Malignant Pleural Effusion and ascites Induce Epithelial-Mesenchymal Transition and Cancer Stem-like Cell Properties via the Vascular Endothelial Growth Factor (VEGF)/Phosphatidylinositol 3-Kinase (PI3K)/Akt/Mechanistic Target of Rapamycin (mTOR) Pathway

Abstract

Malignant pleural effusion (PE) and ascites, common clinical manifestations in advanced cancer patients, are associated with a poor prognosis. However, the biological characteristics of malignant PE and ascites are not clarified. Here we report that malignant PE and ascites can induce a frequent epithelial-mesenchymal transition program and endow tumor cells with stem cell properties with high efficiency, which promotes tumor growth, chemoresistance, and immune evasion. We determine that this epithelial-mesenchymal transition process is mainly dependent on VEGF, one initiator of the PI3K/Akt/mechanistic target of rapamycin (mTOR) pathway. From the clinical observation, we define a therapeutic option with VEGF antibody for malignant PE and ascites. Taken together, our findings clarify a novel biological characteristic of malignant PE and ascites in cancer progression and provide a promising and available strategy for cancer patients with recurrent/refractory malignant PE and ascites.

Keywords: VEGF; ascites; cancer biology; cancer stem cells; epithelial-mesenchymal transition (EMT); malignant pleural effusion; tumor therapy.

FIGURE 1.

Malignant pleural effusion and ascites induce EMT in vivo and in vitro. A, human breast tumor samples (left panel, n = 5) and lung tumor samples (right panel, n = 22) as well as their corresponding exfoliative cells in pleural effusion were immunostained for E-cadherin and vimentin and counterstained with hematoxylin. Scale bars = 20 μm. B–D, representative phase-contrast images of human lung tumor cells (A549, B), human ovarian tumor cells (SKOV3, C) and human breast cancer MCF-7 cells (D) cultured in malignant pleural effusion or ascites from patients with the indicated cancers. Scale bar = 50 μm. Ctrl, control. E, altered expression of E-cadherin and vimentin in MCF-7 cells, as revealed by Western blotting analysis. GAPDH served as the loading control. Pt, patient; BC, breast cancer; LC, lung cancer; GC, gastric cancer; ML, malignant lymphoma; CC, colorectal cancer; PMC, peritoneal mesothelioma.

FIGURE 2.

Malignant pleural effusion and ascites endow cancer cells with a cancer stem cell phenotype. A and B, A549 tumor cells were cultured in malignant pleural effusion and stained with anti-CD133 antibody by direct staining, analyzed, and quantified by flow cytometry. CD133-positive stem cells were quantified. Ctrl, control; Pt, patient; FSC, forward scatter. C and D, flow cytometry analysis of SKOV3 tumor cells cultured in malignant ascites for CD133-positive stem cells. CD133-positive stem cells were quantified. E and F, flow cytometry analysis of MCF-7 cells cultured in malignant pleural effusion and ascites and their parental cells for CD44 and CD24. CD44+/CD24− stem cells were quantified. G, non-cancer stem cells were cell sorted from MCF-7 cells and cultured in malignant pleural effusion and ascites. The CD44+/CD24− cancer stem cell population was analyzed by flow cytometry. H, non-CSCs were cultured in malignant pleural effusion and ascites. The level of vimentin was measured by Western blotting. GAPDH served as the loading control. *, p < 0.05; **, p < 0.01.

FIGURE 3.

