title: Development of a Fluorescent Reporter System to Delineate Cancer Stem Cells in Triple-Negative Breast Cancer.
abstract: Advanced cancers display cellular heterogeneity driven by self-renewing, tumorigenic cancer stem cells (CSCs). The use of cell lines to model CSCs is challenging due to the difficulty of identifying and isolating cell populations that possess differences in self-renewal and tumor initiation. To overcome these barriers in triple-negative breast cancer (TNBC), we developed a CSC system using a green fluorescent protein (GFP) reporter for the promoter of the well-established pluripotency gene NANOG. NANOG-GFP+ cells gave rise to both GFP+ and GFP(-) cells, and GFP+ cells possessed increased levels of the embryonic stem cell transcription factors NANOG, SOX2, and OCT4 and elevated self-renewal and tumor initiation capacities. GFP+ cells also expressed mesenchymal markers and demonstrated increased invasion. Compared with the well-established CSC markers CD24(-) /CD44(+) , CD49f, and aldehyde dehydrogenase (ALDH) activity, our NANOG-GFP reporter system demonstrated increased enrichment for CSCs. To explore the utility of this system as a screening platform, we performed a flow cytometry screen that confirmed increased CSC marker expression in the GFP+ population and identified new cell surface markers elevated in TNBC CSCs, including junctional adhesion molecule-A (JAM-A). JAM-A was highly expressed in GFP+ cells and patient-derived xenograft ALDH+ CSCs compared with the GFP(-) and ALDH(-) cells, respectively. Depletion of JAM-A compromised self-renewal, whereas JAM-A overexpression induced self-renewal in GFP(-) cells. Our data indicate that we have defined and developed a robust system to monitor differences between CSCs and non-CSCs in TNBC that can be used to identify CSC-specific targets for the development of future therapeutic strategies.
authors.affiliation: Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44915, USA.
authors.forename: Praveena S
authors.initials: PS
authors.lastname: Thiagarajan
authors.affiliation: Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44915, USA.|Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH 44195, USA.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Hitomi
authors.affiliation: Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44915, USA.
authors.forename: James S
authors.initials: JS
authors.lastname: Hale
authors.affiliation: Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44915, USA.|Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH 44195, USA.
authors.forename: Alvaro G
authors.initials: AG
authors.lastname: Alvarado
authors.affiliation: Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44915, USA.
authors.forename: Balint
authors.initials: B
authors.lastname: Otvos
authors.affiliation: Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44915, USA.
authors.forename: Maksim
authors.initials: M
authors.lastname: Sinyuk
authors.affiliation: Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44915, USA.
authors.forename: Kevin
authors.initials: K
authors.lastname: Stoltz
authors.affiliation: Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44915, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Wiechert
authors.affiliation: Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44915, USA.
authors.forename: Erin
authors.initials: E
authors.lastname: Mulkearns-Hubert
authors.affiliation: Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44915, USA.
authors.forename: Awad
authors.initials: A
authors.lastname: Jarrar
authors.affiliation: Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44915, USA.
authors.forename: Qiao
authors.initials: Q
authors.lastname: Zheng
authors.affiliation: Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44915, USA.|Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH 44195, USA.
authors.forename: Dustin
authors.initials: D
authors.lastname: Thomas
authors.affiliation: Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44915, USA.|Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH 44195, USA.|Case Comprehensive Cancer Center, Cleveland, OH 44106, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Egelhoff
authors.affiliation: Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH 44195, USA.|Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, OH 44195, USA.|Case Comprehensive Cancer Center, Cleveland, OH 44106, USA.
authors.forename: Jeremy N
authors.initials: JN
authors.lastname: Rich
authors.affiliation: Case Comprehensive Cancer Center, Cleveland, OH 44106, USA.|Department of Pathology and Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, 44106, United States.
authors.forename: Huiping
authors.initials: H
authors.lastname: Liu
authors.affiliation: Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44915, USA.|Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH 44195, USA.|Case Comprehensive Cancer Center, Cleveland, OH 44106, USA.
authors.forename: Justin D
authors.initials: JD
authors.lastname: Lathia
authors.affiliation: Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44915, USA.|Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH 44195, USA.|Case Comprehensive Cancer Center, Cleveland, OH 44106, USA.
authors.forename: Ofer
authors.initials: O
authors.lastname: Reizes
mesh_terms: D000818:Animals; D005260:Female; D017930:Genes, Reporter / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006801:Humans; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D064726:Triple Negative Breast Neoplasms / Q000235:genetics*
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: Cancer stem cell; Fluorescent reporter system; Junctional adhesion molecule-A; NANOG; Triple-negative breast cancer
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