Proteomic comparison of mcf-7 tumoursphere and monolayer cultures

Abstract

Breast cancer is a heterogeneous disease, composed of tumour cells with differing gene expressions and phenotypes. Very few antigens have been identified and a better understanding of tumour initiating-cells as targets for therapy is critically needed. Recently, a rare subpopulation of cells within tumours has been described with the ability to: (i) initiate and sustain tumour growth; (ii) resist traditional therapies and allow for secondary tumour dissemination; and (iii) display some of the characteristics of stem cells such as self-renewal. These cells are termed tumour-initiating cells or cancer stem cells, or alternatively, in the case of breast cancer, breast cancer stem cells. Previous studies have demonstrated that breast cancer stem cells can be enriched for in "tumoursphere" culture. Proteomics represents a novel way to investigate protein expression between cells. We hypothesise that characterisation of the proteome of the breast cancer line MCF-7 tumourspheres compared to adherent/differentiated cells identifies proteins of novel interest for further isolating or targeting breast cancer stem cells. We present evidence that: (i) the proteome of adherent cells is different to the proteome of cells grown in sphere medium from either early passage (passage 2) or late passage (passage 5) spheres; (ii) that spheres are enriched in expression of a variety of tumour-relevant proteins (including MUC1 and Galectin-3); and (iii) that targeting of one of these identified proteins (galectin-3) using an inhibitor (N-acetyllactosamine) decreases sphere formation/self-renewal of MCF-7 cancer stem cells in vitro and tumourigenicity in vivo. Hence, proteomic analysis of tumourspheres may find use in identifying novel targets for future therapy. The therapeutic targeting of breast cancer stem cells, a highly clinically relevant sub-population of tumour cells, has the potential to eliminate residual disease and may become an important component of a multi-modality treatment of cancer.

Figure 1. MCF-7 tumourspheres exhibit self-renewal efficiency and long term culture potential.

Sphere forming efficiency progressively increases over serial passages (P). (a) Between P1 and P2 (t-test p = 0.0013, **), and between P6 and P8 (t-test p<0.0001, ***). (b) Sphere formation was dose dependent. Increasing the number of single cells plated in the sphere assay increased the total number of spheres formed. Sphere sizes range between 50 and 200 μm for day 5 spheres. (c) Growth curve showing total number of cells generated over 8 passages for matched adherent cells (open circles) compared to sphere-cultured cells (closed circles) demonstrating long-term culture capacity for spheres. Error bars represent standard error of the mean (SEM).

Figure 2. MUC1 expression on MCF-7 grown in matched adherent and sphere-promoting conditions.

(a) Proteomics results (y-axis is untransformed label free quantitation (LFQ) values) indicate that the intensity of MUC1 expression is greater on sphere-cultured passage (P) 2 and P5 MCF-7 cells compared to adherent cells (line over columns), ANOVA test, *** p<0.001. (b–d) Surface expression of MUC1 was analyzed by flow cytometry on MCF-7 cells grown as matched adherent (shaded-grey) and sphere (red-line) cultures. The red shaded areas and grey lines represent the isotype controls for sphere and adherent cultures respectively. Sphere cultures were analyzed after 1 (b), 2 (c) and 3 (d) 6-day passages. Live-gated cells were analyzed for the frequency of MUC1-positivity (e) and Δ-median fluorescence intensity (FI) (f). Fold increase values for sphere cultures above the corresponding adherent culture are shown in bars representing the average and SEM of 3 biological repeats. Each biological repeat is the average of three technical repeats. Statistical significance of the change in MUC1 expression for each passage of spheres was calculated compared to adherent cells using a Student's t-test (*** p<0.001; ** p<0.01). Error bars represent SEM.

Figure 3. Galectin-3 expression on MCF-7 adherent cells and tumourspheres.

Galectin-3 (a) and Galectin-1 (b) proteomics results (y-axis is untransformed label free quantitation (LFQ) values) indicate increased expression within spheres compared to adherent cells, (line over columns), ANOVA test, *** p<0.001; ** p<0.01. (c) Comparative quantitation of mRNA expression of Galectin-3 for adherent and P2 spheres. P2 spheres and adherent cells were analysed for expression of galectin-3 by quantitative RT-PCR. Results are displayed as fold increase relative to adherent cells and normalized to β-actin. Error bars represent SEM.

Figure 4. LacNAc effect on phenotype and tumourigenicity of MCF-7 tumourspheres.

(a–e) Representative 4x photomicrographs of passage 2 spheres five days after culture in LacNAc containing sphere media. MCF-7 spheres cultured with LacNAc show a more differentiated morphology than those not cultured with LacNAc with most cells no longer forming spheres and attaching to the culture flask, as is seen under adherent conditions. (a) no treatment (No Tx), (b) 0.1 mM, (c) 0.3 mM, (d) 1 mM, and (e) 3 mM LacNAc. Bars represent 100 μm. LacNAc inhibits sphere forming frequency for MCF-7. Cells were cultured for 5 days in the sphere forming assay, 5000 cells per well. (f) A one-way ANOVA with a post-test for linear trend between number of spheres generated and amount of LacNac added demonstrated a significant decreasing trend (line over columns), *** p≤0.0001. Experiment repeated twice, representative results shown. Passage 2 MCF-7 spheres pre-treated with LacNAc have a slower tumour size enlargement then control cells (g). MCF-7 spheres were cultured in LacNAc at 3 mM for 7 days (closed squares) or left untreated (closed circles). Four immunocompromised mice (per group) were injected subcutaneously with 2×10⁶ cells. Mice were monitored for tumour growth twice weekly. A Gompertz fit model of the data indicated significant differences in tumourigenicity between treated and untreated groups (*** p<0.001). Error bars represent SEM.