Mammosphere Formation in Breast Carcinoma Cell Lines Depends upon Expression of E-cadherin

<div><p>Tumors are heterogeneous at the cellular level where the ability to maintain tumor growth resides in discrete cell populations. Floating sphere-forming assays are broadly used to test stem cell activity in tissues, tumors and cell lines. Spheroids are originated from a small population of cells with stem cell features able to grow in suspension culture and behaving as tumorigenic in mice. We tested the ability of eleven common breast cancer cell lines representing the major breast cancer subtypes to grow as mammospheres, measuring the ability to maintain cell viability upon serial non-adherent passage. Only MCF7, T47D, BT474, MDA-MB-436 and JIMT1 were successfully propagated as long-term mammosphere cultures, measured as the increase in the number of viable cells upon serial non-adherent passages. Other cell lines tested (SKBR3, MDA-MB-231, MDA-MB-468 and MDA-MB-435) formed cell clumps that can be disaggregated mechanically, but cell viability drops dramatically on their second passage. HCC1937 and HCC1569 cells formed typical mammospheres, although they could not be propagated as long-term mammosphere cultures. All the sphere forming lines but MDA-MB-436 express E-cadherin on their surface. Knock down of E-cadherin expression in MCF-7 cells abrogated its ability to grow as mammospheres, while re-expression of E-cadherin in SKBR3 cells allow them to form mammospheres. Therefore, the mammosphere assay is suitable to reveal stem like features in breast cancer cell lines that express E-cadherin.</p> </div

Breast cancer cell lines characteristics in suspension culture.

<p>A) Breast cancer cell lines included in the study showing their origin and main molecular characteristics (IDC, invasive ductal carcinoma; AC, adenocarcinoma; DC, ductal carcinoma; P, primary tumor; M, metastasis; black box, positive; white box, boxes color code stated in the figure). B) Proliferation of breast cancer cell lines in suspension culture. Cell proliferation was measured as the increase in the number of viable cells upon serial non-adherent passages. C) Morphology of the human breast cancer cell lines included in the study. The morphology of the cell lines growing in adhesion is shown in the top row, indicated as T0. Subsequent passages in suspension culture are termed T<i>n</i>, where n represents the passage number (Magnification 120X).</p

Gene expression profiling of the breast cancer cell lines included in the study.

<p>Left panel: clustering of the genes differentially expressed (p-value < 0.001) between the 3 groups of cell lines identified based on their proliferation and morphological characteristics in suspension culture. Blue squares represent transcript levels below the median; white squares represent transcript levels equal to the median; red squares represent transcript levels greater than the median. Right panel: functional annotation of biological processes overrepresented among the genes differentially expressed between the three groups of cell lines.</p

E-cadherin knock-down in MCF7 cells inhibits mammosphere formation.

<p>A) Immunofluorescence staining of MCF7 shE-cad and control cells to show the knockdown of E-cadherin expression. B) Morphology of MCF7 shE-cad and control cells growing in adhesion and suspension culture. C) Proliferation of MCF7 shE-cad and control cells in suspension culture. Proliferation was measured as the increase in the number of viable cells upon serial non-adherent passages. The plot was made using data from 3 independent experiments. N.O.=Non observed.</p

Expression of E-cadherin in SKBR3 cells induces sphere formation.

<p>A) Immunofluorescence staining of SKBR3 E-cadherin and WT cells showing the expression of E-cadherin. B) Morphology of SKBR3 E-cadherin and WT cells growing in adhesion and suspension culture. C) Quantitation of the number of spheres made by SKBR3 E-cadherin and WT cells in T0. The plot was made using data from 3 independent experiments.</p

E-cadherin expression in LTSF (A), STSF (B) and NSF (C) breast cancer cell lines growing in adhesion and suspension culture assessed by immunofluorescence.

<p>E-cadherin expression in LTSF (A), STSF (B) and NSF (C) breast cancer cell lines growing in adhesion and suspension culture assessed by immunofluorescence.</p

ANXA8 expression arrests Kim2A8 cells in G₀.

