Abstract A35: A pluripotency reporter defines lung cancer cells with tumor-initiating properties.

The emerging theory of cancer stem cells (CSC) postulates that cells in a tumor are hierarchically organized, so that only a subpopulation of cells has the capacity for sustaining tumor growth, and regenerating a heterogeneous tumor. Conceptually, CSC can be resistant to therapies that reduce tumor bulk, and therefore responsible for micrometastatic disease and local recurrence. The existence of CSC challenges the current paradigm of cancer treatment by implying that the fundamental test of most current therapies, i.e., their ability to reduce tumor size, may be inadequate unless it is ensured that the subpopulation of cells within a tumor with self-renewal properties is completely eradicated. Thus, their molecular definition is crucial to the development of innovative therapeutic strategies. Specific cell surface markers, analogous to those found in normal stem cells of the corresponding tissue, has proven useful for enrichment of CSC activity in several tissues. Viable human lung CSC have been isolated through their expression of CD133 or CD166 and within cell fractions characterized by intrinsic stem cell properties, such as high ALDH enzymatic activity or dye efflux (side population). CSC can be live-sorted based on these markers and xenotransplantated into immunodeficient mice, the current gold standard assay for identification of CSC. However, cell surface markers are very heterogeneous across lung cancer histological types and do little to functionally define the cells they label. We hypothesize that CSC can be functionally isolated and characterized using a pluripotency reporter. By definition, CSC resemble induced-pluripotent stem cells (iPS), in that they give rise to progeny that lack or have diminished capacity for self-renewal and reconstitution of the tumor bulk. iPS are generated from somatic cells through introduction of four key pluripotency genes: OCT4, SOX2, MYC, and KLF4, all of which are oncogenes (MYC, KLF4) or linked to tumorigenesis (OCT4, SOX2). Moreover, increased expression of OCT4, SOX2, and their downstream target, NANOG was reported in CSC defined by the cell surface marker CD44hi. Teratoma formation, the gold standard assay for adequate cell reprogramming, can occasionally produce teratocarcinomas, which have been proposed to arise from “partially reprogrammed” cells. Furthermore, increased expression of pluripotency genes is also associated with poor lung cancer prognosis. Here, we show that a subpopulation of cells with CSC properties can be isolated using a recently-developed lentivirus-based reporter that provides a readout of SOX2 and OCT4 activation. Reporter-positive cells represent ~8-20% cells in A549 and H460 lung cancer cells under regular growth conditions. Sorted reporter-positive cells form spheres with greater frequency than negative cells in media without serum, and show self-renewal properties through serial passage in matrigel. Reporter-positive cells show higher asymmetric cell division, repopulation, and tumor-initiating activity that can be sustained by passage in vivo. Importantly, the fraction of reporter-positive cells is increased after exposure to the chemotherapeutic agent cisplatin, consistent with the view that inefficient eradication of CSC may be responsible for tumor recurrence. We are currently investigating epigenetic mechanisms that may be responsible for functional heterogeneity and may provide a link between chromatin reprogramming and the CSC phenotype. We expect that characterizing the properties of CSC will reveal innovative strategies to therapeutically target them to treat lung cancer. Citation Format: Ana I. Robles, Brid M. Ryan, Krist Aploks, Binwu Tang, Lalage Wakefield, Curtis C. Harris. A pluripotency reporter defines lung cancer cells with tumor-initiating properties. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr A35.