Background

Cellular heterogeneity in tumors poses tough challenges for cancer therapy (Magee et al., 2012). Certain tumors are arranged hierarchically with the chemotherapy resistant tumor initiating cells (also called cancer stem cells) residing at the bottom of the cellular hierarchy as they maintain the tumor through self-renewal and differentiation (Magee et al., 2012). Plasticity of the tumor initiating cells (TICs) makes them difficult to identify, isolate and study. Heterogeneity and plasticity of energetics of TICs is only beginning to be appreciated and is far from clear (Martinez-Outschoorn et al., 2017) (Intlekofer and Finley, 2019). Moreover, energetics status of TICs appears to be tumor specific, and certain TICs are dependent on mitochondrial energetics and can be eliminated by mitochondrial inhibitors (Pasto et al., 2014; Sancho et al., 2015; Viale et al., 2015; Martinez-Outschoorn et al., 2017; Spurlock et al., 2019).

Mitochondrial energetics is maintained by complex biochemical regulation of multiple bioenergetic parameters, including mitochondrial transmembrane potential maintained across the mitochondrial inner membrane (Δ_Ψ), which is consumed to generate ATP, heat, or reactive oxygen species. Higher mitochondrial transmembrane potential is expected to maintain higher ATP synthesis depending on various other parameters like substrate availability, etc. Previously, stem cells isolated based on mitochondrial transmembrane potential have been found to differ in their stem cell properties (Schieke et al., 2008; Sukumar et al., 2016). Recently, we developed a flowcytometric protocol to simultaneously sort cells for Δ_Ψ, using the red fluorescent potentiometric dye TMRE, and the activity of a functional TIC marker aldehyde dehydrogenase (ALDH), using the AldeFluor reagent (Figures 1-2) (Spurlock et al., 2019). Using such a flow cytometric sorting strategy, we were able to identify a subpopulation of human ovarian TICs with 10 fold higher self-renewing and proliferating capacity, as determined by in vitro limiting dilution assay. Thus, our protocol enables teasing out mitochondrial heterogeneity in stem cell populations.

The role of mitochondrial energetics in maintenance of normal stem cells and TICs is more appreciated as our understanding of stem cell energetics increases (Viale et al., 2015; Chandel et al., 2016; Intlekofer and Finley, 2019). Adult stem cells are majorly quiescent and considered to be highly glycolytic and release from quiescence involves metabolic/energetic shifts that remain poorly understood (Margineantu et al., 2002; Ito and Suda, 2014; Folmes and Terzic, 2016). We proposed that a subpopulation of ovarian TICs with particularly elevated mitochondrial transmembrane potential, associated with other specific mitochondrial properties, are primed for greater self-renewal and proliferation ability (Spurlock et al., 2019). Similar mitochondria based priming has been observed in hematopoietic lineage and has been linked to exit from quiescence and entry into the cell cycle for self-renewal and differentiation (Liang et al., 2020). Our protocol specifically isolates these mitochondria primed stem cells (mpSCs) by combining a TIC marker (Aldh activity) with Δ_Ψ. Aldh activity is a TIC marker for several tumor types, including ovarian, breast, cervical, lung, liver, prostate, bone marrow, skin, colon, and pancreatic (Marcato et al., 2011; Toledo-Guzmán, et al., 2019; Vassalli, 2019). We developed this protocol in human ovarian cancer cell lines; however, we have also performed these assays in human ovarian cancer cells derived from a mouse xenograft model as well as patient ascites. The sorted cells can be studied for stem cell or other properties with relevant methods. We have studied mitochondrial structural and functional properties of the sorted cells using our novel assay mito-SinCe² (Spurlock et al., 2019) (example in Figure 3). This protocol is a simple and valuable tool for the isolation and study of normal and neoplastic stem cells from cell lines as well as primary cell populations. Thus, this protocol will allow a basic understanding of mitochondrial and energetic heterogeneity within stem cell population, and may also prove valuable in translational studies in regenerative medicine and cancer biology.