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

doi:10.1093/jnci/djq361

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 journal › Article › peer-review

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 language	English
Pages (from-to)	1637-52
Number of pages	16
Journal	Journal of the National Cancer Institute
Volume	102
Issue number	21
DOIs	https://doi.org/10.1093/jnci/djq361
Publication status	Published - 2010
Externally published	Yes

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

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

Access to Document

10.1093/jnci/djq361

Cite this

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title = "Prolonged drug selection of breast cancer cells and enrichment of cancer stem cell characteristics",

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.",

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",

author = "Calcagno, {Anna Maria} and Salcido, {Crystal D} and Jean-Pierre Gillet and Chung-Pu Wu and Fostel, {Jennifer M} and Mumau, {Melanie D} and Gottesman, {Michael M} and Lyuba Varticovski and Ambudkar, {Suresh V}",

year = "2010",

doi = "10.1093/jnci/djq361",

language = "English",

volume = "102",

pages = "1637--52",

journal = "Journal of the National Cancer Institute",

issn = "0027-8874",

publisher = "Oxford University Press",

number = "21",

}

TY - JOUR

T1 - Prolonged drug selection of breast cancer cells and enrichment of cancer stem cell characteristics

AU - Calcagno, Anna Maria

AU - Salcido, Crystal D

AU - Gillet, Jean-Pierre

AU - Wu, Chung-Pu

AU - Fostel, Jennifer M

AU - Mumau, Melanie D

AU - Gottesman, Michael M

AU - Varticovski, Lyuba

AU - Ambudkar, Suresh V

PY - 2010

Y1 - 2010

N2 - 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.

AB - 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.

KW - Animals

KW - Antibiotics, Antineoplastic

KW - Antigens, CD

KW - Antigens, CD24

KW - Antigens, CD44

KW - Breast Neoplasms

KW - Cell Line, Tumor

KW - Cell Movement

KW - Cell Proliferation

KW - Cell Survival

KW - Collagen

KW - Confounding Factors (Epidemiology)

KW - Disease Models, Animal

KW - Doxorubicin

KW - Drug Combinations

KW - Drug Resistance, Multiple

KW - Drug Resistance, Neoplasm

KW - Female

KW - Flow Cytometry

KW - Gene Expression Regulation, Neoplastic

KW - Glycoproteins

KW - Humans

KW - Laminin

KW - Lung Neoplasms

KW - Mice

KW - Mice, SCID

KW - Microarray Analysis

KW - Neoplastic Stem Cells

KW - Ovarian Neoplasms

KW - P-Glycoprotein

KW - Peptides

KW - Proteoglycans

KW - RNA, Messenger

KW - Reverse Transcriptase Polymerase Chain Reaction

KW - Transplantation, Heterologous

KW - Up-Regulation

KW - Uterine Cervical Neoplasms

U2 - 10.1093/jnci/djq361

DO - 10.1093/jnci/djq361

M3 - Article

C2 - 20935265

SN - 0027-8874

VL - 102

SP - 1637

EP - 1652

JO - Journal of the National Cancer Institute

JF - Journal of the National Cancer Institute

IS - 21

ER -

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

Abstract

Keywords

UN SDGs

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