daunorubicin To address this need we have developed a functi
To address this need, we have developed a functional imaging approach for CSC identification. The stem cell phenotype in embryonic stem daunorubicin (ESCs) is maintained by a central triad of master transcriptional regulators, OCT4, SOX2, and NANOG, which promote stemness by upregulating genes involved in pluripotency and self-renewal while suppressing genes involved in differentiation (Young, 2011). Indeed, ectopic expression of just three factors, OCT4, SOX2, and KLF4, is sufficient to induce pluripotency and stem-like characteristics in differentiated somatic cells (Schmidt and Plath, 2012), suggesting that reactivation of stem cell transcription factors might be an efficient mechanism for transformed cells to acquire the ability to self-renew. We therefore hypothesized that OCT4 and SOX2, the two most upstream regulators of the stem cell phenotype, would be active in CSCs and could be used to drive a reporter construct that would mark the CSCs. In support of this hypothesis, embryonic stem-like gene expression signatures are found to be enriched in many aggressive tumors (Ben-Porath et al., 2008), and myeloid leukemia stem cells have been shown to employ a transcriptional program that is more similar to embryonic than adult stem cells (Somervaille et al., 2009). Promoter-reporter constructs based on portions of the promoters of OCT4, SOX2, or NANOG have been widely used in monitoring the reprogramming of somatic cells to the induced pluripotent state (Hotta et al., 2009) but have had only limited application in identifying CSCs (Levings et al., 2009), where expression levels of these transcription factors are likely to be much lower. In addition, the relatively large promoter regions used in such constructs invariably contain response elements for additional transcription factors, which may reduce reporter specificity.
Discussion It is increasingly appreciated that a tumor represents a whole ecosystem of mutually interacting cellular and acellular components that generate a continually evolving tumor microenvironment (Quail and Joyce, 2013). Many aspects of this dynamic and complex microenvironment, such as hypoxia and inflammation, can modulate CSC properties and response to therapy (Conley et al., 2012; Cui et al., 2013; Korkaya et al., 2012) and function in different spatial contexts within the tumor. Thus, it would be desirable to observe the behavior of the CSCs in their native habitat with all microenvironmental cues intact. Here, we have developed and validated a flexible and powerful lentiviral-based reporter system for direct visualization, quantitation, and isolation of the cells with CSC properties in multiple preclinical tumor models in vitro and in vivo. Cells detected by this method are relatively undifferentiated, can self-renew and give rise to phenotypically heterogeneous offspring, show enhanced asymmetric division, and are enriched for tumor-initiating and metastasis-initiating ability in vivo. Importantly, the marked cells are also relatively resistant to chemotherapeutics, suggesting that a highly clinically relevant tumor cell subpopulation is being detected with this reporter. Our approach depends on the presence of the stemness transcription factors SOX2 and/or OCT4 in the CSC. SOX2 is expressed in immature cells of many self-renewing epithelial tissues in the adult animal (Arnold et al., 2011), and it has been detected in a variable percentage of cells in many malignant tissues, some of which clearly depend on SOX2 for their tumor-initiating ability (Gangemi et al., 2009). Detection of OCT4 is complicated by the existence of alternate transcripts and pseudogenes, and evidence is convincing that OCT4 is not expressed in adult somatic stem cells (Lengner et al., 2008). However, ectopic expression of OCT4 in the intestinal epithelium and epidermis blocks differentiation and leads to uncontrolled proliferation of progenitor cells (Hochedlinger et al., 2005), and forced overexpression of OCT4 in primary breast epithelial cells generated tumor-initiating cells (Beltran et al., 2011), suggesting that reactivation of epigenetically silenced OCT4 would be a parsimonious route to tumor formation. Ionizing radiation was recently shown to reprogram differentiated breast cancer cells into cells with CSC characteristics associated with reexpression of OCT4 and SOX2, further supporting an intimate connection between stemness and OCT4/SOX2 expression (Lagadec et al., 2012). So far, our reporter has identified a minority cell population in all the primary and established breast cancer cells we have studied, suggesting that the presence of functional SOX2/OCT4 in a subpopulation of tumor cells may be a relatively widespread phenomenon.