CSCs of breast tumors are commonly enriched by combinations

CSCs of breast tumors are commonly enriched by combinations of several cell-surface antigens, such as CD44/CD24/EPCAM (Al-Hajj et al., 2003), or by high ALDH (aldehyde dehydrogenase) activity (Ginestier et al., 2007). However, existing markers lack specificity, also reflective of a substantial proportion of non-CSCs. Furthermore, the applicability of existing markers is often limited to specific breast cancer subtypes (Nakshatri et al., 2009) in addition to interindividual intrinsic differences (Visvader and Lindeman, 2012). Previous studies have investigated the CSC content in different breast cancer subtypes (Harrison et al., 2013; Kim et al., 2012; Ricardo et al., 2011); however, thus far it is not exactly known whether distinct subtypes harbor the same or dissimilar CSCs. The large multitude of assays currently employed indicates either a lack of universal markers or reflects the heterogenic and dynamic nature of CSCs. The exact characterization of putative CSC pools is a pivotal requirement for clinical identification, monitoring, and targeting of these cells. To elucidate the heterogeneity of the CSC pool and to study the CSC compartment in ERα+ and ERα− breast cancer subtypes, we set up a single-cell quantitative real-time PCR (qPCR) approach, profiling the expression of well-established key regulators involved in differentiation, stemness, epithelial-to-mesenchymal transition (EMT), and cell-cycle regulation. Three functional assays for CSC enrichment were applied: (1) growth in anchorage-independent culture; (2) growth in hypoxia; and (3) cell selection based on label retention in mammosphere culture. All methods have previously been shown to enrich for mps1 kinase that exhibit increased cancer-initiating potential in mouse model systems (Harrison et al., 2010, 2013; Ponti et al., 2005; Richichi et al., 2013). By extensive single-cell analyses of breast cancer cells, we now define hierarchically organized CSC pools and modes of cell state transitions.
Results To study CSC heterogeneity and cellular composition in breast cancer, we applied three established techniques to modulate the CSC pool; growth in anchorage-independent culture (Harrison et al., 2010), hypoxia (Harrison et al., 2013), and a combination of the lipophilic PKH26 dye and the mammosphere assay to select for lowly proliferative, mammosphere-initiating cells (Ponti et al., 2005; Richichi et al., 2013) (Figures 1A–1C). All CSC enrichment methods have previously been demonstrated to enrich for cells displaying various CSC features, such as increased in vivo tumor-initiating capacity. Although in vivo data are not reported in this study, for simplicity we refer to enriched cell fractions as CSCs. The expression of key markers associated with differentiation (CDH1, CD24, EPCAM, ESR1, PGR), breast cancer stemness (CD44, ITGA6, DNER, ALDH1A3, ABCG2), pluripotency (POU5F1, NANOG, SOX2), EMT/metastasis (SNAI1, SNAI2, FOSL1, VIM, CDH2, ID1), and proliferation (CCNA2, MKI67, ERBB2) was quantitatively assessed at the single-cell level (Figures 1D and 1E). Detailed gene and qPCR assay information is provided in Table S1.
Discussion The CSC model suggests that tumors are driven by a small subset of cells with self-renewing and differentiation capacity, giving rise to phenotypically diverse, hierarchically organized tumors. CSCs display activated signaling pathways associated with normal stem cells and increased tumor-initiating capacity in xenograft models (Visvader and Lindeman, 2012), and have been shown to mediate metastasis (Liu et al., 2010) and increased resistance against radiotherapy and chemotherapy, contributing to relapse following therapy (Li et al., 2008; Yu et al., 2007; Zhang et al., 2010). The CSC concept has pivotal clinical implications for effective cancer treatments, since specific subpopulations in a tumor need to be targeted and monitored in order to better control tumor progression. However, diversity of breast cancer phenotypes as well as cellular plasticity complicate categorization of CSCs and, as a consequence, effective targeting of critical subpopulations of cancer cells.