Ultrasound-stimulated microbubble enhancement of radiation treatments: endothelial cell function and mechanism

Endothelial cell death caused by novel microbubble-enhanced ultrasound cancer therapy leads to secondary tumour cell death. In order to characterize and optimize these treatments, the molecular mechanisms resulting from the interaction with endothelial cells were investigated here. Endothelial cells (HUVEC) were treated with ultrasound-stimulated microbubbles (US/MB), radiation (XRT), or a combination of US/MB+XRT. Effects on cells were evaluated at 0, 3, 6, and 24 hours after treatment. Experiments took place in the presence of modulators of sphingolipid-based signalling including ceramide, fumonisin B1, monensin, and sphingosine-1-phosphate. Experimental outcomes were evaluated using histology, TUNEL, clonogenic survival methods, immuno-fluorescence, electron microscopy, and endothelial cell blood-vessel-like tube forming assays. Fewer cells survived after treatment using US/MB+XRT compared to either the control or XRT. The functional ability to form tubes was only reduced in the US/ MB+XRT condition in the control, the ceramide, and the sphingosine-1-phosphate treated groups. The combined treatment had no effect on tube forming ability in either the fumonisin B1 or in the monensin exposed groups, since both interfere with ceramide production at different cellular sites. In summary, experimental results supported the role of ceramide signalling as a key element in cell death initiation with treatments using US/MB+XRT to target endothelial cells.