Stem Cells. 2015 Jul;33(7):2114-25.
Development of a Fluorescent Reporter System to Delineate Cancer Stem Cells in Triple-Negative Breast Cancer.
- 1Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA.
- 2Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio, USA.
- 3Case Comprehensive Cancer Center, Cleveland, Ohio, USA.
- 4Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Ohio, USA.
- 5Department of Pathology and Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, Ohio, USA.
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.
KEYWORDS: Cancer stem cell; Fluorescent reporter system; Junctional adhesion molecule-A; NANOG; Triple-negative breast cancer
A new paradigm to delineate cancer stem cells in triple-negative breast cancer
Praveena S. Thiagarajan1, Justin D. Lathia1-3, Ofer Reizes1-3
Department of 1Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44915, USA; 2Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH 44195, USA; 3Case Comprehensive Cancer Center, Department of Pathology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA
Dr. Ofer Reizes
Department of Cellular and Molecular Medicine
Lerner Research Institute
9500 Euclid Ave., NC10
Cleveland, OH 44195, USA
Phone – 216.445.0880; Fax – 216.444.8359
Triple negative breast cancers (TNBCs) are highly heterogeneous tumors and are characterized by the lack in expression of estrogen, progesterone and HER-2 receptors (1, 2). Advanced tumors including the aggressive TNBC subtype are hierarchically organized with a subpopulation of cancer cells at the apex with enhanced ability to induce tumorigenesis (3, 4). These cells have stem-like properties including self-renewal and expression of the embryonic stem cell transcription factors NANOG, SOX2, and OCT4 thus termed cancer stem cells (CSCs) (5). CSCs are able to drive heterogeneity and considered to underlie therapeutic resistance and relapse. Due to their ability to self-renew and to differentiate into multiple lineages, CSCs can generate a diverse progeny of cells leading to intra-tumoral heterogeneity (4, 5). Chemotherapy is still the main stay of TNBC treatment due to the lack of hormone and HER2 receptor expression in this subtype. TNBC does respond remarkably well to chemotherapy compared with other cancer subtypes initially, this subtype also has the highest rates of early recurrence (6). The poor prognostic features of recurrence and therapeutic resistance are postulated due to the enrichment of CSCs upon treatment (7). Current treatment options do not target CSCs so warrant development of effective treatment options to specifically target these cells. Owing to complex CSC behavior, isolating and characterizing CSCs in TNBC continues to be a challenge.
Isolation of breast CSCs have been primarily based on TNBC non-specific enrichment markers such as CD44+/CD24- and/or ALDH+ phenotype is widely employed to identify CSCs in many different cancer types but these markers do not robustly distinguish CSCs from non-CSCs in real-time (3). To study CSC characteristics, it necessitates design of a cancer stem cell model that addresses the heterogeneity in TNBC, enables reliable enrichment of CSCs, and provides the ability to track multi-potency over time. The limitations of the available cancer stem models have excluded their application in reliably enriching for CSCs in breast cancer cells and tumors. To address all the features of CSCs, we developed a novel TNBC CSC reporter system using a green fluorescent protein (GFP) driven by the promoter for the embryonic stem cell transcription factor NANOG (8). Our system relies on the role of NANOG as a master regulator of stem cell self-renewal and pluripotency; a protein that has emerged as a pro-carcinogenic factor in cancer cell lines (9, 10). The reporter provides an opportunity to visualize and monitor TNBC CSCs in real time.
To generate the reporter as proof of concept for utility of this approach, we transduced two TNBC cell lines (MDA-MB-231 and HCC70) with the NANOG-GFP promoter reporter in which flow cytometry sorted GFP-positive (GFP+) and GFP-negative (GFP-) cells enrich for CSCs and non-CSCs, respectively (8). Of note, GFP-positive cells were able to self-renew to generate GFP-positive cells but also divide asymmetrically to generate GFP-negative cells. This demonstrates the development of cellular heterogeneity over time in tissue culture. Moreover, CSCs (GFP+) exhibited increased NANOG mRNA and protein expression compared with non-CSCs (GFP-). GFP+ cells were enriched for the CSC markers CD49f and CD24–/CD44+. Further, CSCs demonstrate increased expression of the stem cell transcription factors NANOG, SOX2 and OCT4. Based on limiting dilution analyses, GFP+ cells demonstrate increased self-renewal and significantly higher stem cell frequencies compared with GFP- cells. CSCs (GFP+) initiate tumors with at high frequency compared to non-CSCs (GFP-) cells after transplant into NOD-SCID gamma (NSG) mice. Cells dissociated from both the CSCs and non-CSCs-injected tumors displayed a high degree of heterogeneity (8).
We demonstrated the use of this reporter system in a high-throughput flow cytometry screen as a discovery approach to identify new CSC markers for therapeutic targeting. To validate the utility of our reporter system to identify CSC-specific molecular pathways, we performed a high-throughput flow cytometry screen (8). Previous screening methods for CSCs in TNBC have proven challenging due to the inability to isolate a pure population of CSCs. Using a commercially available panel of cell surface antibodies, we observed an increase in the expression of well-established CSC cell-surface receptors including CD29 (integrin β1), CD44, and CD49f in CSCs in both cell lines. CSCs showed increased expression of junctional adhesion molecule – A (JAM-A) compared with the non-CSCs not only in both MDA-MB-231 and HCC70 cell lines but also in TNBC patient-derived xenograft cells (8). Expression of JAM-A has been shown to positively correlate with poor prognosis in patients with invasive breast cancer (11). We observed that JAM-A is necessary and sufficient for CSC maintenance (8).
In summary, we developed a novel TNBC CSC system using a GFP reporter driven by the NANOG promoter (8). Understanding CSC biology and maintenance are absolutely crucial for the development of effective therapeutics against aggressive cancers. Integration of this reporter with novel discovery and drug screening platforms could open new doors for research in developing new anti-CSC therapeutics. We have validated this robust system wherein we characterized and monitored the role of CSCs and non-CSCs in TNBC tumor initiation and progression in a systematic fashion as shown in Figure 1 (8). Using this approach, specific targeting of CSCs in TNBC could unravel the potential for development of innovative anti-CSC therapeutic strategies to be integrated into clinical paradigms.
Figure 1. The NANOG promoter-driven GFP reporter enriches for cancer stem cells (CSCs). Schematic shows the lentiviral transduction of the NANOG-GFP promoter reporter construct in two triple-negative breast cancer (TNBC) cell lines (MDA-MB-231 and HCC70). Following lentiviral transduction, the transduced cells were sorted based on GFP expression into GFP+ CSCs and GFP- non-CSCs. GFP+ CSCs enriched for the expression of cancer stem cell markers (CD24lo/ CD44hi, CD49f) and stem cell transcription factors (NANOG, OCT4, SOX2). Compared with the GFP- non-CSCs, GFP+ CSCs also enriched in a significant fashion for the ability to invade, develop cellular heterogeneity both in-vitro and in-vivo, self-renew, generate tumorspheres, undergo asymmetric cell-division and induce initiation of tumor formation. A high throughput flow cytometry screen was performed using the NANOG-GFP promoter reporter system and identified that the junctional adhesion molecule-A (JAM-A) to be essential for TNBC CSC maintenance. Hence, we demonstrate that our NANOG promoter reporter system can be utilized for discovery approaches and designing new screening strategies to identify and develop CSC-specific therapeutics for therapy-refractory TNBC.
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