Vascular Effects Of Rat Walker-256 Tumor Stimulated Bylow-intensity Ultrasound: A Preliminary Study

Background:Malignant tumor has become the leading cause of death in China.Chemotherapy is often the preferred method of treatment for most cancers.However,tumor response to chemotherapy is different,some tumors appear to be resistant or not sensitive to chemotherapy,especially those with poor blood supply,such as pancreatic cancer.When a tumor is poorly perfused,the drug delivery to tumor tends to be restricted and so the drug concentration.Consequently the cytotoxic effect of chemotherapy on tumor cells is decreased.Therefore,it is of clinical significance to increase the blood supply and vascular permeability of tumor during chemotherapy.In recent years,with the further research on the therapeutic effect of ultrasound(US)contrast agent,microbubble(MB)enhanced ultrasound cavitation has been used in promoting drug delivery and tumor ablation.Sonoporation induced by acoustic cavitation can increase tissue permeability by disrupting tissue or cell barrier.In previous experiments we found that pulsed ultrasound combined with microbubbles can destruct tumor microvasculature at the peak negative pressure of 2.6-4.8 MPa,resulting in hematoma,edema and tumor circulation blockage.We also found that low pressure amplitude US of 1.0MPa may enhance the blood perfusion of VX2 tumor in rabbits.For better understanding of tumor vascular effects induced by low pressure US and microbubbles,Walker-256 tumors of rat were treated with four different US pressure levels combined with microbubbles in this experiment.Contrast-enhanced ultrasound(CEUS)and histological examination were performed to evaluate the blood perfusion change and the vascular morphology of tumor.In order to explore the effects of high pressure amplitude pulsed US on tumor vascular structure and permeability,we treated Walker-256 tumor with 4.6MPa US followed by intravenous injection of doxorubicin(Dox).All of the tumors were assessed by CEUS and pathology.In addition,were used to visualize of Dox.The Dox concentration and distribution from the tumor samples were further quantified by fluorescent microscopy and high-pressure liquid chromatography tandem mass spectrometry(LC-MS/MS).Objectives:1.To investigate the vascular effects of Walker-256 tumor treated by four different US pressure levels.2.To investigate the circulation blockage effect of high pressure US on Walker-256 tumor of rats and the effects on Dox concentration of tumor.Materials and Methods:1.Materials(1)Instruments:A diagnostic US system(Vevo 2100,Visual Sonics,Canada)equipped with 20 MHz linear probe was used to image the tumors.The therapeutic US of the first study was generated by a digital US device manufactured by Shenzhen Welld Medical Electronics Co.The frequency of the therapeutic transducer was 1.0 MHz and the peak negative pressure ranged from 100 to 1500 k Pa.The peak negative pressure was calibrated by an acoustic detection system with a needle hydrophone(HNC-0400,ONDA).The therapeutic US instrument used in the second study,CZ-960,was manufactured by Mianyang Sonic Electronics Co.,Ltd.The weak focused transducer was operated with a frequency of 831 KHz and the acoustic pressure of 4.6 MPa.(2)Animals:A total of 33 healthy male SD rats,with an body weight between 150 g to 180 g were used.The laboratory animal center of Xinqiao hospitalprovided all the rats.(3)Reagents:Zhifuxian,a lipid-coated microbubble with perfluoropropane gas core,was used as both US contrast agent and cavitation nuclei.The microbubble concentration is(4~9)×109/ml and the mean particle diameter is 2?m.Doxorubicin(Dox)is produced by Hu Bei Zhonglong Company.2.MethodsIn the first part of study,fifty-two tumors implanted on male SD rats by cell inoculation,were randomly assigned to four therapy groups treated with ultrasound at 200kPa(n=12),400 k Pa(n=10),600kPa(n=12)and 800kPa(n=12)respectively and one control group.The duty cycle was 1% and the ultrasound was delivered to the tumor for 5 minutes.In each of the therapy groups,0.1ml MBs were injected via the tail vein during the ultrasound explosion for half of the tumors.The other half of the tumors received injection of the same volume of saline.The blood perfusion of tumor was quantified by CEUS before and after treatment and the gray scale value(GSV)of tumor CEUS were analyzed.Finally,the tumors were removed for histological examination.In the second part of study,fourteen tumors,built in 7 healthy male SD rats by cell inoculation,were randomly assigned into two groups,the control group and the experimental group.The pressure of ultrasound used for treatment was 4.6MPa.The blood perfusion of tumor was quantified by CEUS before and after treatment.The animals received intravenous infusion of Dox via the tail vein immediately after ultrasound treatment and Dox infusion duration was 30 mins.Finally,the tumors were removed for histological examination,fluorescent microscopy examination and high-pressure LC-MS/MS analysis.Results:In the first part of study,CEUS showed that there was no significant difference in GSV before and after treatment in all groups(P > 0.05).Pathological examination showed plasma extravasation and slight tissue edema in the tumors treated with 600 kPa ultrasound.Significant hemorrhage and tissue edema were observed in the tumors treated with 800 kPa ultrasound,with or without MB administrated during US exposure.In the second part of study,pathological examination showed large volume of fresh bleeding and slight tissue edema in the tumors treated with 4.6MPa ultrasound.CEUS showed that tumor perfusion was blocked significantly when treated with US at 4.6MPa,with the average GSV reduced from(18.1±5.0)(before treatment)to(6.7±3.8)(after treatment).Compared with the control group,the fluorescence distribution of Dox in the treatment group was significantly decreased.High-pressure LC-MS/MS analysis showed that the concentration of Dox in the tumor tissue of the experimental group(4.68±1.01)?g/g was significantly lower than that in the control group(7.49±0.81)?g/g(P<0.05).Conclusions:In the first part of study,we find thatlow-pressure-amplitude US cannot induce significant changes of blood perfusion in rat Walker-256 tumor,but the tumor microvascular permeability is increased at the acoustic pressure of 600-800 kPa.In the second part of study,we can make a conclusion that the blood perfusion and drug concentration in rat Walker-256 tumor is decreased at the acoustic pressure of 4.6MPa.