Disruption Of Tumor Angiogenesis Using Microbubble Enhanced Ultrasound At Different Peak Negative Pressures

Background:Angiogenesis is an important condition for tumor growth, invasion and metastasis. It isknown that solid tumor greater than1-2mm cannot grow without tumorangiogenesis. Tumor angiogenesis displays many structural and functional abnormalities:such as thin vessel wall, incomplete basement membrane without smooth muscle,hyperpermeability, pericytes osteoporosis, etc when compared with normal vessel. Theseabnormalities make tumor vessels sensitive targets of anti-angiogenesis therapy. Targetedanti-angiogenesis methods achieve the purpose of cancer treatment by inhibiting ordestroying tumor angiogenesis. However, the existing targeted anti-tumor angiogenesismethods, such as angiogenesis inhibitors, can also affect the body’s physiological processes(wound healing, tissue repair, etc.) during anti-angiogenesis process, and cannot avoid drugresistance.Intravascular microbubble (MB) enhanced ultrasound (MEUS) cavitation canphysically destroy the tumor microvessels and block tumor perfusion. The mechanism liesin reversible or irreversible damages of tumor vascular wall caused by mechanical effectssuch as shock waves and micro-jets generated by cavitation. In preliminary experiments wefound that pulsed focused ultrasound with a peak negative pressure (PNP) of4.8MPa canirritate microbubbles and thus block tumor microcirculation for24hours. When PNP wasreduced to2.6MPa, microcirculation blockage can only lasts for one hour. Microbubbleenhanced ultrasound (MEUS) cavitation treatment has good prospects such as safety,efficiency, simple operation, and easily repeatability. To further understand the relationshipbetween the intensity of ultrasonic cavitation and tumor microvascular injury, we treatedrabbit VX2tumors with MEUS at three different levels of peak negative pressure.Contrast-enhanced ultrasound (CEUS) and immunohistochemistry were performed toassess tumor circulation and microvessel density (MVD). Objectives:1. To observe the therapeutic effects of MEUS at different PNP levels on VX2tumoraccording to the changes of tumor blood perfusion, and preliminarily analyze the influenceof ultrasound peak negative pressure on blocking degrees of tumor microcirculation.2. To investigate the dose-effect relationship between PNP and MVD by comparingtumor MVD of different groups.Materials and Methods:1. Materials⑴Instrument and equipment:①Therapeutic ultrasound instrument: DCT-700digitalultrasonic cavitation treatment device. It was designed and manufactured by ShenzhenWilder Medical Electronics limited-liability company. Ultrasonic frequency is1.0MHz,with a pulse repetition frequency of100Hz, a duty cycle of1.5%, and adjustablepeak negative pressure.②Diagnostic ultrasound imaging system: Siemens S2000ultrasound system with contrast pulse sequence (CPS) imaging mode was used to performcontrast enhanced ultrasound (CEUS). The9L4linear probe with the emission frequency of7~9MHz and the center frequency at8MHz was applied.⑵Animals: A total of fourty-six healthy male New Zealand white rabbits were used.All the rabbits were provided by the laboratory animal center of Xinqiao hospital, with anaverage body weight between1.5kg to2.0kg.⑶Reagents:①Microbubbles:“Zhifuxian” ultrasound contrast agent made byperfluoropropane gas core with a lipid shell was used for the nucleation of acousticcavitation as well as the contrast agent of CEUS. Its diameter was2~8um with meandiameter2um, and its concentration was approximately4~9×109/ml.②The mouseanti-human CD31monoclonal antibody (Platelet endothelial cell adhesion molecule-1,PECAM-1/CD31) was purchased from Abcam company, stored at4°refrigerator.2. methods⑴Treatment experiment of VX2tumors by MEUS at different PNPs:Thirty intramuscular VX2tumors (sixteen New Zealand rabbits bearing muscle VX2 tumor) were randomly assigned to three groups treated by MEUS at2184kPa,1785kPa,and1019kPa PNPs respectively. The peak negative pressure groups receivedcorresponding ultrasonic parameters treatment. The rabbits were subjected to ultrasoundirradiation for5minutes and injected with0.2ml MBs. Before and after treatment, thetumor blood perfusion was using contrast-enhanced ultrasound (CEUS) for analysis. To getpeak intensity (PI) value and area under curve (AUC) value we need to analyse thetime-intensity curve (TIC). Finally, the tumors were harvested for histological examination.⑵Dose-effect relation study:Thirty New Zealand rabbits bearing muscle VX2tumor were randomly assigned intofour groups: the three different PNP groups(n=8) and the control group(n=6). Thecontrol group received no treatment. The other three groups received treatments similar asthat described above. After the treatment, the tumors were immediately harvested, fixed,embedded, and sliced. Then immunohistochemical staining was conducted to calculatemicrovessel density (MVD). MVD values of different groups were compared statistically.Results:1. Treatment experiment of VX2tumors by MEUS at different PNPs:CEUS shows that tumor perfusion almost vanished when treated by MEUS at2184kPa PNP, with the average PI value reduced from (40.5±9.9)%(before treatment) to(11.6±7.8)%(after treatment), and the average AUC value reduced from (1299.1±512.6)%sto (280.1±186.1)%s (P<0.05); Significant declined of tumor blood perfusion can also befound in the tumors treated by MEUS at1785kPa PNP, with the average PI reduced from(42.5±7.8)%to (24.2±14.8)%, and the average AUC reduced from (1378.1±494.6)%s to(549.4±463.4)%s (P<0.05); Tumor blood perfusion slightly increased in the1019kPa PNPgroup, but the difference shows no statistic significance (P>0.05). Pathological examinationshowed signs of tumor vascular damages in the three treatment groups, such as rupturedvascular wall, overflowed erythrocytes, microvascular bleeding, red blood cells in tissueinterspace, and edema.2. Dose-effect relation study:The average MVD values of2184kPa,1785kPa, and1019kPa PNP groups were(157.15±124.37),(141.12±60.45), and (311.64±73.93), respectively, all lower than theMVD value of the control group tumor (619.73±282.78). Statistical analysis shows that the differences were statistically significant (P<0.05), while the differenced between any twotreatment groups were not statistically significant (P>0.05).Conclusions:1. The blood perfusion of VX2tumor can be blocked by MEUS treatment at the PNP of1785kPa and2185kPa. However, MEUS treatment at1019kPa PNP may cause increasedblood perfusion.2. Intravenous microbubble mediate ultrasound cavitation can reduce the number oftumor microvessels. It may be related to the destruction of tumor microvessel structure byultrasound cavitation. Besides, tumor MVD value declined more in the2184kPa and1785kPa PNP groups than in the1019kPa PNP group.