Single cell gene expression analysis in injury-induced collective cell migration

Abstract

Collective cell behavior in response to mechanical injury is central to various regenerative and pathological processes. Using a double-stranded locked nucleic acid probe for monitoring real-time intracellular gene expression, we examined the spatiotemporal response of epithelial cells during injury-induced collective migration and compared to the blocker assay with minimal injury as control. We showed that cells ~150 µm from the wound edge exhibit a gradient in response to mechanical injury, expressing different genes depending on the wounding process. While release of contact inhibition is sufficient to trigger the migratory behavior, cell injury additionally induces reactive oxygen species, Nrf2 protein, and stress response genes, including heat shock protein 70 and heme oxygenase-1, in a spatiotemporal manner. Furthermore, we show that Nrf2 has an inhibitory role in injury-induced epithelial-mesenchymal transition, suggesting a potential autoregulatory mechanism in injury-induced response. Taken together, our single-cell gene expression analyses reveal modular cell responses to mechanical injury, manipulation of which may afford novel strategies for tissue repair and prevention of tumor invasion in the future.

Spatiotemporal dynamics of microRNA during epithelial collective cell migration

“…To study the dsLNA probe’s ability to measure the spatial distribution of miRNA, MCF-7 epithelial cells were seeded in a 24-well plate and grown to confluence before a model wound was created in the monolayer with a pipette tip. β-actin mRNA has been previously shown to increase at the wound edge as the cells manage cell growth and migration [20]. The random control probe again signified the background level inherent in the experiment.…”

“…Of these modifications, 2′-sugar modifications, such as locked nucleic acids (LNA), are some of the most effective RNA-binding oligonucleotides due to their increased binding affinity and resistance to degradation [17–19]. In particular, alternating DNA/LNA bases has been shown to not only optimize the balance between specificity and stability for intracellular detection, but it has also proven accurate enough to distinguish gene expression in a population of cells down to the single-cell level [10, 20]. …”

NRF2 activates a partial Epithelial-Mesenchymal Transition and is maximally present in a hybrid Epithelial/Mesenchymal phenotype

“…NRF2 has been well-studied in the context of cellular redox status, where it acts as a regulator in response to diverse forms of stress [28], for instance by activating antioxidant response within the cell to respond to oxidative stress, thus promoting cell survival [29]. However, its role in mediating clustered cell migration -a proposed outcome of partial EMT [1] -has been only recently explored [12,30].…”

Advances in Techniques for Probing Mechanoregulation of Tissue Morphogenesis

“…Careful experimental designs with multiple techniques are often required to study collective cell behavior. In addition to mechanical perturbation techniques, novel biochemical and mechanical techniques (e.g., intracellular probes 100,128 and traction force microscopy 129,130 ) should be developed and used to study morphogenic processes. Another key challenge in studying the emergent complexity of biological tissues is the difficulty in understanding the high-order structures and functions resulting from local interactions of individual cells.…”

“…98 In addition, physical stimulation with a mechanical probe may also be applied to create local mechanical stress and injury to individual cells in the tissue. [99][100][101] A mechanical probe with comb-drive (capacitive) force measurement, for instance, has been used to study mechanical stimulation-induced intercellular calcium communication in microengineered endothelial networks. 102,103 Endothelial cells collectively regulated their calcium levels against a large range of probing forces and repeated stimulations.…”