Developing ovarian cancer stem cell models: laying the pipeline from discovery to clinical intervention

Abstract

Despite decades of research, ovarian cancer is still associated with unacceptably high mortality rates, which must be addressed by novel therapeutic approaches. One avenue through which this may be achieved is targeting of tumor-initiating 'Cancer Stem Cells' (CSCs). CSCs are sufficient to generate primary and recurrent disease through extensive rounds of asymmetric division, which maintain the CSC pool while producing the tissues that form the bulk of the tumor. CSCs thrive in the harsh tumor niche, are generally refractory to therapeutic intervention and closely-linked to the Epithelial-Mesenchymal Transition process, which facilitates invasion and metastasis. While it is well-accepted that CSC-targeting must be assessed as a novel therapeutic avenue, few ovarian CSC models have been developed due to perceived and actual difficulties associated with the process of 'CSC Discovery'. In this article we review contemporary approaches to CSC Discovery and argue that this process should start with an understanding of the specific challenges associated with clinical intervention, laying the pipeline backwards towards CSC Discovery. Such an approach would expedite the bridging of the gap between laboratory isolation and clinical targeting of ovarian CSCs.

Figure 1

The defining properties stem cells (SC) and cancer stem cells (CSCs). SCs and CSCs can both be defined and validated via three properties. Self-renewal: (C)SCs can divide in a potency preserving fashion, producing two daughter stem cells. Differentiation: When necessary, (C)SCs can differentiate to produce daughter cells of reduced potency. Tissuegenesis: (C)SCs can derive the multiple cell types needed to form their given tissue. In the case of CSCs this is referred to as tumorigenesis (‘malignant tissuegenesis’).

Figure 2

The undifferentiated stem cell (SC) state is regulated by multiple factors in the stem cell niche. Studies of the SC niche have shown that multiple factors regulate (C)SC activity. The most prominent factors range from chemokine/cytokine signaling and metabolite gradients to basement membrane and stromal cell interactions. Together, these factors regulate the undifferentiated state of SCs and CSCs.

Figure 3

Cancer Stem Cell (CSC) Discovery pipeline. This figure illustrates the CSC discovery process. A) First, CSCs must be identified within a heterogeneous population of cells. As outlined in this review, CD133+, ALDH+ and HSP+ are the most successfully used ovarian CSC markers to date. Any CSCs identified must be considered putative CSCs (pCSCs) until they are validated as being capable of self-renewal, differentiation and the generation of a tumor with similar histology to that from which they were derived. B) Such pCSCs must be isolated to facilitate their validation. Fluorescence-activated and/or magnetic-activated cell sorting are commonly used to isolate pCSCs from non-pCSCs. C) Isolated cells can be validated by being assayed for tumorigenesis properties via xenograftment into mice, while their ability to self-renew and differentiate can be assayed via single cell asymmetric division assays.

Figure 4

The single cell asymmetric division assay (SCAD assay). Utilizing cancer stem cell (CSC) markers via fluorescence-activated cell sorting, putative (pCSCs) and non-pCSCs are plated as a single cell per well in a 96-well plate. Such cells are allowed to form colonies, which are cultured to generate sufficient cells to re-analyze for the presence of the original CSC markers. If a clone reconstitutes the parent phenotype, it has demonstrated the CSC-like capacity to self-renew and differentiate. If a clone fails to grow or does not reconstitute the parent phenotype, it has demonstrated a reduced differentiation potential characteristic of non-CSCs. The SCAD assay used in parallel with the xenograft tumorigenicity assay can efficiently validate CSCs and non-CSCs.

Figure 5

Clonal Cancer Stemness. i) CSC Theory states that tumors are composed of multiple cell types produced by CSCs via multiple rounds of self-renewal and differentiation. ii) The Genetic Heterogeneity viewpoint states that cumulative acquired genetic mutations lead to genetically divergent cell lineages. CSC Theory and Genetic Heterogeneity are not mutually exclusive concepts. In fact, growing evidence suggests both are correct. iii) This points towards the Clonal Cancer Stemness model of malignancy, where both CSCs and genetic divergence account for the heterogeneous traits of malignancy (e.g. chemoresistance and metastasis). Additionally, CSC hierarchical arrangement allows for CSCs to enter periods of quiescence, which can complicate current therapeutic approaches.