Abstract P2-06-01: Development of a fluorescent reporter system to delineate self-renewing cells in triple negative breast cancer
Authors: Thiagarajan Praveena S , Hitomi Masahiro , Hale James S , Alvarado Alvaro G , Otvos Baltin , Stoltz Kevin , Sinyuk Maksim , Jarrar Awad , Zheng Qiao , Thomas Dustin , Egelhoff Thomas , Rich Jeremy N , Lathia Justin D , Reizes Ofer 
Published: May 2015

Background : Advanced cancers including triple-negative breast cancer (TNBC), the most aggressive breast cancer subtype, contain a self-renewing, tumorigenic cancer stem cell (CSC) population. CSCs contribute to tumor progression and therapeutic resistance. Despite an effective early response to chemotherapy, TNBC relapses with a highly heterogeneous and refractory metastatic disease enriched in CSCs. Apart from being chemo- and radio-resistant, CSCs display a high degree of multipotency, heterogeneity and plasticity. Due to the complex nature of CSCs, elucidating the characteristics of CSCs in TNBC progression continues to be a challenge. Thus, developing anti-CSC therapies to be integrated into clinical paradigms represents an immediate priority.

Rationale : A major obstacle to the identification of CSC regulatory mechanisms is the lack of experimental systems that enable the reliable enrichment of CSCs from non-CSCs for comparative analysis. While CSCs have been isolated from TNBC using CD44+/CD24- and/or ALDH+ phenotype, this enrichment paradigm requires refinement as it is not universally applicable and lacks the ability to study CSCs in real time. The limitations of these systems have excluded their application in studying the molecular heterogeneity among breast cancer tumors. CSCs are molecularly characterized based on the expression of cell surface receptors and the embryonic stem cell transcription factors NANOG, OCT4 and SOX2. NANOG, the master regulator of stem cell self-renewal, has emerged as a pro-carcinogenic factor in cancer cell lines with CSC behavior. Our previous studies have shown that silencing NANOG in cancer cells leads to reduced proliferation and self-renewal based on in vitro and in vivo experiments.

Hypothesis : We hypothesized that a Nanog promoter could be used to effectively enrich for TNBC CSCs.

Results : We generated two TNBC cell lines (MDA-MB-231 and HCC70) harboring NanogGFP reporter gene by lentiviral transductions. Increased NANOG mRNA and protein expression was observed in flow cytometry-sorted GFP+ cells compared with GFP- cells. GFP+ cells were enriched for the CSC markers CD49f and CD44+/CD24-. GFP+ cells also demonstrated an increased protein expression of the stem cell transcription factors NANOG, SOX2 and OCT4. Limiting dilution analyses revealed increased self-renewal and significantly higher stem cell frequencies in GFP+ cells. GFP+ cells demonstrated a mesenchymal phenotype with increased invasive capacity. Subcutaneous injections of GFP+ cells showed significantly higher in vivo tumor initiation and progression than GFP- cells. Flow cytometry-sorted GFP+ and GFP- cells enriched for CSCs and non-CSCs, respectively. Using this system, we performed a high-throughput flow cytometry screen and identified an additional novel CSC marker for TNBC.

Conclusion : We have developed a novel TNBC CSC reporter system using a GFP reporter driven by the Nanog promoter and identified a novel CSC marker. We have defined and validated this robust system wherein we have characterized and monitored the role of CSCs and non-CSCs in TNBC tumor initiation and progression. Using this approach, identifying CSC targets in TNBC could unravel the potential for development of innovative therapeutic strategies.

Citation Format: Praveena S Thiagarajan, Masahiro Hitomi, James S Hale, Alvaro G Alvarado, Baltin Otvos, Kevin Stoltz, Maksim Sinyuk, Awad Jarrar, Qiao Zheng, Dustin Thomas, Thomas Egelhoff, Jeremy N Rich, Justin D Lathia, Ofer Reizes. Development of a fluorescent reporter system to delineate self-renewing cells in triple negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-06-01.