, and prolonged overall survival. Additionally, Tie1 deletion in experimental murine metastasis models prevented extravasation of tumor cells into the lungs and reduced metastatic foci. Taken together, the data support Tie1 as a therapeutic target by defining its regulatory functions during angiogenesis and vascular abnormalization and identifying its role during metastasis.

Keywords: Angiogenesis; Cancer; Oncology; endothelial cells.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Tie1 loss reduces angiogenesis and delays tumor growth at late stages.
(A) Growth curves of LLC tumors in WT and Tie1iECKO mice (n = 9 WT; n = 8 Tie1iECKO). ***P < 0.001, by 2-way ANOVA. Data are expressed as the mean ± SEM. (B and C) Quantification of vessel area (B) and vessel density (C) in LLC tumors from WT and Tie1iECKO mice (n = 9–10). **P < 0.01 and ***P < 0.001, by 2-tailed Mann-Whitney U test. Error bars represent mean ± SD. (D) Representative images of LLC tumor hypoxia (stained for anti-HIF1α). Arrowheads indicate hypoxic areas. Scale bar: 1 mm. (E) Quantification of HIF1α-positive areas (n = 10 WT; n = 9 Tie1iECKO). ***P < 0.001, by 2-tailed Mann-Whitney U test. Error bars represent mean ± SD. (F) Representative images of necrotic LLC primary tumors (arrowheads indicate H&E-stained light pink areas). Scale bar: 1 mm. (G) Quantification of necrotic areas from WT and Tie1iECKO tumors (n = 10 mice). Error bars represent mean ± SD. ***P < 0.001, by 2-tailed Mann-Whitney U test.
Figure 2
Figure 2. Deletion of endothelial Tie1 leads to increased vascular coverage and improved vessel perfusion.
(A) Quantification of desmin-positive blood vessels in WT and Tie1iECKO LLC primary tumors (n = 10 WT; n = 9 Tie1iECKO). *P < 0.05, by 2-tailed Mann-Whitney U test. (B) Representative confocal images of blood vessels (stained with anti-CD31) and pericytes (stained with anti-desmin). Scale bar: 50 μm. (C) Quantification of αSMA-positive blood vessels in LLC tumor vasculature (n = 10). *P < 0.05, by 2-tailed Mann-Whitney U test. (D) Representative microscopic images of blood vessels (stained for anti-CD31) and smooth muscle cells (stained for anti-αSMA). Scale bar: 50 μm. (E) Quantification of vessel sprouting (n = 5). **P < 0.01, by 2-tailed Mann-Whitney U test. (F) Quantification of lectin-perfused vessel areas (n = 5). *P < 0.05, by 2-tailed Mann-Whitney U test. (G) Representative microscopic images of blood vessels (stained for anti-CD31) perfused with lectin from WT and Tie1iECKO tumors. Images are representative of 5 mice. Scale bar: 100 μm. Error bars represent mean ± SD.
Figure 3
Figure 3. The intratumoral vasculature in Tie1iECKO mice progressively becomes antiangiogenic and normalizes over time during primary tumor growth.
(AD) Quantification of vessel area (A), vessel density (B), and desmin-positive (C) and αSMA-positive (D) blood vessels 9 and 12 days after inoculation with LLC cells (n = 5). **P < 0.01 and *P < 0.05, by 2-tailed Mann-Whitney U test. (E and F) Quantification of primary tumor necrosis 9 and 12 days after inoculation of LLC cells (n = 5) (E) and analysis of the lectin-perfused vessel area (n = 5) (F). *P < 0.05, by 2-tailed Mann-Whitney U test. Error bars represent mean ± SD.
Figure 4
Figure 4. Deletion of endothelial Tie1 reduces tumor cell intravasation and limits metastasis.
