Research Papers:

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

Stephen J.P. Pratt _, Erick O. Hernández-Ochoa, Rachel M. Lee, Eleanor C. Ory, James S. Lyons, Humberto C. Joca, Ashley Johnson, Keyata Thompson, Patrick Bailey, Cornell J. Lee, Trevor Mathias, Michele I. Vitolo, Matt Trudeau, Joseph P. Stains, Christopher W. Ward, Martin F. Schneider and Stuart S. Martin

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Oncotarget. 2018; 9:25008-25024. https://doi.org/10.18632/oncotarget.25186

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Stephen J.P. Pratt1,2,3, Erick O. Hernández-Ochoa1, Rachel M. Lee2,3, Eleanor C. Ory2,3, James S. Lyons4, Humberto C. Joca4, Ashley Johnson2, Keyata Thompson2,3, Patrick Bailey1,2,3, Cornell J. Lee2,3, Trevor Mathias2,3, Michele I. Vitolo2,3, Matt Trudeau2, Joseph P. Stains4, Christopher W. Ward4,5, Martin F. Schneider1 and Stuart S. Martin1,2,3

1Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, USA

2Department of Physiology, University of Maryland School of Medicine, Baltimore, MD, USA

3Marlene and Stewart Greenebaum NCI Comprehensive Cancer Center, University of Maryland School of Medicine, Baltimore, MD, USA

4Department of Orthopaedics, University of Maryland School of Medicine, Baltimore, MD, USA

5School of Nursing, University of Maryland, Baltimore, MD, USA

Correspondence to:

Stephen J.P. Pratt, email: [email protected]

Stuart S. Martin, email: [email protected]

Keywords: human breast cancer; calcium; mechanotransduction; purinergic receptor; intercellular signaling

Received: October 26, 2017     Accepted: April 03, 2018     Published: May 18, 2018


Aggressive cellular phenotypes such as uncontrolled proliferation and increased migration capacity engender cellular transformation, malignancy and metastasis. While genetic mutations are undisputed drivers of cancer initiation and progression, it is increasingly accepted that external factors are also playing a major role. Two recently studied modulators of breast cancer are changes in the cellular mechanical microenvironment and alterations in calcium homeostasis. While many studies investigate these factors separately in breast cancer cells, very few do so in combination. This current work sets a foundation to explore mechano-calcium relationships driving malignant progression in breast cancer. Utilizing real-time imaging of an in vitro scratch assay, we were able to resolve mechanically-sensitive calcium signaling in human breast cancer cells. We observed rapid initiation of intracellular calcium elevations within seconds in cells at the immediate wound edge, followed by a time-dependent increase in calcium in cells at distances up to 500μm from the scratch wound. Calcium signaling to neighboring cells away from the wound edge returned to baseline within seconds. Calcium elevations at the wound edge however, persisted for up to 50 minutes. Rigorous quantification showed that extracellular calcium was necessary for persistent calcium elevation at the wound edge, but intercellular signal propagation was dependent on internal calcium stores. In addition, intercellular signaling required extracellular ATP and activation of P2Y2 receptors. Through comparison of scratch-induced signaling from multiple cell lines, we report drastic reductions in response from aggressively tumorigenic and metastatic cells. The real-time scratch assay established here provides quantitative data on the molecular mechanisms that support rapid scratch-induced calcium signaling in breast cancer cells. These mechanisms now provide a clear framework for investigating which short-term calcium signals promote long-term changes in cancer cell biology.

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