Ca2+ sonotransfer into breast cancer cells in a suspension, 3-D spheroid and subcutaneous tumor models

Calcium-based treatments have gained considerable attention in the field of electroporation, primarily, due to their comparable efficacy to conventional electro-chemotherapy. However, their applications in sonoporation remain under-investigated, despite its high potential for site-specific and temporally-controlled drug delivery. Current study examines the curative potential of calcium sonoporation across multiple experimental models, including: i) cell suspension, ii) 3-D spheroid culture and iii) subcutaneous murine breast cancer tumors. Murine breast cancer is an established analogue of stage IV human breast cancer. For comparison, parallel experiments, using classical anticancer drug bleomycin were performed. Ca²⁺ sonoporation efficiently enhanced 4 T1 cell death in a suspension in the absence of microbubbles, under relatively low acoustic pressure (100-200 kPa). In contrast, efficient spheroid growth reduction required microbubble-mediated inertial cavitation at higher (700 kPa) acoustic pressure. In vivo, Ca²⁺ sonoporation demonstrated similar tumor growth reduction as bleomycin sonoporation. Both treatments reduced tumor growth from the third day after the onset of treatment. Successful cancer treatment was achieved even at lower values of cavitation dose metrics. Our study presents a multi-level validation of Ca²⁺ sonoporation as an effective treatment strategy for murine breast cancer. Importantly, complete tumor eradication and prolonged animal survival up to one month were observed even at significantly reduced cavitation activity, indicating clinical safety of the treatment.