Snail1 expression in endothelial cells controls growth, angiogenesis and differentiation of breast tumors

Abstract

Snail1 is a transcriptional factor required for epithelial to mesenchymal transition and activation of cancer-associated fibroblasts (CAF). Apart from that, tumor endothelial cells also express Snail1. Here, we have unraveled the role of Snail1 in this tissue in a tumorigenic context. Methods: We generated transgenic mice with an endothelial-specific and inducible Snail1 depletion. This murine line was crossed with MMTV-PyMT mice that develop mammary gland tumors and the consequence of Snail1 depletion in the endothelium were investigated. We also interfere Snail1 expression in cultured endothelial cells. Results: Specific Snail1 depletion in the endothelium of adult mice does not promote an overt phenotype; however, it delays the formation of mammary gland tumors in MMTV-PyMT mice. These effects are associated to the inability of Snail1-deficient endothelial cells to undergo angiogenesis and to enhance CAF activation in a paracrine manner. Moreover, tumors generated in mice with endothelium-specific Snail1 depletion are less advanced and show a papillary phenotype. Similar changes on onset and tumor morphology are observed by pretreatment of MMTV-PyMT mice with the angiogenic inhibitor Bevacizumab. Human breast papillary carcinomas exhibit a lower angiogenesis and present lower staining of Snail1, both in endothelial and stromal cells, compared with other breast neoplasms. Furthermore, human breast tumors datasets show a strong correlation between Snail1 expression and high angiogenesis. Conclusion: These findings show a novel role for Snail1 in endothelial cell activation and demonstrate that these cells impact not only on angiogenesis, but also on tumor onset and phenotype.

Figure 1

Snail1 is expressed in human tumor endothelial cells. A, Representative images of hematoxylin and eosin staining (HE) and Snail1 and CD31 immunohistochemistry in blood vessels of human neoplasms: colorectal carcinomas (first row), pancreatic adenocarcinomas (second row) and human fibromatosis (third row). Black arrowheads point Snail1+ cells. Scale bars: 100 µm (HE) and 20 µm (others). B, Double immunohistochemical staining of human breast tumors with Snail1 (brown) and the endothelium-specific protein von Willebrand factor (vWF) (red). Black arrowheads indicate examples of Snail1+ cells. Scale bar: 25 µm. C, Western blot analysis of Snail1 in HMEC-1 cells. MEF Wild-Type and KO for Snail1 were used as control. D, Snail1 (green) immunofluorescence analysis in cultured HMEC-1 cells. Actin cytoskeleton was stained with Alexa 555-conjugated Phalloidin (red) and nuclei were counterstained with DAPI (blue). Scale bar: 25 µm.

Figure 2

Snail1 controls angiogenesis in vivo. A, Number of endothelial cells in lungs or livers of VE-Cadh^Snail1KO or VE-Cadh^Snail1CT mice. B-C, Matrigel plugs containing vehicle, VEGFA or FGF2 were xenografted in the flanks of VE-Cadh^Snail1CT or VE-Cadh^Snail1KO mice and analyzed one week later. B, Representative results of CD31 immunohistochemical analysis. Scale bar: 100 µm. C, Quantification of CD31 staining in Matrigel plugs. Data represent the mean ± SEM of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001.

Figure 3

Snail1 depletion in endothelial cells retards tumor onset and stromal activation in the MMTV-PyMT breast cancer model. Comparison of survival (A), tumor size (B), tumor onset (C) and tumor burden at 18 weeks (D) between PyMT-VE-Cadh^Snail1CT and PyMT-VE-Cadh^Snail1KO mice. E-I, Representative images of Ki67 (E), CD31 (F), Vimentin (G), Masson's trichromic (H) or phosphoSmad2 (I) stainings of PyMT-VE-Cadh^Snail1CT and PyMT-VE-Cadh^Snail1KO at hyperplasic (E) or premalignant ducts (F-I). The quantification of the stainings was carried out as indicated in Suppl. Methods and is presented below or at the right. Bars correspond to 20 µm (CD31), 40 µm (Ki67, Vimentin and PSmad2) or 25 µm (Masson's). Data represent mean ± SEM of at least three mice per group. * P < 0.05; ** P < 0.01; *** P < 0.001.

