Proteomic Comparison of MCF-7 Tumoursphere and Monolayer Cultures

<div><p>Breast cancer is a heterogenous 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 <em>in vitro</em> and tumourigenicity <em>in vivo</em>. 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.</p> </div

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

<p>Sphere forming efficiency progressively increases over serial passages (P). (a) Between P1 and P2 (<i>t</i>-test <i>p</i> = 0.0013, **), and between P6 and P8 (<i>t</i>-test <i>p</i><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).</p

Molecules of interest up-regulated in spheres compared to adherent cells.

<p>1) ANOVA p-values comparing adherent, passage2 spheres and passage 5 spheres.</p

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

<p>Galectin-3 (a) and Galectin-1 (b) proteomics results (<i>y</i>-axis is untransformed label free quantitation (LFQ) values) indicate increased expression within spheres compared to adherent cells, (line over columns), ANOVA test, *** <i>p</i><0.001; ** <i>p</i><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.</p

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

<p>(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), *** <i>p</i>≤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 (*** <i>p</i><0.001). Error bars represent SEM.</p

Schematic representation of <i>in</i><i>vitro</i> prenylation assay.

<p>(A) Cell lysates are subjected to <i>in </i><i>vitro</i> prenylation using a recombinant Rab prenylation machinery and the biotin-labeled analogue of GGPP, BGPP. Only those Rabs unprenylated in the cell will be labeled in the assay and can be visualized or purified through the biotin-tag. (B) Western blot analysis of cell lysate from untreated and compactin-treated BHK cells after <i>in </i><i>vitro</i> prenylation with BGPP. Unprenylated Rabs are detected via the biotin-tag and actin serves as a loading control. BGPP: biotin-geranyl-pyrophosphate, GGPP: geranylgeranyl-pyrophosphate. Note that compactin treated sample is underloaded and the actual level of unprenylated RabGTPases are higher than they appear from comparison of the band’s intensities. </p

Rescue of underprenylation in REP1 knockdown cells by REP1 but not by REP2.

<p>(A) REP1 knockdown cells were transfected with rat REP1-myc, REP2-myc or the vector control. The cell lysate was subjected to the <i>in </i><i>vitro</i> prenylation with BGPP to quantitate the levels of unprenylated Rabs. (B) Quantification of unprenylated Rabs in REP1 knockdown cells transfected with REP1 or REP2. The Western Blot signal of the unprenylated Rabs was normalized to the β-actin signal to account for differences in loading. REP1 knockdown cells transfected with vector only were taken as reference representing maximal underprenylation of Rabs in this model and therefore shown as 100%.The graph represents the mean of three independent experiments (± SEM). P-levels are denoted above the bars and were determined with the two-tailed Student’s t-test.</p

Analysis of Rab prenylation rates <i>in</i><i>vivo</i> and <i>in</i><i>vitro</i>.

<p>(A-B) Analysis of Rab prenylation status after blocking and releasing prenylation <i>in </i><i>vivo</i>. HeLa cells were treated with compactin for 24h and then incubated with GGOH for different time periods. Cells were lysed and subjected to <i>in </i><i>vitro</i> prenylation with BGPP and recombinant RabGGTase and REP for 6h. (A) Degree of prenylation for each Rab was determined by mass spectrometry. The decrease in signal from the timepoint 0h to 5h was determined by label-free spectral counting and converted into the degree of prenylation for each Rab 5 hours after GGOH addition. The graph represents the mean of three independent experiments (±SEM). (B) Streptavidin-HRP Western blot detection of unprenylated Rabs in the cellular lysates at different timepoints after GGOH addition to compactin-treated cells. The cellular lysates were prenylated with BGPP and RabGGTase as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081758#pone-0081758-g003" target="_blank">Figure 3 (C)</a><i>In </i><i>vitro</i> prenylation of lysate from compactin-treated HeLa cells transfected with different GFP-Rabs. <i>In </i><i>vitro</i> prenylation reaction was stopped after an hour and subjected to Western blot analysis visualized by infrared Odyssey scanning for total GFP-Rab (GFP/red) and for prenylated biotin-labeled GFP-Rab (biotin/green). Representative blots are shown. (D) The graph represents the percentage of prenylated GFP-Rabs after an hour <i>in </i><i>vitro</i> prenylation reaction normalized to complete (overnight) prenylation. Means of three independent experiments are shown (±SEM).</p

Localization is an indicator of prenylation.

<p>MALDI mass spectrometry comparison of unmodified (gray) and <i>in </i><i>vitro</i> prenylated (black) Citrine-Rab7 (A) and Citrine-Rab27a (B). Average protein masses are indicated at the peaks. Prenylated Citrine-Rab7:REP1 (C) and prenylated Citrine-Rab27a:REP1 (D) were microinjected into compactin treated A431 cells. Membrane targeting of Rab proteins was detected by imaging the cells 20 min post-injection. Scale bars represent 10 µm.</p

Rates of <i>in</i><i>vivo</i> prenylation and localization for Rab1a, Rab7a and Rab27a.

<p>Unprenylated, fluorescently-tagged recombinant CFP-Rab1a (upper panel), YFP-Rab7a (middle panel) and Cherry-Rab27a (lower panel) were microinjected into A431 cells. Cells were imaged over 22h and cellular localization of the injected Rab proteins detected by their fluorescent tags. The timepoints after microinjection are displayed above the images in hours or overnight (ON). Cells were pre-incubated with compactin where stated. Scale bars represent 20 µm.</p