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 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

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