Prolonged mammosphere culture of MCF-7 cells induces an EMT and repression of the estrogen receptor by microRNAs

Abstract

Mammosphere culture has been used widely for the enrichment of mammary epithelial stem cells and breast cancer stem cells (CSCs). Epithelial-to-mesenchymal transition (EMT) also induces stem cell features in normal and transformed mammary cells. We examined whether mammosphere culture conditions per se induced EMT in the epithelial MCF-7 breast cancer cell line. MCF-7 cells were cultured as mammospheres for 5 weeks, with dispersal and reseeding at the end of each week. This mammosphere culture induced a complete EMT by 3 weeks. Return of the cells to standard adherent culture conditions in serum-supplemented media generated a cell population (called MCF-7(M) cells), which displays a stable mesenchymal and CSC-like CD(44+)/CD(24-/low) phenotype. EMT was accompanied by a stable, marked increase in EMT-associated transcription factors and mesenchymal markers, and a decrease in epithelial markers and estrogen receptor α (ERα). MCF-7(M) cells showed increased motility, proliferation and chemoresistance in vitro, and produced larger tumors in immunodeficient mice with or without estrogen supplementation. MicroRNA analysis showed suppression of miR-200c, miR-203, and miR-205; and increases in miR-222 and miR-221. Antisense hairpin RNA inhibitor targeting miR-221 resulted in re-expression of ERα in MCF-7(M) cells. This study provides the first example of mammosphere culture conditions inducing EMT and of EMT regulating microRNAs that target ERα.

Configurable 2D and 3D spheroid tissue cultures on bioengineered surfaces with acquisition of epithelial–mesenchymal transition characteristics

“…27,28,30,32 This correlation is supported by our data, which shows that 3D on-chip culture conditions induced EMT from spheroids cultured using the epithelial SKOV3 ovarian cancer cell line. Similar evidence with the MCF7 breast cancer cell line 27 and the human oral squamous cancer cell line 30 has also been reported.…”

“…25 The measurements of the relative mRNAs from 3D on-chip and on-dish cultures (see Supplementary Figure S5) provided further indication that these cells derived from the chip exist in a mesenchymal-like cell state, which more closely resembles those that have undergone EMT than those from 3D on-dish culture. Moreover, Figure 5d presents results of expressions of CD326/EpCAM (broadly expressed on cells of epithelial origin and on epithelium-derived tumor cells 26 ) and vimentin (often used as a marker of mesenchymally derived cells 27 ). For 3D on-chip culture, the reduction in epithelial cell adhesion is evident by a marked decrease in CD326 expression and a gain in vimentin expression, which are two classic features of EMT, compared with that obtained from 2D on-dish culture.…”

“…EMT has a crucial role during cancer invasion and metastasis, and it purportedly generates cells with properties of stem cells; 25,[27][28][29] the present microfluidic platform appears to be unique in this regard. Several studies have demonstrated that EMT may be induced in cellular spheres cultured in serum-free medium supplemented with adequate mitogens, such as the basic fibroblast growth factor and epidermal growth factor, using a low-attachment culture dish; however, this approach appears to be costly and ineffective.…”

“…Several studies have demonstrated that EMT may be induced in cellular spheres cultured in serum-free medium supplemented with adequate mitogens, such as the basic fibroblast growth factor and epidermal growth factor, using a low-attachment culture dish; however, this approach appears to be costly and ineffective. 27,28,30 These drawbacks are partially overcome in this study, in which the 3D microscale on-chip environment selects clones that are EMT transmitted. Moreover, the present approach does not require growth factors and possesses the potential to handle rare cells, which is especially important for clinical samples and circulating tumor cells.…”

MicroRNA expression profiling identifies decreased expression of miR-205 in inflammatory breast cancer

“…60 Downregulation of miR-205 is also seen in epithelial-mesenchymal transition induced by mammosphere culture of breast cancer cell line MCF-7. 78 Interestingly, expression of miR-205 is downregulated in breast cancer cell lines that belong to claudin-low subtype, which is known to be associated with high epithelial-mesenchymal transition, 79 as well as in triple-negative breast cancer human samples. 74,80 Of note, the commonly used triple-negative inflammatory breast cancer cell line SUM149, unlike the claudin-low subtype of breast cancer cell lines, does not contain a detectable mesenchymal subpopulation 81,82 and demonstrates a much higher expression level of miR-205 than the triple-negative breast cancer cell lines with a mesenchymal phenotype.…”