Generation of MCF-7 cells with aggressive metastatic potential in vitro and in vivo

Abstract

Epithelial-mesenchymal transition (EMT) is a cellular development program characterized by loss of cell adhesion and increased cell mobility. It is essential for numerous processes including metastasis. In this study we have generated "aggressive" MCF-7 breast cancer cells (MCF-7-EMT), which show significantly increased invasion in contrast to wild type MCF-7 (MCF-7 WT) cells. In addition, we have analyzed, whether these cell lines differ in their metastatic behavior in vivo and in expression of invasion and/or EMT-relevant genes. Invasive behavior of different human breast cancer cell lines was tested. "Aggressive" MCF-7 cells (MCF-7-EMT) were generated using coculture and mammosphere culture techniques. To analyze whether or not MCF-7-EMT cells in contrast to MCF-7 WT cells form metastases in vivo, we assessed metastases in a nude mouse model. mRNA expression profiles of MCF-7 WT cells and MCF-7-EMT cells were compared using the Affymetrix micro array technique. Expression of selected genes was validated using real-time PCR. In addition, protein expression of epithelial marker E-cadherin (CDH1) and mesenchymal markers N-cadherin (CDH2), Vimentin (VIM), and TWIST was compared. The breast cancer cell lines showed different invasive behavior from hardly any invasion to a stronger cell movement. Coculture with osteoblast-like MG63 cells led to significantly increased cell invasion rates. The highest increase was shown using MCF-7 WT cells. Generated MCF-7-EMT cells showed significantly increased invasion as compared to MCF-7 WT cells. In 8 of 10 mice bearing orthotopically growing MCF-7-EMT tumors, we could detect metastases in liver and lung. In mice bearing MCF-7 WT tumors (n = 10), no metastases were found. MCF-7 WT cells and MCF-7-EMT cells were different in expression of 325 genes. Forty-four of the most regulated 50 invasion and/or EMT-related genes were upregulated and 6 genes were downregulated in MCF-7-EMT cells. Protein expression of mesenchymal markers CDH2, VIM, and TWIST was clearly increased in MCF-7-EMT cells. Protein expression of epithelial marker CDH1 was clearly decreased. With the breast cancer cell lines, MCF-7-EMT and MCF-7 WT cells, we have an excellent model of cells for further studies of EMT and invasion in vitro and in vivo.

Analysis of the thrombotic and fibrinolytic activities of tumor cell–derived extracellular vesicles

“…33,34 Exosomes from MCF7 cells or MCF7 cells treated with PMA to increase their malignant potential failed to display thrombotic activity with fibrinogen or human platelet-poor plasma (supplemental Figure 1B). However, thrombotic activity was detected with MVs from MCF7 cells with fibrinogen or human platelet-poor plasma (supplemental Figure 1A), whereas MVs from PMA-treated MCF7 cells displayed thrombotic activity only in the presence of fibrinogen (supplemental Figure 1A).…”

New Insights into the Role of Podoplanin in Epithelial–Mesenchymal Transition

“…Rif is distantly related to other GTPases (32-49% identity), but retains the common switch 1 and 2 regions that allows the function of small GTPases. The mechanism of Rif induced filopodia has been studied by Goh et al (2011), who showed a direct interaction of Rif with mDia1 without involving the Cdc42 effectors N-WASP In a recent study (Ziegler et al, 2014), MCF7 cells with increased invasiveness ("aggressive" MCF7) were selected, but these cells did not express podoplanin. In another study with MCF7 cells, Yang and Kim (2014) showed that inhibition of ROCK activity leads to MCF7 cells activation, with loss of epithelial characteristics (loss of cell junctions, loss of E-cadherin and β-catenin in the membrane and increased proliferation, migration and invasion).…”

Inhibition of SDF-1/CXCR4-induced epithelial–mesenchymal transition by kisspeptin-10

“…For better clearness invasion is shown as % of control (=100 %). Actual numbers of invaded cells of different human breast cancer cell lines were published by Ziegler et al [20].…”

“…Mesenchymal transformed MCF-7 cells (MCF-7-EMT) were generated as described by Ziegler et al [20]. Briefly, single-cell suspensions of wild type MCF-7 cells (MCF-7 WT) were suspended at a density of 40,000 cells/mL in DMEM/F-12 containing 5 lg/mL insulin (Sanofi-Aventis, Frankfurt, Germany), 0.5 mg/mL hydrocortisone, 2 % B27 supplement (Invitrogen, Darmstadt, Germany), 20 ng/mL epidermal growth factor (EGF; Sigma, Deisenhofen, Germany), 20 ng/ml fibroblast growth factor-2 (FGF-2; Sigma), and 1 % penicillin/streptomycin (PAA Laboratories) before incubation on ultralow adherence six-well plates (2.5 mL/plate; Corning, Lowell, MA, USA).…”

“…In addition, we have generated mesenchymal transformed MCF-7 breast cancer cells (MCF-7-EMT), which show significantly increased invasion in contrast to wild type MCF-7 (MCF-7 WT) [20]. Using these models we have analyzed whether SDF-1 is responsible for human breast cancer cell invasion and EMT.…”