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 journalArticle

Abstract

BACKGROUND: Cancer 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.

METHODS: Cancer 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.

RESULTS: Pathway 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.

CONCLUSION: The cell population with cancer stem cell characteristics increased after prolonged continuous selection for doxorubicin resistance.

Original languageEnglish
Pages (from-to)1637-52
Number of pages16
JournalJournal of the National Cancer Institute
Volume102
Issue number21
DOIs
Publication statusPublished - 2010
Externally publishedYes

Keywords

  • Animals
  • Antibiotics, Antineoplastic
  • Antigens, CD
  • Antigens, CD24
  • Antigens, CD44
  • Breast Neoplasms
  • Cell Line, Tumor
  • Cell Movement
  • Cell Proliferation
  • Cell Survival
  • Collagen
  • Confounding Factors (Epidemiology)
  • Disease Models, Animal
  • Doxorubicin
  • Drug Combinations
  • Drug Resistance, Multiple
  • Drug Resistance, Neoplasm
  • Female
  • Flow Cytometry
  • Gene Expression Regulation, Neoplastic
  • Glycoproteins
  • Humans
  • Laminin
  • Lung Neoplasms
  • Mice
  • Mice, SCID
  • Microarray Analysis
  • Neoplastic Stem Cells
  • Ovarian Neoplasms
  • P-Glycoprotein
  • Peptides
  • Proteoglycans
  • RNA, Messenger
  • Reverse Transcriptase Polymerase Chain Reaction
  • Transplantation, Heterologous
  • Up-Regulation
  • Uterine Cervical Neoplasms

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  • Cite this

    Calcagno, A. M., Salcido, C. D., Gillet, J-P., Wu, C-P., Fostel, J. M., Mumau, M. D., Gottesman, M. M., Varticovski, L., & Ambudkar, S. V. (2010). Prolonged drug selection of breast cancer cells and enrichment of cancer stem cell characteristics. Journal of the National Cancer Institute, 102(21), 1637-52. https://doi.org/10.1093/jnci/djq361