Up-modulation of PLC-β2 reduces the number and malignancy of triple-negative breast tumor cells with a CD133+/EpCAM+ phenotype: a promising target for preventing progression of TNBC

Abstract

Background: The malignant potential of triple negative breast cancer (TNBC) is also dependent on a sub-population of cells with a stem-like phenotype. Among the cancer stem cell markers, CD133 and EpCAM strongly correlate with breast tumor aggressiveness, suggesting that simultaneous targeting of the two surface antigens may be beneficial in treatment of TNBC. Since in TNBC-derived cells we demonstrated that PLC-β2 induces the conversion of CD133^high to CD133^low cells, here we explored its possible role in down-modulating the expression of both CD133 and EpCAM and, ultimately, in reducing the number of TNBC cells with a stem-like phenotype.

Methods: A magnetic step-by-step cell isolation with antibodies directed against CD133 and/or EpCAM was performed on the TNBC-derived MDA-MB-231 cell line. In the same cell model, PLC-β2 was over-expressed or down-modulated and cell proliferation and invasion capability were evaluated by Real-time cell assays. The surface expression of CD133, EpCAM and CD44 in the different experimental conditions were measured by multi-color flow cytometry immunophenotyping.

Results: A CD133⁺/EpCAM⁺ sub-population with high proliferation rate and invasion capability is present in the MDA-MB-231 cell line. Over-expression of PLC-β2 in CD133⁺/EpCAM⁺ cells reduced the surface expression of both CD133 and EpCAM, as well as proliferation and invasion capability of this cellular subset. On the other hand, the up-modulation of PLC-β2 in the whole MDA-MB-231 cell population reduced the number of cells with a CD44⁺/CD133⁺/EpCAM⁺ stem-like phenotype.

Conclusions: Since selective targeting of the cells with the highest aggressive potential may have a great clinical importance for TNBC, the up-modulation of PLC-β2, reducing the number of cells with a stem-like phenotype, may be a promising goal for novel therapies aimed to prevent the progression of aggressive breast tumors.

Keywords: Breast cancer stem cell (BCSC); CD133; EpCAM; Invasiveness; PLC-β2; Proliferation; Triple-negative breast cancer (TNBC).

Fig. 1

Expression of CD133 and EpCAM in MDA-MB-231 cells. In a representative cytofluorimetrical evaluation of CD133 and EpCAM surface levels in MDA-MB-231 cells after labelling with a PE-conjugated anti-CD133 antibody or with a FITC-conjugated anti-EpCAM antibody. The expression of each antigen is shown, on the left, on a frequency distribution histogram (count vs. PE or FITC signal) in which the mean fluorescence intensity (MFI) of the entire population is reported. The red filled histograms represent positive staining for CD133 or EpCAM and the open histograms, outlined by gray lines, show staining with isotype matched antibodies. On the right, surface expression of each antigen is shown on a biparametric dot plot and the percentage and MFI of positive cells are indicated. In b representative surface expression of both CD133 and EpCAM in MDA-MB-231 cells after double labelling with a PE-conjugated anti-CD133 and with a FITC-conjugated anti-EpCAM antibodies is shown on a biparametric dot plot and the percentage of cells in all the derived quadrants is indicated

Fig. 2

CD133 and EpCAM surface levels in MDA-MB-231 sub-populations. MDA-MB-231 cells were subjected to positive immunomagnetic separation after labeling with MicroBeads-conjugated anti-CD133 antibody followed by the positive selection through column of cells labeled with MicroBeads-conjugated anti-EpCAM antibody. Surface levels of CD133 and EpCAM were evaluated in all sub-populations after double labelling with a PE-conjugated anti-CD133 and with a FITC-conjugated anti-EpCAM antibody. The expression of each antigen is shown on a frequency distribution histogram (count vs. PE or FITC signal) in which the MFI of the entire population is reported. The red filled histograms represent positive staining for CD133 or EpCAM and the open histograms, outlined by gray lines, show staining with matched isotype antibodies. The dot plot analysis was used to assess the enrichment in CD133⁻/EpCAM⁻, CD133⁻/EpCAM⁺, CD133⁺/EpCAM⁻ and CD133⁺/EpCAM⁺ cells in all subsets and the percentage of the main cell phenotype in each enriched sub-population is indicated. The data are indicative of three separate experiments

