Mesenchymal traits and cancer stem cells in mammospheres: chicken or egg?

The cancer stem cell (CSC) hypothesis states that tumors are initiated and maintained by a subset of cancer cells with the ability to self-renew and to differentiate into cells of various lineages. Putative CSCs have been identified in a variety of human malignancies including breast cancer. Breast CSCs expressing the cell surface markers CD44high/CD24low were able to generate tumors when transplanted in small numbers in immunocompromised NOD/SCID mice. In addition to the cell sorting method, breast CSCs have been identified in vitro by their ability to grow as mammospheres (MS) in non-adherent, serum-free conditions. MS obtained in these pro-stem culture conditions have been shown to be enriched in cells expressing CD44high/CD24low markers.

Beside stem-like features, the epithelial-mesenchymal transition (EMT) appears to play a critical role in breast cancer progression. EMT is a process wherein epithelial cells undergo multiple changes that enable them to adopt mesenchymal features, including enhanced capacity for migration, invasiveness and elevated resistance to apoptosis. EMT programs are orchestrated by a set of pleiotropic transcription factors, including Slug, Snail, Twist and Zeb1, which can directly repress levels of E-cadherin, the hallmark of the epithelial state. N-cadherin, the mesenchymal intermediary filament vimentin and extracellular matrix (ECM) components such as fibronectin are mesenchymal markers. Mani et al. and Morel et al. independently found that the activation of the EMT program in both normal and transformed mammary epithelial cells is associated with the acquisition of stem cell properties, including the ability to form spheres in non-adherent cultures. In addition, recent studies have shown that the subtypes of breast carcinomas enriched in stem features, such as claudin-low tumors, also express EMT markers.

Borgna et al. have studied distinct subtypes of breast cancer cell lines (luminal, HER2-positive, basal-like and claudin-low) under MS-proficient conditions (suspension). Their results suggest that the growth in suspension as MS favors the expansion of cells with mesenchymal traits (Fig. 1). For instance, an increase in the expression of vimentin has been observed in seven out of 10 cell lines, along with a significant decrease in the expression of the E-cadherin epithelial marker CDH1. Moreover, Borgna et al. have also observed a global increment in the expression of EMT transcription factors Zeb1, Zeb2, Snail2 and Twist 1. Overall, their data provide evidence for a shift toward a mesenchymal phenotype, along with stemness features, under proficient culture conditions. This supports the use of MS as an in vitro model for the study of therapeutic approaches targeting mesenchymal phenotypes.

Figure 1. Borgna et al. have shown that breast cancer cells grown as MS are enriched in both SC features and EMT markers.

The enrichment in mesenchymal features under MS-proficient conditions may be due to an increase in the proportion of a pre-existing subpopulation of cells with mesenchymal phenotype, or by de novo induction of EMT. Interestingly, Borgna et al. have observed the gain of mesenchymal features in short-term MS in MCF7 cells (a luminal cell line) lacking enrichment in the CD44high/CD24low fraction, suggesting that the gain of mesenchymal phenotype may precede the acquisition of stem-like features. This is an important observation that can be further validated by a functional assessment of MS under different culture conditions, as cells acquire an EMT and then a stem cell profile. We have recently developed a quantitative approach to validate different possible scenarios that can lead to the enrichment of stem cell activity following induction of EMT., Additionally, we have suggested the utility of comparing mammosphere data to computational mammosphere simulations in elucidating the growth characteristics of mammary CSCs. I anticipate that this “modus operandi” of using quantitative modeling (grounded in experimental data) to gain new insights may well provide a rational means for predicting the timeline of the acquisition of mesenchymal and stem features. The process of EMT and its relationship to CSCs are novel and rapidly converging directions of research, and many related questions may be considered. For instance, it would be interesting to use the suggested MS culture conditions to develop and validate novel and effective therapeutic strategies, followed by in vivo validations. Moreover, in vitro MS studies under tumor microenvironmental conditions, such as hypoxia, may provide further information, which can be also validated by in vivo experiments.