Ultrasound and microbubbles (USMB) potentiated doxorubicin penetration and distribution in 3D breast tumour spheroids

Abstract

Ultrasound and microbubbles (USMB) has been shown to improve chemotherapy in vitro cell suspension and monolayer models. These models are limited in their ability to study the potential of USMB in enhancing drug penetration within a three-dimensional (3D) tissue model which can in part reproduce the organization of a microtumour recapitulating cell-cell interactions and diffusion of molecules through the tissue. The 3D tumour spheroid models are considered as bridge for filling the gap between 2D in vitro monolayer cultured cells and animal models. In this study, we developed a 3D MDA-MB-231 breast cancer spheroid model and investigated the potential of USMB in enhancing drug penetration and distribution within the spheroid. The 3D spheroids were exposed to USMB (770 kPa Peak negative pressure, 1 MHz pulse frequency, 8 μs pulse duration and 2 m s pulse repetition period; and Definity at 1.7% v/v concentration) and doxorubicin (Dox) at 50 μM concentration. The short- and long-term effects on the spheroids were assessed using flow cytometry and Laser scanning confocal microscopy (LSCM). The application of USMB enhanced penetration of Dox into deeper regions of tumour spheroid. The Dox distribution was assessed using 3D optical sectioning and radial profiling at different z-planes of the spheroids. The flow cytometry data confirms that the USMB increases the Dox uptake per cell by 25% with 1.2 fold increase in total Dox fluorescence. No significant differences were observed in the long-term spheroid size with Dox + USMB compared to Dox alone. However, the spheroids appear more dispersed with the combined treatment of Dox + USMB compared to Dox alone. The tumour model show the enhanced chemotherapeutic effect in 3D tumour spheroids of USMB along with increased penetration and accumulation of chemotherapeutic molecules into deeper spheroid regions.