Supplementary MaterialsSupp FigureS1-S3. power of this system as a screening platform, we performed a circulation cytometry screen that confirmed increased CSC marker expression in the GFP+ populace 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 rescued self-renewal in GFP? cells. Our Prilocaine data show 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. . TNBC constitutes 15%-20% of all breast cancers and is characterized by poor prognosis and the lack of effective specific therapeutic options . TNBC patients show higher rates of early relapse due to refractory drug-resistant local and/or metastatic disease even after an initial effective response to cytotoxic standard chemotherapy, which remains the mainstay of TNBC treatment . The hypothesis that a populace of self-renewing malignancy stem cells (CSCs) drives tumor recurrence and metastasis and underlies TNBC heterogeneity is usually well supported [9-11]. CSCs are characterized by their ability to propagate tumors and recapitulate the heterogeneity present in the original lesion [12, 13]. TNBCs are resistant to chemotherapy, and recurrence HESX1 has been postulated to be a result of the chemo- and radio-resistance exhibited by CSCs [14, 15]. Due to confounding factors such as cellular heterogeneity and an evolving epigenetic state of CSCs, the mechanisms underlying their self-renewal and role in tumor progression are being Prilocaine actively pursued . While CSCs have been postulated to be crucial for TNBC maintenance and progression, studying the characteristics of TNBC CSCs remains a challenge. A major obstacle to the identification of CSC regulatory mechanisms is a lack of experimental systems that enable the reliable enrichment of CSCs from non-CSCs for comparative analysis . Many groups have isolated TNBC CSCs using CD24-unfavorable/CD44-positive (CD24?/CD44+) cells and/or through high aldehyde dehydrogenase I activity (ALDH+) [18, 19]. These enrichment paradigms require refinement, as they are not universally relevant to all breast tumors [20-22]. Additionally, many Prilocaine CSC studies have been performed primarily studies have used high passage TNBC cell lines that have not Prilocaine been well-characterized for CSC studies. Further complicating the study of CSCs in TNBC is the lack of a well-defined system to analyze these cells in real time. To interrogate the molecular heterogeneity of TNBC cells, we developed a novel CSC reporter system using a GFP reporter driven by the promoter of the embryonic stem cell transcription factor is usually a stem cell transcription factor and a grasp regulator of stem cell self-renewal [23, 24]. has emerged as a pro-carcinogenic factor , and immunostaining Prilocaine data show a strong correlation between NANOG and other malignancy stem cell markers [25-28]. silencing in malignancy cells prospects to reduced proliferation, self-renewal based on tumorsphere assays, and tumor initiation in xenograft transplant studies [23, 29]. We generated two TNBC cell lines (MDA-MB-231 and HCC70) in which GFP+ and GFP? cells show differences in CSC marker expression and function [30, 31]. The cell surface signature of both GFP+ and GFP? cells was evaluated using a high-throughput screening method validated by our group, and we found that NANOG promoter-driven GFP also enriches for TNBC cells positive for CSC surface markers. The screen revealed additional receptors enriched in CSCs. Our approach has the ability to enrich for any populace of CSCs, enabling interrogations to understand the key functions of CSCs in TNBC initiation and progression. Materials and Methods Cell culture MDA-MB-231 and HCC70 breast malignancy cells (American Type Culture Collection; Manassas, VA) were cultured in log-growth phase in altered Eagle’s medium (MEM) supplemented with 1 mM sodium pyruvate (Cellgro, Kansas City, MO) and 10% heat-inactivated fetal calf serum (FCS) at.