Podoplanin increases the migration of human fibroblasts and affects the endothelial cell network formation: A possible role for cancer-associated fibroblasts in breast cancer progression

<div><p>In our previous studies we showed that in breast cancer podoplanin-positive cancer-associated fibroblasts correlated positively with tumor size, grade of malignancy, lymph node metastasis, lymphovascular invasion and poor patients’ outcome. Therefore, the present study was undertaken to assess if podoplanin expressed by fibroblasts can affect malignancy-associated properties of breast cancer cells. Human fibroblastic cell lines (MSU1.1 and Hs 578Bst) overexpressing podoplanin and control fibroblasts were co-cultured with breast cancer MDA-MB-231 and MCF7 cells and the impact of podoplanin expressed by fibroblasts on migration and invasiveness of breast cancer cells were studied in vitro. Migratory and invasive properties of breast cancer cells were not affected by the presence of podoplanin on the surface of fibroblasts. However, ectopic expression of podoplanin highly increases the migration of MSU1.1 and Hs 578Bst fibroblasts. The present study also revealed for the first time, that podoplanin expression affects the formation of pseudo tubes by endothelial cells. When human HSkMEC cells were co-cultured with podoplanin-rich fibroblasts the endothelial cell capillary-like network was characterized by significantly lower numbers of nodes and meshes than in co-cultures of endothelial cells with podoplanin-negative fibroblasts. The question remains as to how our experimental data can be correlated with previous clinical data showing an association between the presence of podoplanin-positive cancer-associated fibroblasts and progression of breast cancer. Therefore, we propose that expression of podoplanin by fibroblasts facilitates their movement into the tumor stroma, which creates a favorable microenvironment for tumor progression by increasing the number of cancer-associated fibroblasts, which produce numerous factors affecting proliferation, survival and invasion of cancer cells. In accordance with this, the present study revealed for the first time, that such podoplanin-mediated effects can affect tube formation by endothelial cells and participate in their pathological properties in the tumor context. Our experimental data were supported by clinical studies. First, when IDC and DCIS were analyzed by immunohistochemistry according to the presence of podoplanin-expressing cells, the numbers of cancer-associated fibroblasts with high expression of this glycoprotein were significantly higher in IDC than in DCIS cases. Second, using immunofluorescence, the co-localization of PDPN-positive CAFs with blood vessels stained with antibody directed against CD34 was observed in tumor stroma of IDC samples.</p></div

Migratory capability of human breast cancer cell lines co-cultured with human fibroblasts expressing different amounts of podoplanin.

<p><b>(A)</b> MSU1.1 PDPN fibroblasts overexpressing podoplanin and podoplanin-negative control MSU1.1 NC cells co-cultured with fluorescently labeled MDA-MB-231 breast cancer cells, <b>(B)</b> MSU1.1 PDPN fibroblasts overexpressing podoplanin and podoplanin-negative control MSU1.1 NC cells co-cultured with fluorescently labeled MCF7 breast cancer cells. The migratory properties of breast cancer cells were studied using transwell migration assay. Data are present as mean ±SD.</p

Effect of podoplanin expressed in fibroblastic MSU1.1 cells on the pseudo tub formation properties of endothelial HSkMEC cells in co-culture.

<p><b>(A)</b> Pseudo tube formation Matrigel^TM assay of <b>(a)</b> fluorescently labeled (DiD) endothelial HSkMEC cells, <b>(b)</b>fluorescently labeled (DiO) MSU1.1 PDPN fibroblasts overexpressing podoplanin,<b>(c)</b> fluorescently labeled (DiD) endothelial HSkMEC cells co-cultured with fluorescently labeled (DiO) control podoplanin-negative MSU1.1 NC fibroblasts and <b>(d)</b> fluorescently labeled (DiD) endothelial HSkMEC cells co-cultured with fluorescently labeled (DiO) MSU1.1 PDPN fibroblasts overexpressing podoplanin. Images were obtained in the red channel for DiD labelling of HSkMEC cells, in the green channel for the DiO labelling of MSU1.1 NC and MSU1.1 PDPN and the merge for the co-localization. Images were analyzed after 12 h of assay duration. <b>(B)</b> Number of nodes and <b>(C)</b> number of meshes formed by HSkMEC endothelial cells co-cultured with MSU1.1 PDPN fibroblasts overexpressing podoplanin and podoplanin-negative control MSU1.1 NC fibroblasts. <b>(D)</b> Co-localization of MSU1.1 PDPN and MSU1.1 NC fibroblastic cells with endothelial HSkMEC cells. The number of nodes, meshes and percentage of fibroblasts co-localized with endothelial cells were estimated using ImageJ software.</p

