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Real-time scratch assay reveals mechanisms of early calcium signaling in breast cancer cells in response to wounding


The connections between mechanically-activated ATP signaling, purinergic receptors, calcium signaling, and EMT in vitro cancer biology are mounting, but are still not well-defined.

Dr. Stephen J.P. Pratt and Dr. Stuart S. Martin research team said that "By first experimentally defining rapid mechanically-induced calcium signaling in cancer cells, this work sets a foundation to explore mechano-calcium relationships driving malignant progression"

There is growing evidence supporting the notion that calcium signaling is affected at multiple levels in cancer and that calcium, calcium permeable channels, and calcium-binding proteins may play an important role in tumor progression.

Here the research team went on to describe early signaling mechanisms in human breast cancer cells in response to mechanical wounding.

Figure 1: Real-time scratch assay reveals early calcium signaling in MCF-7 cells in response to wounding. (A) Human breast MCF-7 cancer cells were loaded with the calcium sensitive dye Fluo-4 and mechanically stimulated using a blunt fire-polished glass microprobe. Time series imaging shows that mechanically-induced increases in intracellular calcium occurred in cells that were directly stimulated and was followed by rapid changes in calcium from neighboring cells. This wave-like signal propagation occurred in radial fashion (up to 10 cells away, ~200μm) and was relatively transient compared with a directly stimulated cell showing persistent cytosolic calcium (arrow). (B) Fluo-4 loaded MCF-7 cell monolayers were simultaneously scratched with a glass pipette and imaged for 400 seconds (6.7 minutes). Similar to mechanical touch, this real-time scratch assay revealed mechanically-induced increases in intracellular calcium at the wound edge followed by a time-dependent signal propagation to neighboring cells at far distances (~30 cells away, ~500μm). Y-axis vs. intensity traces represent data from corresponding x-y frames (positioned directly above). Pixel intensity was plotted for all y-axis points (i.e. 512 pixels or 1.3mm image height) from a single frame, however these values were derived from averaging the pixel intensity across all x-axis points (i.e. direction of scratch for all 512 pixels). These assays indicate intercellular communication in response to changes in the mechanical environment or to wounding. Scale bars equal 200μm.

They were able to resolve mechanically-stimulated calcium signaling at the wound edge and the resulting intercellular communication to distant cells using a real-time scratch assay.

Propagation of calcium signaling to distant cells resolved within seconds, while cells at the wound edge demonstrated persistent elevation of calcium for up to 50 minutes.

Calcium, a ubiquitous second messenger, is involved in many cellular processes identified as hallmarks of cancer such as regulation of the cell cycle, invasion, migration and cell death.

By first experimentally defining rapid mechanically-induced calcium signaling in cancer cells, this work sets a foundation to explore mechano-calcium relationships driving malignant progression.

The Pratt/Martin research team concluded "These mechanisms now provide a clear framework for investigating which short-term calcium signals promote long-term changes in cancer cell biology."

Full text - https://doi.org/10.18632/oncotarget.25186

Correspondence to - Stephen J.P. Pratt - sjppratt@umaryland.edu and Stuart S. Martin - SSMartin@som.umaryland.edu

Keywords - human breast cancer, calcium, mechanotransduction, purinergic receptor, intercellular signaling

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