Figure 4

Endothelium-specific Snail1 depletion alters tumor cell content of MMTV-PyMT tumors. A, Immunohistochemical Ki67 analysis (images, left panel; and quantification, right panel) of PyMT-VE-Cadh^Snail1CT and PyMT-VE-Cadh^Snail1KO carcinomas at 18 and 22 weeks, respectively. Scale bar: 40 µm. B, Percentage of epithelial, endothelial, immune and other stromal cells in PyMT-VE-Cadh^Snail1CT and PyMT-VE-Cadh^Snail1KO carcinomas determined by FACS. C-D, CD45 (C), CK14 and p63 (D) analysis (of the indicated carcinomas. Scale bar: 40 µm (C); 200 µm (D). In the quantifications, data represent mean ± SEM of at least three mice per group. * P < 0.05; *** P < 0.001.

Figure 5

Tumors with an endothelial-specific Snail1-depletion show an alteration in tumor differentiation. A, Hematoxylin and eosin staining of tumors obtained from PyMT-VE-Cadh^Snail1CT and PyMT-VE-Cadh^Snail1KO mice at the indicated time points. Scale bars: 200 µm. B-C, Quantification of tumor stage (B) and carcinoma morphology (C) in PyMT-VE-Cadh^Snail1CT and PyMT-VE-Cadh^Snail1KO tumors obtained at the indicated time points. D, Her2, ERα and Laminin α analysis of carcinomas generated in PyMT-VE-Cadh^Snail1CT and PyMT-VE-Cadh^Snail1KO mice. Scale bar: 50 µm. Staining quantification was performed as indicated in the Supplemental Information and is presented below. ** P < 0.01; *** P < 0.001.

Figure 6

Alterations in tumor vasculature associated to Snail1 depletion. A, CD31 analysis of PyMT-VE-Cadh^Snail1CT and PyMT-VE-Cadh^Snail1KO tumors collected at the indicated times. Scale bars: 200 µm (upper row); 40 μm (bottom row). B, Quantification of CD31+ area (upper graph) and vessel lumen size (bottom graph). C, Masson's trichrome staining of MMTV-PyMT tumors. Scale bar: 50 μm. Data in B and C represent mean ± SEM of at least three independent experiments. D, Quantification of vessel Collagen coverage as the percentage of vessels displaying full, intermediate or absent coating by Collagen fibers. E, Transmission electron microscopy images of PyMT-VE-Cadh^Snail1CT and PyMT-VE-Cadh^Snail1KO tumor vessels. Arrowheads label vesiculo-vacuolar organelles (VVO) in the endothelial cells of the capillaries. Scale bars: 2 μm (PyMT-VE-Cadh^Snail1CT mice); 5 μm (PyMT-VE-Cadh^Snail1KO mice); 1 μm (magnifications of both). F, Quantification of vessel lumen integrity, number of VVO and presence of filopodia in MMTV-PyMT carcinoma vessels. More than fifty vessels of different tumors per group were analyzed. Data represents the percentage of the vessels at the defined categories. * P < 0.05; *** P < 0.001.

Figure 7

Reduced angiogenesis is associated to papillary carcinoma morphology in a pre-clinical murine model and human breast tumor samples. A-C, Tumor size (A), tumor onset (B) and carcinoma morphology (C) of MMTV-PyMT mice treated with Bevacizumab. D, Tumor stage in a cohort of patients with non-specific type (NST) or papillary breast carcinomas. Data are represented as percentage of cases according to breast cancer TNM classification. E-I, Analysis of CD31 and Snail1 expression in breast tumors; representative images of the immunochemical staining of an NST and a papillary carcinoma are presented in E. Bars in E corresponds to 100 μm. The quantification of CD31+ areas per tumor case (F), the percentage of tumor vessels expressing Snail1 per tumor case (G) and the percentage of Snail1+ stromal areas in the vicinity of vessels per tumor cases (H) was represented in normal breast tissue adjacent to tumors, NST carcinomas and papillary carcinomas. I, Percentage of human tumor blood vessels expressing or not Snail1 with Snail1+ stromal cells in the vicinity. J-K, Regression analysis of CD31 protein versus Snail1 protein expression (J) and PECAM1 (CD31) mRNA versus VIM mRNA expression (K) in human breast cancer tumor samples from TCGA consortium data. Regression lines between the indicated parameters and corresponding r2 goodness-of-fit values are shown for each graph, as well the Pearson and Spearman correlation coefficients with their corresponding P value. L, Kaplan-Meier survival curves of patients with breast tumors mimicking those obtained in PyMT-VE-Cadh^Snail1CT and PyMT-VE-Cadh^Snail1KO. Log rank test P value is shown in the graph. * P < 0.05; ** P < 0.01; *** P < 0.001.