Strategy of Isolating ‘Primed’ Tumor Initiating Cells Based on Mitochondrial Transmembrane Potential

Abstract

Various stem cells have been found to be dependent on mitochondrial energetics. The role of mitochondria in regulating the self-renewal of normal stem cells and stem-like tumor initiating cells (TICs) is increasingly being appreciated. We proposed that TIC populations have a sub population of cells that are "primed" by mitochondria for self-renewal. Using ovarian cancer model, we have developed a protocol to identify and isolate these "primed" cells using Fluorescence-Assisted Cell Sorting (FACS). We combined live cell stains for a functional marker of TICs and for mitochondrial transmembrane potential to enrich TICs with higher mitochondrial potential that form in vitro spheroids 10-fold more than the other TICs with lower mitochondrial potential. This protocol can be directly used or modified to be used in various cell types. Thus, this protocol is anticipated to be invaluable for the 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.

Spheroid architecture strongly induces miR-221/222 expression and promotes oxidative phosphorylation and survival of mobile tumor cells through a mechanism that includes restriction of miR-9 expression

“…Adaptation to glucose-deprived condition of the peritoneum by switching to oxidative metabolism is thought to maximize the survival of ovarian cancer stem cells and promote metastasis and tumor growth at distal sites. Consistent with this hypothesis, segregation of ovarian cancer stem cells with higher relative mitochondrial transmembrane potential yields cells with more potent tumor-initiating and spheroid-forming properties [48]. Using an immunodeficient murine xenograft model, we directly compared the survival of CAP cells injected into the peritoneum after growth over ULP as spheroids with the survival of TOV-LT cells injected into the peritoneum after growth over ULP as loose clumps.…”

Enrichment of carcinogen-driven “mitochondria-primed” human skin stem cells and their identification using single-cell analyses

“… 1 Quantify the abundance of neoplastic stem cells using a tumorsphere assay with Extreme Limiting Dilution Analysis statistics, as described in our previously published step by step protocol. 2 Enrichment of neoplastic stem cells in the TF1 population can be confirmed by comparing the quantified abundance of stem cells (frequency) obtained in step 15 between clones transformed with 1 nM TCDD (TF1) or 10 nM TCDD (TF10) and the Parental population. To confirm the unique mitochondrial structure related to mitochondrial priming in the TF1 population, perform the single cell mitochondrial structural analyses (described in our previously published step by step mito-SinCe 2 protocol 4 ) on Mitotracker green stained cells (described in our previously published step by step protocol 5 ).…”

“… 2 Enrichment of neoplastic stem cells in the TF1 population can be confirmed by comparing the quantified abundance of stem cells (frequency) obtained in step 15 between clones transformed with 1 nM TCDD (TF1) or 10 nM TCDD (TF10) and the Parental population. To confirm the unique mitochondrial structure related to mitochondrial priming in the TF1 population, perform the single cell mitochondrial structural analyses (described in our previously published step by step mito-SinCe 2 protocol 4 ) on Mitotracker green stained cells (described in our previously published step by step protocol 5 ). Note: From this step onwards TF1 population will be considered as the mpSC enriched population, while the TF10 and P populations will be considered as neoplastic and parental non-neoplastic controls, respectively.…”

“…We have previously described flow cytometry dependent isolation of neoplastic mitochondria primed stem cells (mpSCs) from ovarian cancer cells. 2 Here, we describe the protocol for a series of experiments demonstrating enrichment and identification of neoplastic mpSCs from skin keratinocytes. In the main publication, we showed that low dose of the environmental carcinogen 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) enriches the mpSCs with greater self-renewal ability that are sustained by fine-tuned repression of mitochondrial fission.…”

Fine-tuned repression of Drp1-driven mitochondrial fission primes a ‘stem/progenitor-like state’ to support neoplastic transformation

“…In contrast, an elevated mitochondrial fusion state, sustained by mitofusin, can drive immortalization of neoplastic stem cells to support tumorigenesis in a Drosophila model (Bonnay, Veloso et al, 2020). Using analyses of single cells, we reported that neoplastic ovarian cells with minimum mitochondrial fission are found within a (Aldh marked) neoplastic stem cell sub-population that has >10 folds self-renewal and proliferation ability compared to the other (Aldh marked) neoplastic stem cell sub-population (Spurlock, Gupta et al, 2019, Spurlock, Hanumanthu et al, 2021. Thus, we proposed, that a repressed mitochondrial fission state may prime neoplastic stem cells towards maximizing their potential of self-renewal and proliferation.…”