Disruption Of Tumor Neovasculature By Microbubbles Enhanced Ultrasound Cavitation

Background:Tumor angiogenesis is of vital importance to the growth and metastasis of solid tumors. Anti-angiogenesis, widely considered as an emerging technique in cancer therapy, usually attack or inhibit certain targets of tumor angiogenesis using specific chemical or biological substances, thus may have certain side effects as a method of pharmacologic therapy. High-intensity focused ultrasound (HIFU), at the intensity of 2000～10000 W/cm2, usually inducing permanent coagulative necrosis damage to tissues, has become a new method for tumor thermal ablation. The clinical applications of HIFU, however, were limited because of rare adequate acoustic window to access the tumors, or inevitable injuries to the adjacent structures. Physical therapy applications using ultrasound cavitation on the disruption of tumor neovasculature had seldom been reported.Microbubbles (MBs) can generate significant physical effects from cavitation under ultrasound excitation. The mechanical energy released by cavitation (non-thermal effects) has a potential ability to eliminate targeted living tissues. This hypothesis suggests a possibility of disrupting the immature, leaky and fragile tumor microvasculature. In addition, being a simple physical therapeutic method, MBs enhanced ultrasound cavitation to obstruct tumor microcirculation would have a good repeatability, and would be able to prevent the thermal side effects of HIFU treatment.Objective:To investigate the possibility of MBs enhanced ultrasound cavitation on tumor microcirculation obstruction, as well as the mechanism underneath through Pathological histology study on the neoplasm, especially the morphological changes of the tumor micro-vasculature. This new method might be a potential noninvasive physical therapeutic method on malignant tumor neovasculature. Materials and Methods:(1) Materials:①Therapeutic ultrasound instrument: specifically designed pulsed focused ultrasound (PFUS) devices, with the transducers frequency of 1.2 MHz & 831 KHz, adjustable emission/pause time, pulse length, pulse recurrence frequency, and peak acoustic pressure.②Diagnostic ultrasound imaging system: GE Logiq 9 ultrasound system, 9L probe, eligible to perform contrast enhanced ultrasonography (CEUS) with low MI.③Microbubbles: Zhifuxian, a formulation of lipid-encapsulated MBs with perfluoropropane gas, with the concentra1tion of 4~9×109 /ml. The mean diameter of MBs was measured to be 2μm, and 98% of the MBs less than 8μm in diameter.(2) Methods:①26 New Zealand rabbits bearing subcutaneous VX2 tumor were randomly assigned into 3 groups for the factors including MBs infusion and ultrasound exposure. PFUS was delivered directly to the tumor surface for 10 minutes during intravenous infusion of MBs at 0.2 ml/kg in the experimental group(PFUS +MBs). The control groups were applied with only PFUS exposure or MBs injection. The tumor perfusion was imaged using CEUS before and 0 minute, 30 minutes, and 60 minutes after treatment. The gray scale values (GSV) of tumor contrast perfusion were compared statistically.②32 male Wistar rats bearing subcutaneous Walker-256 tumor were randomly assigned into 4 different groups (Low acoustic pressure-PFUS +MBs, High acoustic pressure-PFUS +MBs, PFUS +NS, and Sham +MBs), and received respective treatments similar as described above. CEUS were performed before and after treatment, and prior to the scheduled point-of-time for animals sacrifice. After a 100 ml 4% paraformaldehyde intravascular perfusion fixation, the tumors were removed, fixed, sectioned, and stained for review with a light microscope (LM) and a transmission electron microscope (TEM). Qualitative and quantitative descriptions of the histological changes of the neoplasm, especially the morphological changes of the tumor micro-vasculature were noted and compared. A Spearman rank correlation analysis was performed to establish whether there was a relationship between the quantitative histological change and the visual score of the pre-sacrifice CEUS of the tumors. Results:①The contrast perfusion of VX2 tumors almost vanished immediately after treatment in the PFUS +MBs group, with the GSV reduced from 67.8±13.3 (before treatment) to 29.7±20.1 (0 minute post treatment, P﹤0.01), 37.3±19.9 (30 minutes post treatment), and 34.3±20.7 (60 minutes post treatment). No statistically significant were found before and after treatment (P >0.05) in both control groups (PFUS +NS & Sham +MBs).②The predominant pathological findings of insonating the subcutaneous Walker-256 neoplasms appeared to be microvascular dilation, destruction of tumor capillaries associated with hemorrhage and increased intercellular edema, and in situ thrombosis. Necrosis of neoplastic cells occurred mainly in the areas around the damaged vasculatures. Injury of the endothelial cells of tumor capillaries was confirmed by TEM. A high correlation between the quantitative histological change and the visual score of the CEUS of the tumors were demonstrated with a Spearman rank correlation analysis (rs = -0.890, P﹤0.01).Conclusions:The tumor micro-circulation can be blocked by MBs enhanced ultrasound cavitation.The main effects of MBs enhanced ultrasound cavitation over tumor micro-circulation was histological changes in the tumor vasculature, including damage of the endothelia, hemorrhage, increased intercellular fluid, and in situ thrombosis.