<p>(<b>A</b>) Kim2A8 and Kim2RTS cells were grown in 6-well plates with or without 100ng/ml of dox for 48 hours. The graph shows the average percentage numbers of cells in G₀/G₁, S and G₂/M as quantified by FACS from three independent experiments. (<b>B</b>) Kim2A8 and Kim2RTS cells grown in chamber slides with or without 100ng/ml dox for six days were fixed and stained for Ki67 antigen. EGFP was used as a reporter of ANXA8 expression. (<b>C</b>) Graph showing the percentage of Ki67-positivity in the EGFP positive and negative populations of Kim2A8 cells grown with or without 100ng/ml dox. At least 1000 cells were analyzed in each population. (<b>D</b>) Western blot showing Ki67 and ANXA8 protein expression in cells after six days in culture. Actin was used as a loading control. Numbers show the relative intensities of ANXA8 and Ki67 bands respectively (normalised to actin) determined by measuring area pixel intensities using AIDA Image Analyzer software. The reduction of Ki67 levels (∼50%) is consistent with the reduced number of Ki67+ve Dox-treated Kim2A8 cells seen in (<b>B</b>).</p

ANXA8 expression inhibits proliferation of Kim2A8 cells.

<p>(<b>A</b>) Kim2A8 and Kim2RTS cells were seeded in 24-well plates, allowed to attach and grown in the presence or absence of 100ng/ml dox (first treatment at time point 0). At each time point protein extracts were prepared (in duplicates). The graph shows the amount of protein as determined by BCA assay against time. This assay was performed in triplicate and the graph shows a representative result from one experiment. (<b>B</b>) Cells were seeded in 96-well plates and treated with dox for 48 hours, labelled with BrdU and the incorporation of BrdU was quantified and plotted for each condition (six wells per condition). ***p< 0.001 (<b>C</b>) Equal amount of cells (250–300) were grown in the presence or absence of 100ng/ml dox. After 14 days cells were fixed, stained and the plates photographed. A representative plate per condition is shown. The experiment was carried out in triplicate. (<b>D</b>) Graph showing the number of colonies per plate from the experiment (<b>C</b>) as quantified by Image J (**p<0.003).</p

ANXA8 expression induces morphological changes in Kim-2 cells.

<p>Kim2A8 and Kim2RTS cells were grown in the presence or absence of 100ng/ml dox. Pictures were taken after 48 hours (<b>A</b>) or six days (<b>B</b>) of treatment. EGFP was used as a reporter of ANXA8 expression. Both proteins are expressed from opposite sides of a bidirectional promoter. (<b>C</b>) Nuclear sizes were analysed after 6 days by measuring the nuclear area (stained with DAPI) of at least 90 individual cells from each dox-treated and untreated populations using ImageJ. There was a significant difference between Kim2A8 cells expressing and not expressing ANXA8 (ANOVA: p<0.05).</p

ANXA8 is expressed in ERα−ve/c-kit+ve luminal progenitor cells.

<p>(<b>A</b>) Primary mammary epithelial cells were sorted and RNA extracted as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119718#pone.0119718.ref039" target="_blank">39</a>]. qRT-PCR was used to measure <i>AnxA8</i> mRNA abundance in four different populations. <i>AnxA8</i> was not detected in either mammary stem cells (MaSC) or myoepithelial cells. Although very low levels were detected in differentiated ERα+ve luminal epithelial cells, ERα−ve luminal epithelial progenitor cells showed a 17-fold higher abundance. The graph shows the abundance relative to the levels of expression in differentiated cells and 95% confidence limits. (<b>B</b>) Bar graph showing the proportion of c-kit+ve/AnxA8+ve and c-kit+ve/AnxA8−ve cells during puberty (V6), early (P4.5) and late (P12.5) pregnancy. 1,000 cells were assessed per developmental time point. Error Bars denote standard error of the mean. (<b>C</b>) Co-immunofluorescence staining for ANXA8 and c-kit in mouse mammary gland from an early-pregnant (day 4.5) mouse.</p