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

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.

Mechanoactivation of NOX2-generated ROS elicits persistent TRPM8 Ca 2+ signals that are inhibited by oncogenic KRas

“…In nontumorigenic cells, some of the earliest signaling events that can occur in response to a mechanical stimulus are the unfolding of proteins ( 26 – 29 ), initiation of ionic currents (e.g., calcium) ( 30 , 31 ), or generation of reactive oxygen species (ROS) signaling ( 32 ). Indeed, our prior work demonstrating a rapid calcium signaling response to scratch wounding in nontumorigenic breast epithelial and breast cancer cells supports a role for rapid mechanochemical signaling in breast cancer ( 33 ).…”

“…Therefore, we developed an assay to test the earliest responsiveness of epithelial cells to mechanical stimuli. Our previously described real-time scratch assay ( 33 ) demonstrated the responsiveness of breast epithelial and breast cancer cells to acute mechanical scratch through the rapid initiation (within seconds) of cytoplasmic calcium signaling. However, scratch assays are routinely performed on rigid plastic or glass surfaces that greatly exceed the stiffness of the tumor microenvironment, are inherently damaging to cells, and can introduce confounding elements resulting from the nonspecific dumping of cellular contents.…”

“…We previously reported that breast epithelial cells are responsive to mechanical scratch through the initiation of cytoplasmic calcium signaling, a mechanism both spatially and temporally regulated ( 33 ), whereby signaling away from the scratch area to neighboring cells was an intercellular communication mediated by extracellular ATP and activation of purinergic P2Y2 GPCRs, but cells at the immediate scratch edge showed persistent increases in cytoplasmic calcium from extracellular sources for up to 50 min. Our data from applying scratch on dishes of increasing stiffness isolate these mechanisms, ultimately showing that signaling to neighboring cells is also mediated by cell damage ( Fig.…”

Alterations of Ion Homeostasis in Cancer Metastasis: Implications for Treatment

“…For the latter, extracellular ATP and activation of P2Y2 receptors are necessary. Aggressively tumorigenic and metastatic cells displays drastic reductions in the calcium signal changes in response to loss of cell-cell contact ( 29 ).…”

Calcium signaling: breast cancer’s approach to manipulation of cellular circuitry

“…However, future studies may reveal even more complexity beyond mere changes in protein expression, as genomically unstable cancer cells inherently have a high probability for mutagenesis that could alter protein activity and calcium signaling. Indeed, some studies report that cancer cell lines which harbor many different mutations, as well as specific cancer mutations such as KRas, can alter breast epithelial calcium signaling responses (Pratt et al 2018(Pratt et al , 2020.…”