Fig. 3

CD133 and EpCAM related proliferation and invasion capability of MDA-MB-231 sub-populations. Cells derived from immunomagnetic separations were immediately grown in culture medium for 96 h and daily counted by hemocytometer after Trypan Blue staining (a). After 24 h from separation, the cellular subsets were subjected to dynamic monitoring of proliferation (b) and invasion through Matrigel (d) using the xCELLigence RTCA system. Cell Index (CI) is reported and error bars indicate ±SD. The correspondent Slope analysis, that describes the steepness, incline, gradient, and changing rate of the CI curves over time, is shown in c and e respectively. In f a representative image of ECM invading cells in a Boyden Chamber assay, whose number is reported in g. The data are the mean of three separate experiments ± SD. The asterisks indicate statistically significant differences (P < 0.05)

Fig. 4

PLC-β2-related features in sub-populations enriched in CD133⁺ and/or EpCAM⁺ cells. In a representative fluorescence microscopy images of MDA-MB-231 sub-populations enriched in CD133⁻/EpCAM⁻, CD133⁻/EpCAM⁺, CD133⁺/EpCAM⁻ and CD133⁺/EpCAM⁺ cells subjected to immunocytochemical analysis with the anti-PLC-β2 antibody. The fluorescence intensity of PLC-β2 staining was calculated in digitized images by the ImageJ software and reported in b as arbitrary units. In c immunocytochemical analysis of the indicated sub-populations after simultaneous staining with the anti-PLC-β2 antibody (green fluorescence) and with the anti-CD133 or anti- EpCAM antibody (red fluorescence). In d the enriched CD133⁺/EpCAM⁺ sub-population was transfected with siRNAs specific for PLC-β2 (PLC-β2 siRNAs) or with a construct expressing the human PLC-β2 (Over PLC-β2) and subjected to simultaneous flow cytometry analysis of CD133 and EpCAM surface expression. Non-silencing scramble siRNAs or an empty vector were used as controls (Ctrl). In each experimental condition, fold change is compared with Ctrl, taken as 1. Proliferation and invasiveness of cells in the same experimental conditions were measured by the xCELLigence system (e). All the data are the mean of three separate experiments performed in triplicate ± SD. *P < 0.05. Bar = 20 μm

Fig. 5

Relationship between PLC-β2 and MDA-MB-231 sub-populations with a stem-like phenotype. In a MDA-MB-231 cells transfected with siRNAs specific for PLC-β2 (PLC-β2 siRNAs) or with a construct expressing human PLC-β2 (Over PLC-β2) were subjected to a bi-parametric flow cytometry by direct staining with the anti-CD133 and anti-EpCAM fluorescent antibodies to evaluate the number of CD133⁻/EpCAM⁻, CD133⁻/EpCAM⁺, CD133⁺/EpCAM⁻ and CD133⁺/EpCAM⁺ cells. Non-silencing scramble siRNAs or an empty vector were used as control (Ctrl). The data, representative of three separate experiments, are presented on pie charts and the percentage of each cellular subset is reported. In b MDA-MB-231 cells in which PLC-β2 was forcedly silenced (PLC-β2 siRNAs) or over-expressed (Over PLC-β2) were simultaneously stained with APC-conjugated anti-CD44, FITC-conjugated anti-EpCAM and PE-conjugated anti-CD133 and subjected to flow cytometry evaluation of CD44, EpCAM and CD133 surface levels. Data are the mean of three separate experiments performed in triplicate ± SD. *P < 0.05