(A) Schematic representation of the experimental design. LLC cells were injected s.c., and tamoxifen was administrated as indicated. Tumors were surgically removed after 14 days, and mice were sacrificed 3 weeks after primary tumor resection. Shown are representative images of H&E-stained lungs from WT and Tie1iECKO mice. Arrowheads indicate metastases. Scale bar: 3 mm. (B) Number of mice that developed lung metastases (black) after primary tumor removal (n = 14). (C) Kaplan-Meier survival curve of WT and Tie1iECKO mice after primary tumor removal (n = 10 WT; n = 6 Tie1iECKO). *P < 0.05 , by Gehan-Breslow-Wilcoxon test. Mice with less than 50% endothelial Tie1 deletion were excluded from the analysis. (D) Blood was drawn from WT and Tie1iECKO tumor-bearing mice at the time of tumor removal and plated in culture dishes. The formation of tumor cell colonies was traced over time. Graph shows the number of blood samples that developed tumor cell colonies (n = 11 WT; n = 10 Tie1iECKO). (E) ECs were seeded onto gelatin-coated Transwell inserts, and PKH67-labeled LLC cells were allowed to transmigrate for 8 hours. Thereafter, the cells were counted (percentage of LLC cells transmigrated through siTie1-KD HUVECs vs. control; n = 3 independent experiments, with each experiment performed in triplicate). **P < 0.01, by 2-tailed Mann-Whitney U test. (F) Relative VE-cadherin (Cdh5) expression in isolated tumor ECs (n = 4 WT; n = 5 Tie1iECKO). *P < 0.05, by 2-tailed Mann-Whitney U test. (G) Representative confocal microscopic images of blood vessels from WT and Tie1iECKO tumors stained with VE-cadherin and DAPI. Scale bar: 100 μm. (H) Quantification of VE-cadherin mean fluorescence intensity (MFI) (n = 5). **P < 0.01, by 2-tailed Mann-Whitney U test. Error bars represent mean ± SD.
Figure 5
Figure 5. Deletion of endothelial Tie1 reduces tumor cell extravasation and seeding but does not affect seeded micrometastasis.
(A) Schematic representation of the experimental metastasis model. Mice were preinjected with 4 doses of tamoxifen, and B16F10 cells were injected i.v. 1 week after the last tamoxifen dose. Mice were sacrificed, and their lungs were analyzed 2 weeks after tumor cell inoculation. The graph shows the number of WT and Tie1iECKO mice that developed lung metastases (black) 2 weeks after i.v. injection of B16F10 cells (n = 5). (B) Representative H&E-stained images of lung tissue from WT and Tie1iECKO mice. Arrowheads indicate metastatic foci. Scale bar: 3 mm. (C) Quantification of the number of lung metastases per mouse (n = 5). *P < 0.05, by 2-tailed Mann-Whitney U test. (D) Schematic representation of the experimental design. LLC cells were injected s.c., and tumors were resected on day 14. Endothelial Tie1 was thereafter deleted via tamoxifen administration. Mice were sacrificed, and lungs were analyzed 3 weeks after the primary tumor resection. Graph shows the number of WT and Tie1iECKO mice that developed lung metastases (black) 3 weeks after surgical removal of the primary tumor (n = 8). (E) Representative H&E-stained images of lung tissue from WT and Tie1iECKO mice. Arrowheads indicate metastases. Scale bar: 3 mm. (F) Quantification of the metastatic areas in lungs from WT and Tie1iECKO mice (n = 6 WT; n = 7 Tie1iECKO). Statistical significance was determined by a 2-tailed Mann-Whitney U test. Error bars represent mean ± SD.
Figure 6
Figure 6. Endothelial Tie1 deletion induces vessel stabilization through Ang1/Tie2 signaling.
(A) Representative scans of the cytokine array performed on WT and Tie1iECKO LLC tumor lysates showing Ang1 and Ang2 protein expression (n = 4). (B) Quantitation of the cytokine array intensities in A, with quantitation of the Ang1/Ang2 ratio (n = 4). *P < 0.05, by 2-tailed Mann-Whitney U test. (C) Relative Kdr mRNA expression in isolated tumor ECs (n = 4 WT; n = 5 Tie1iECKO). *P < 0.05, by 2-tailed Mann-Whitney U test. (D) Relative Tek expression in isolated tumor ECs (n = 4 WT; n = 5 Tie1iECKO). (E) Scans of Western blots performed on tumor lysates with Abs against Tie2 and actin as a loading control. Representative images are from 2 independent experiments. (F) Quantitation of the Tie2 and actin blot intensities shown in E (n = 5; 2-tailed Mann-Whitney U test). (G) Representative images of CD31-positive tumor blood vessels stained for Tie2. Scale bar: 100 μm. (H) Quantification of CD31 and Tie2 colocalization (n = 6). *P < 0.05, by 2-tailed Mann-Whitney U test. Error bars represent mean ± SD.

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