Inhibition of Carbonic Anhydrase IX Suppresses Breast Cancer Cell Motility at the Single-Cell Level

Protein Carbonic Anhydrase IX (CA IX), which is expressed in various hypoxic solid tumors in order to maintain proper pH, is also related to cancer cell adhesion, invasion, and metastasis processes. Here, we investigated whether CA IX inhibition by a highly CA IX selective agent benzenesulfonamide VD11-4-2 triggers changes in individual cell motility. We seeded breast cancer cells on an extracellular matrix-coated glass-bottomed dish and in a microfluidic device with a gradient flow of epidermal growth factor (EGF), tracked individual cell movement, calculated their migration speeds, and/or followed movement direction. Our results showed that the inhibitor VD11-4-2 decreased the speed of CA IX positive breast cancer cells by 20-26% while not affecting non-cancerous cell migration. The inhibitor suppressed the cell migration velocity increment and hindered cells from reaching their maximum speed. VD11-4-2 also reduced CA IX, expressing cell movement towards the growth factor as a chemoattractant. Such a single cell-based migration assay enabled the comprehensive investigation of the cell motility and revealed that VD11-4-2 shows the ability to suppress breast cancer cell migration at a lower concentration than previously tested CA IX inhibitors.