Abstract
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.
Highlights
Altered calcium handling and the mechanical properties of tumors are both emerging as possible modulators of breast cancer
Mechanically-induced calcium signaling has been established in many epithelial cell types [27, 28] including mammary epithelial cells [29], the mechanical induction of calcium has not been well characterized in cancer
This was followed by a time-dependent increase in intracellular calcium in cells at much greater distances from the wound edge compared to mechanical touch
Summary
Altered calcium handling and the mechanical properties of tumors are both emerging as possible modulators of breast cancer. Tumor rigidity and extracellular matrix (ECM) stiffness are increasingly recognized as important contributors to disease progression [1,2,3,4]. Preclinical studies show that ECM stiffness can disrupt adherens junctions [7], alter cellular differentiation [8], and increase cell proliferation [9] and migration [10]. Many of these malignant cell phenotypes are associated with altered calcium signaling [11, 12]. There may be a critical intersection between calcium signaling and tumor mechanics in driving aggressive cellular phenotypes, the precise link remains unclear
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