Prolonged drug selection of breast cancer cells and enrichment of cancer stem cell characteristics

Anna Maria Calcagno, Crystal D. Salcido, Jean Pierre Gillet, Chung Pu Wu, Jennifer M. Fostel, Melanie D. Mumau, Michael M. Gottesman, Lyuba Varticovski, Suresh V. Ambudkar

Research output: Contribution to journalArticlepeer-review

Abstract

BackgroundCancer stem cells are presumed to have virtually unlimited proliferative and self-renewal abilities and to be highly resistant to chemotherapy, a feature that is associated with overexpression of ATP-binding cassette transporters. We investigated whether prolonged continuous selection of cells for drug resistance enriches cultures for cancer stem-like cells.MethodsCancer stem cells were defined as CD44+/CD24- cells that could self-renew (ie, generate cells with the tumorigenic CD44+/CD24- phenotype), differentiate, invade, and form tumors in vivo. We used doxorubicin-selected MCF-7/ADR cells, weakly tumorigenic parental MCF-7 cells, and MCF-7/MDR, an MCF-7 subline with forced expression of ABCB1 protein. Cells were examined for cell surface markers and side-population fractions by microarray and flow cytometry, with in vitro invasion assays, and for ability to form mammospheres. Xenograft tumors were generated in mice to examine tumorigenicity (n = 52). The mRNA expression of multidrug resistance genes was examined in putative cancer stem cells and pathway analysis of statistically significantly differentially expressed genes was performed. All statistical tests were two-sided. ResultsPathway analysis showed that MCF-7/ADR cells express mRNAs from ABCB1 and other genes also found in breast cancer stem cells (eg, CD44, TGFB1, and SNAI1). MCF-7/ADR cells were highly invasive, formed mammospheres, and were tumorigenic in mice. In contrast to parental MCF-7 cells, more than 30% of MCF-7/ADR cells had a CD44+/CD24- phenotype, could self-renew, and differentiate (ie, produce CD44+/CD24- and CD44+/CD24+ cells) and overexpressed various multidrug resistance-linked genes (including ABCB1, CCNE1, and MMP9). MCF-7/ADR cells were statistically significantly more invasive in Matrigel than parental MCF-7 cells (MCF-7 cells = 0.82 cell per field and MCF-7/ADR = 7.51 cells per field, difference = 6.69 cells per field, 95% confidence interval = 4.82 to 8.55 cells per field, P <. 001). No enrichment in the CD44+/CD24- or CD133+ population was detected in MCF-7/MDR.ConclusionThe cell population with cancer stem cell characteristics increased after prolonged continuous selection for doxorubicin resistance.

Original languageEnglish (US)
Pages (from-to)1637-1652
Number of pages16
JournalJournal of the National Cancer Institute
Volume102
Issue number21
DOIs
StatePublished - Nov 3 2010
Externally publishedYes

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

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