Immunohistochemical staining of podoplanin expressing CAFs in DCIS and IDC.

<p><b>(A)</b> The lack of podoplanin expression (D2-40) in CAFs of ductal carcinoma in situ (DCIS). In the center numerous lymphatic vessels D2-40+ are seen. <b>(B)</b> Strong podoplanin expression in stromal CAFs of invasive ductal carcinoma (IDC). <b>(C)</b> The intensities of podoplanin expression in stromal CAFs of DCIS (<i>n</i> = 27) and IDC (<i>n</i> = 95).****<i>p</i><0.0001 for podoplanin-expressing IDC as compared with DCIS (Mann-Whitney <i>U</i>-test). Reaction intensities with murine monoclonal anti-human D2-40 antibody were calculated on the basis of the semi-quantitative IRS scale of Remmele and Stegner [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184970#pone.0184970.ref033" target="_blank">33</a>] and are presented as means.</p

Ezrin and ROCK affects the migratory properties of human MSU1.1 fibroblasts overexpressing podoplanin.

<p><b>(A)</b> Expression of ezrin in podoplanin-rich native MSU1.1 fibroblasts (MSU1.1 PDPN), podoplanin-rich MSU1.1 fibroblasts transfected with scrambled siRNA (MSU1.1 PDPN/siC), podoplanin-rich MSU1.1 fibroblasts transfected with siRNA directed against ezrin mRNA (MSU1.1 PDPN/siE), podoplanin-negative native MSU1.1 fibroblasts (MSU1.1 NC), podoplanin-negative MSU1.1 fibroblasts transfected with scrambled siRNA (MSU1.1 NC/siC), and podoplanin-negative MSU1.1 fibroblasts tran/siE, and MSU1.1 PDPN fibroblasts, <b>(C)</b> Migratory properties of podoplanin-negative MSU1.1 NC/siC, MSU1.1 NC/siE, and MSU1.1 NC fibroblasts. Transwell migration assay. Data are presented as mean ±SD, <b>(D)</b> Expression of ezrin and its phosphorylated form (Thr567) in podoplanin-rich MSU1.1 PDPN and Hs578 PDPN fibroblasts in comparison to controlMSU NC and Hs578 NC cells. Ezrin and phosphorylated ezrin (pThr5567) were detected by Western blotting using specific rabbit polyclonal antibodies. β-Actin served as an internal control. (<b>E</b>) Western blotting analysis of active RhoA-GTPases relative to the total expression levels of RhoA in podoplanin-rich MSU1.1 PDPN and Hs578 PDPN fibroblasts in comparison to control MSU1.1 NC and Hs578 NC cells. The levels of β-actin were determined as a control for protein loading. (<b>F</b>) Expression of phosphorylated ezrin (Thr567) in podoplanin-rich MSU1.1 PDPN and Hs578 PDPN fibroblasts treated with specific Y-27632 inhibitor of ROCK. Phosphorylated ezrin (pThr5567) was detected by Western blotting using specific rabbit polyclonal antibodies. (<b>G</b>) Migratory properties of podoplanin-rich MSU1.1 PDPN fibroblasts grown in the presence of Y-27632 inhibitor. <b>(H)</b> Migratory properties of podoplanin-rich Hs578 PDPN fibroblasts grown in the presence of Y-27632 inhibitor. Transwell migration assay. Data are presented as mean ±SD.</p

Migratory capability of human fibroblasts expressing different amounts of podoplanin co-cultured with human breast cancer cell lines or cultured alone.

