Mesenchymal stem cells promote mammosphere formation and decrease E-cadherin in normal and malignant breast cells.

Ann H Klopp,Massimo Cristofanilli,Rogério Gaspar,Anshul Gupta,Travis Solley,Bisrat G Debeb,Wendy A Woodward,Michael T Lewis,Xiaomei Zhang,Mauro Ferrari,Thomas A Buchholz,Li Li,James M Reuben,Michael Andreeff,Wei Xu,Savitri Krishnamurthy,Lara Lacerda,Erika L Spaeth ,Frank C Marini

doi:10.1371/journal.pone.0012180

Abstract

IntroductionNormal and malignant breast tissue contains a rare population of multi-potent cells with the capacity to self-renew, referred to as stem cells, or tumor initiating cells (TIC). These cells can be enriched by growth as “mammospheres” in three-dimensional cultures.ObjectiveWe tested the hypothesis that human bone-marrow derived mesenchymal stem cells (MSC), which are known to support tumor growth and metastasis, increase mammosphere formation.ResultsWe found that MSC increased human mammary epithelial cell (HMEC) mammosphere formation in a dose-dependent manner. A similar increase in sphere formation was seen in human inflammatory (SUM149) and non-inflammatory breast cancer cell lines (MCF-7) but not in primary inflammatory breast cancer cells (MDA-IBC-3). We determined that increased mammosphere formation can be mediated by secreted factors as MSC conditioned media from MSC spheroids significantly increased HMEC, MCF-7 and SUM149 mammosphere formation by 6.4 to 21-fold. Mammospheres grown in MSC conditioned media had lower levels of the cell adhesion protein, E-cadherin, and increased expression of N-cadherin in SUM149 and HMEC cells, characteristic of a pro-invasive mesenchymal phenotype. Co-injection with MSC in vivo resulted in a reduced latency time to develop detectable MCF-7 and MDA-IBC-3 tumors and increased the growth of MDA-IBC-3 tumors. Furthermore, E-cadherin expression was decreased in MDA-IBC-3 xenografts with co-injection of MSC.ConclusionsMSC increase the efficiency of primary mammosphere formation in normal and malignant breast cells and decrease E-cadherin expression, a biologic event associated with breast cancer progression and resistance to therapy.

Highlights

Normal and malignant breast tissue contains a rare population of multi-potent cells with the capacity to selfrenew, referred to as stem cells, or tumor initiating cells (TIC)
Normal human mammary epithelial cells (HMEC) with or without 2, 5 or 10% mesenchymal stem cells (MSC) labeled with red fluorescent protein (RFP) were plated into low-density suspension cultures in serumfree growth factor supplemented media into ultra-low attachment plates
MSC were found to interact as spheres and as single cells with HMEC as soon as one day after plating (Figure 1A)

Summary

Introduction

Normal and malignant breast tissue contains a rare population of multi-potent cells with the capacity to selfrenew, referred to as stem cells, or tumor initiating cells (TIC). These cells can be enriched by growth as ‘‘mammospheres’’ in three-dimensional cultures. Like normal tissues, are composed of a heterogenous population of cells with variable capacity for self-renewal. Multipotent tumor cells with the capacity to self-renew and recapitulate the tumors from which they were derived following transplantation into immunocompromised mice are referred to as tumor initiating cells (TIC) or cancer stem cells. Tumors grown as mammospheres ( known as tumorspheres) are enriched with stem cells markers, lin2/CD44+/CD242 and ALDH1, and have increased capacity for tumor initiation in xenograft models [6]

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