Introduction: The transient receptor potential cation channel subfamily V member 4 (TRPV4) is elevated in the basal molecular subtype of breast cancer. These breast cancers have poor prognosis and significantly overlap with the triple negative breast cancers. TRPV4 appears to contribute to the migration potential of breast cancer cells. However, the consequences of pharmacological activation of TRPV4 using the TRPV4 activator GSK1016790A have not been fully explored, particularly in the context of single cell Ca²⁺ imaging.

Aims: To assess temporal and spatial changes in cytoplasmic free Ca²⁺ ([Ca²⁺]_CYT) induced by the TRPV4 activator GSK1016790A in MDA-MB-468 basal breast cancer cells.

Methods: MDA-MB-468 cells were plated onto 96-well microplates and loaded with the Ca²⁺ sensitive indicator Fluo-4 or Fura-2. Fluorescence changes induced by 0, 1 or 100 nM of GSK1016790A were detected using an automated epifluorescence microscope (ImageXpress). Image segmentation analysis was used to assess changes in [Ca 2+]_CYT as assessed by Fluo-4, and ratiometric imaging was used to assess relative levels of [Ca²⁺]_CYT in Fura-2 loaded MDA-MB-468 breast cancer cells.

Results: MDA-MB-468 breast cancer cells exhibited spontaneous [Ca²⁺]_CYT oscillations. GSK1016790A at 100 nM induced pronounced, rapid and sustained increases in [Ca²⁺]_CYT in MDA-MB-468 breast cancer cells. Pronounced single cell heterogeneity was observed in [Ca²⁺]_CYT changes.

Discussion: These studies provide further evidence that MDA-MB-468 cells express functional TRPV4 channels and suggest that there may be significant heterogeneity in MDA-MB-468 breast cancer cell responses to TRPV4 activation