Malignant pleural effusion and ascites promote tumor growth and therapy resistance. A, SKOV3 cells cultured in ascites were subcutaneously injected into athymic nude BALB/C mice (n = 3–5). Shown are representative pictures of tumors and quantification of tumor weight. Ctrl, control; Pt, patient. B, representative images of ki-67 stains showing that cancer cells in the malignant ascites group had a higher proliferation index. Scale bar = 50 μm. C, immunofluorescent images showing that tumors in the malignant PE and ascites group had more tumor vasculature. Scale bar = 50 μm. D, MCF-7 and malignant PE and ascites-treated MCF-7 cells were exposed to the chemotherapeutic agents cisplatin and paclitaxel. Cell viability was determined by flow cytometry using Annexin V/PI staining. Shown is quantification of cell viability. E, the level of the multidrug resistance genes ABCB1 and ABCG2 was analyzed with an RT-PCR assay. F, IL-2/IL-15 activated NK cell cytotoxicity against MCF-7 cells cultured in malignant PE and ascites at a range of E:T ratios compared with their parental cells. G, flow cytometry analysis of MHC-I expression on the surface of MCF-7 tumor cells. Data are presented as mean ± S.D. *, p < 0.05; **, p < 0.01.

FIGURE 4.

Malignant PE and ascites functionally activate the PI3K/Akt/mTOR pathway. A, MCF-7 cells were treated with malignant PE and ascites at the indicated times. Cell lysates were analyzed by Western blotting with antibodies against p-Akt and p-P70S6K. GAPDH served as the loading control. B, Western blotting for p-P70S6K, p-Smad1, p-NFκB p65, Notch, Hes-1, p-β-catenin, p-GSK3β, p-MAPK p44/p46, and GAPDH after stimulation with malignant PE and ascites for 30 min. GAPDH served as the loading control (Ctrl). Pt, patient. C and D, MCF-7 cells were treated with malignant PE and ascites for 30 min. Shown is quantitative PCR analysis of hedgehog (C) and nodal/activin (D) pathway-associated genes. Data were normalized to human GAPDH expression and are presented as -fold change relative to parental MCF-7 cells. E, representative images of the morphology of MCF-7 cells after treatment with malignant PE/ascites as well as antagonists of pan-PI3K (LY294002) and mTOR (rapamycin) inhibitors. Scale bar = 50 μm. F, Western blotting analysis of vimentin expression of MCF-7 cells after treatment with malignant PE/ascites as well as the indicated antagonists. G, flow cytometry analysis and quantification of CD44+/CD24− cancer stem cells in MCF-7 cells after treatment with malignant PE/ascites as well as the indicated antagonists. *, p < 0.05.

FIGURE 5.

VEGF correlates with cancer stem cell increase induced by malignant PE and ascites. The -fold increase in cancer stem cells was plotted against the concentration of VEGF, IGF-I, TGF-β, β2-microglobumin, IL-6, IL-8, TNF-α, osteopontin, angiopoietin-2, endoglin, HGF and HB-EGF. Linear regressions were traced according to the distribution of points.

FIGURE 6.

Targeting VEGF effectively antagonizes protumor effects of malignant PE and ascites. A, Western blotting assessed the expression of VEGFRs on the MCF-7, A549, and SKOV3 cancer cell lines. HUVECs were the positive control. B, the expression of VEGFRs was evaluated in primary breast cancer samples and their corresponding exfoliative cells in pleural effusion (n = 5). Scale bar = 50 μm. C, flow cytometry analysis of CD44+/CD24− cancer stem cells in malignant PE/ascites-cultured MCF-7 cells in the presence of neutralizing VEGF antibodies. *, p < 0.05. Pt, patient. D–F, MCF-7 tumor cells were cultured in ascites before and after bevacizumab (Beva) therapy, respectively, from a 50-year-old male with a history of metastatic colorectal cancer. Cell morphology was photographed under a microscope (D). Scale bar = 50 μm. CD44+/CD24− cancer stem cells (E) and vimentin expression (F) in D was assessed by flow cytometry and Western blotting, respectively. G and H, a 57-year-old lung cancer patient with refractory malignant PE received intravenous bevacizumab therapy. The pleural effusion volume in G was tracked. Shown are representative images from computed tomography scans of the chest documenting responses to the bevacizumab therapies in H. Images revealed the massive right-sided PE on admission. PE was obviously reduced after treatment with two doses of bevacizumab.