Abstract
Isolation of tumor-initiating cells currently relies on markers that do not reflect essential biologic functions of these cells. We proposed to overcome this limitation by isolating tumor-initiating cells based on enhanced migration, a function tightly linked to tumor-initiating potential through epithelial-to-mesenchymal transition (EMT). We developed a high-throughput microfluidic migration platform with automated cell tracking software and facile recovery of cells for downstream functional and genetic analyses. Using this device, we isolated a small subpopulation of migratory cells with significantly greater tumor formation and metastasis in mouse models. Whole transcriptome sequencing of migratory versus non-migratory cells from two metastatic breast cancer cell lines revealed a unique set of genes as key regulators of tumor-initiating cells. We focused on phosphatidylserine decarboxylase (PISD), a gene downregulated by 8-fold in migratory cells. Breast cancer cells overexpressing PISD exhibited reduced tumor-initiating potential in a high-throughput microfluidic mammosphere device and mouse xenograft model. PISD regulated multiple aspects of mitochondria, highlighting mitochondrial functions as therapeutic targets against cancer stem cells. This research establishes not only a novel microfluidic technology for functional isolation of tumor-initiating cells regardless of cancer type, but also a new approach to identify essential regulators of these cells as targets for drug development.
Highlights
IntroductionMarker-based approaches for TICs suffer from several limitations: i) a modest enrichment for TICs with a large portion of recovered cells lacking the ability to form new tumors[10]; ii) inconsistency across different cancer types and even within the same type of cancer[9,10,11,12]; and iii) limited relation to actual functions of TICs or patient prognosis[13,14]
1/stem cell frequency with confidence intervals and CD24−/low/CD44+), we compared differentially expressed genes we identified for migratory cells with those reported in literature[16]
The results show that phosphatidylserine decarboxylase (PISD) regulates several in vitro phenotypes associated with epithelial-to-mesenchymal transition (EMT) and TICs in breast cancer
Summary
Marker-based approaches for TICs suffer from several limitations: i) a modest enrichment for TICs with a large portion of recovered cells lacking the ability to form new tumors[10]; ii) inconsistency across different cancer types and even within the same type of cancer[9,10,11,12]; and iii) limited relation to actual functions of TICs or patient prognosis[13,14]. To advance studies of TICs, we developed a high-throughput microfluidic platform to isolate TICs in breast cancer by the EMT property of enhanced cell migration. This approach enriches TICs based on an essential function rather than empirically-defined markers. The strong relationship between EMT and TICs across almost all epithelial cancers suggests that our approach may become a general technology to isolate TICs in multiple malignancies beyond breast cancer
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