<p>Transwell migration assay with fluorescently labeled MSU1.1 PDPN fibroblasts overexpressing podoplanin and podoplanin-negative control MSU1.1 NC fibroblasts co-cultured with MDA-MB-231 breast cancer cells <b>(A)</b> or MCF7 breast cancer cells <b>(B)</b>; transwell migration assay with MSU1.1 PDPN fibroblasts overexpressing podoplanin and podoplanin-negative control MSU1.1 NC fibroblasts <b>(C)</b>, transwell migration assay with Hs578 PDPN fibroblasts overexpressing podoplanin and podoplanin-negative control Hs578 NC fibroblasts <b>(D)</b>. Data are presented as mean ±SD. Bonferroni multiple comparison test, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, ****<i>p</i><0.0001. Wound-healing assay with MSU1.1 PDPN fibroblasts overexpressing podoplanin and podoplanin-negative control MSU1.1 NC fibroblasts. Cell–free gap (500 μm ± 50 μm) was generated in the monolayers of fibroblasts, photographs were taken each hour up to 48 hours after the injury, always from the same place (3 pictures per well) <b>(E)</b>, and analyzed using ImageJ software (National Institute of Health) <b>(F)</b>. Cell-free area was calculated. Data were analyzed using the Bonferroni multiple comparison test, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, ****<i>p</i><0.0001.</p

Podoplanin does not affect the invasiveness of breast cancer cell lines.

<p><b>(A)</b> Invasiveness of breast cancer cell lines co-cultured with fibroblastic cells expressing different amounts of podoplanin. Invasiveness of fluorescently labeled MDA-MB-231 and MCF7 breast cancers analyzed by Matrigel^TM invasion assay in co-cultures with control podoplanin-negative MSU1.1 NC cells and MSU1.1 PDPN cells overexpressing podoplanin. Data are presented as mean ±SD. <b>(B)</b> Expression of MMPs and TIMP1 in fibroblastic cells expressing different amounts of podoplanin. Relative expression of MMPs and TIMP1 gene was normalized against expression of <i>ACTB</i> gene and MSU1.1 NC cells were assigned as a calibrator sample. Results are expressed as mean ± SD.</p

Immunofluorescent co-localization of PDPN-positive CAFs and CD34-positive blood vessels in IDC stroma.

<p><b>(A)</b> IDC with high expression of podoplanin, <b>(B)</b> IDC with low expression of podoplanin. Anti-podoplanin antibody was stained with secondary donkey anti-mouse antibodies conjugated with Alexa-Fluor 594 (red), and anti-CD34 antibody was detected with donkey anti-rabbit Alexa-Fluor 488 conjugated secondary antibody (green).</p

Expression of proangiogenic factors mRNA in fibroblastic cell lines: MSU1.1 PDPN overexpressing podoplanin and control MSU1.1 NC transduced with vector alone.

<p><b>(A)</b> VEGF-A, <b>(B)</b> FGF-1, <b>(C)</b> ANGPT-1, <b>(D)</b> ANGPT-2, <b>(E)</b> VE-cadherin. Real-time RQ-PCR was used to analyze proangiogenic factors mRNA. Proangiogenic factors levels were normalized against <i>ACTB</i> gene expression and cell line MSU1.1 NC was assigned as a calibrator sample. Each bar represents mean ± SEM, * <i>p</i> < 0.05, ** <i>p</i> < 0.01, **** <i>p</i>< 0.0001.</p

Immunofluorescent analysis of podoplanin-regulated ezrin and Rho-GDI presence in plasma membrane of MSU1.1 and HS578T cells.

<p>Podoplanin-negative and podoplanin-overexpressing MSU1.1 and HS578T cells were immunofluorescently labelled to visualize distribution of ezrin and Rho-GDI proteins. Based on plasma membrane and cytoplasm co-staining with WGA-Alexa Fluor 647 and CTV, respectively, ezrin <b>(A)</b> and Rho-GDI <b>(B)</b> content in plasma membrane was quantified with confocal microscopy and image analysis using ImageJ software. Data are presented as mean fluorescence intensities in identified plasma membrane regions (single dots). Data were analyzed using the Mann–Whitney test, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, ****<i>p</i><0.0001.</p