Inhibitory Effect Of Microbubble Enhanced Ultrasound Combining Endostar On Walker-256 Tumor In Rats

BackgroundTumor growth,progression and metastasis are dependent on angiogenesis,anti-angiogenic therapy is a method to suppress tumor growth by disrupting and inhibiting angiogenesis.Tumor vasculature is structurally and functionally abnormal,with irregular sizes,shapes and branching patterns,in lack of the normal hierarchy and recognizable features of arterioles,capillaries or venules.This vulnerability provides a sensitive target to mechanical disruption.Previous studies have found microbubble-enhanced ultrasound?MEUS?could generate microvasculature disruption and blood perfusion blockage and therefore might become a non-invasive physiotherapy for anti-angiogenesis.However,MEUS treatment can only arrest tumor perfusion for 24 hours but it cannot prevent a slow blood flow recovery after 24 hours.Perfusion recovery usually indicates angiogenesis.Therefore,repeated treatments were required for a persistent effect.Endostatin is an angiogenesis inhibitor which inhibits endothelial proliferation,angiogenesis and tumor growth.Endostar?human recombinant endostatin?is a derivative of human endostatin approved for the treatment of non-small-cell lung cancer in China,which has the similar blockage effect on VEGF signaling pathway and improved stability and bioactivity compared to endostatin.Previously,Zhang et al.has found the anti-tumor effect combining ultrasonic cavitation with Endostar loaded microbubbles in colon cancer.However,drug-loaded microbubbles have many limitations.The payload is usually limited and the stability is not guaranteed.It may also take a long period to develop a drug-loaded microbubble as a new drug.Recently,sonochemotherapy by combining routine chemotherapy and microbubble enhanced diagnostic ultrasound has been showed promising in experimental studies and clinical researches.Thus,we hypothesized by combining regular anti-angiogenic therapy and physical anti-vascular effect of MEUS,a potentiated therapy with double anti-vascular effects might be achieved.This combination therapy may prevent the long and expensive development of drug-loaded microbubbles.ObjectivesTo investigate the feasibility and effectiveness of combining MEUS and intravenous Endostar injection for inhibition on Walker-256 tumor in rats.Materials and methods1.Experimental apparatusVINNO 70 Color Doppler Ultrasound system?manufactured by Vinno Technology Co.Ltd?with a high frequency linear array probe?X4-12L?was operated at a frequency range of4?12 MHz.The CBI mode was used for CEUS imaging with a mechanical index of 0.08.For MEUS therapy,a therapeutic ultrasound?TUS?device specially designed for microbubble cavitation was used.The device was composed of a TUS transducer,a sinusoidal wave generator and a pulse-receiving power amplifier?CZ960,Mianyang Sonic Electronic Ltd,Mianyang,China?.The transducer operated at a frequency of 831 k Hz was built with an air-backed,spherically concave disk that was 25 mm in diameter,with a 160-mm radius of curvature.Tumors were exposed for 6s to a tone burst of 400 cycle with a 9 Hz pulse repetition frequency.The acoustic burst was intermitted by a 6 seconds interval permitting circulation to refill with microbubbles.The total sonication lasted for 5 minutes.The ultrasound peak negative pressure was 4.3 MPa measured with a calibrated hydrophone.The actual duty cycle turned out to be approximately 0.18%,which corresponded to the acoustic intensity(I_SPTA)of 1.13W/cm².2.Experimental animals60 healthy male SD rats,8 weeks old weighing about 180-200g,were provided by the Laboratory Animal Center of the Second Affiliated Hospital of Army Medical University.3.Experimental reagentsSonazoid^?,a phospholipid-coated perfluorobutane microbubbles?GE Healthcare,Oslo,Norway?were used both as an ultrasound contrast agent and as cavitation nuclei.Microbubble?MB?suspension was made by diluting 16?l of microbubbles drawn from vial to 4 ml saline solution.The MB had a mean diameter of 2.1?m and concentration of 6×10⁸/ml.Endostar?Shandong Simcere-Medgenn Bio-Pharmaceutical Co,Shandong,China,15mg:3ml,2.4×10⁵U?was used as the anti-angiogenic drug.4.Methods?1?Animal model:A Walker 256 cell suspension was diluted with phosphate-buffered saline into 1-2×10⁷ cells/ml and a 0.2 ml suspension was injected subcutaneously into a unilateral hind limb.The rats were imaged when the resulting tumor reached the size of approximately 1cm in diameter.Rats were anesthetized with an intraperitoneal injection of1%pentobarbital sodium at 30mg/kg and placed in a supine position.A 25-gauge needle was inserted into the tail vein for intravenous injection.?2?Experimental groups and protocols:60 tumor-bearing rats were randomly divided into 4 groups,each had 15 rats,to receive Endostar+MEUS combined treatment,Endostar,MEUS,or Sham ultrasound respectively.For rats in the combination group,Endostar was administrated intravenously at 5mg/kg per day,and then the tumor was exposed to 5 mins of therapeutic ultrasound?TUS?with continuous injection of 1 ml MB suspension during insonation.During the TUS exposure,the transducer was hand-held against the tumor percutaneously coupling with gel.Rats in the Endostar group received Endostar intravenous injection at 5mg/kg per day.Rats in the MEUS group underwent the same MEUS treatment procedure as the combination group but without injection of Endostar.For rats receiving sham US,same amount of MB injection was given and the transducer was applied to the tumor but not powered on.All the tumors were treated once a day and for three consecutive days.?3?Two-dimensional ultrasound?2D-US?,contrast-enhanced ultrasound?CUES?and quantitative analysis:Tumor size was determined by 2D-US every day for 7 consecutive days starting from the first day and tumor volume was calculated by the following equation:₆⁷?)?l=length,w=width,h=height,referring to the three maximal diameters perpendicular to each other?.Tumor blood perfusion was evaluated by CEUS before treatment,1 day and 4 days after the treatment.CEUS imaging was analyzed by the internal quantification software CBI?VINNO Technology Co.Ltd,Suzhou,China?to determine the peak intensity?PI?and area under curve?AUC?.A region of interest?ROI?covering the entire tumor was delineated along the border referring to the 2-D imaging of tumor.?4?HistologyAt the end of the study,all mice were sacrificed 4 days after the last treatment,ie,the7th day starting from the first treatment,and tumors were harvested.Sections were sent to perform CD31,CD34 and VEGFA immunohistological staining.All slides were first observed at 100×magnification to identify an area containing the highest microvessel density,which was called a“hot spot”.Then at 400×magnification,MVD was determined by the average number in three fields of the“hot spot”.Apoptosis analysis of tumor cells was performed after the treatment by TUNEL assay using a detection kit.All slides were observed under microscopy and for apoptosis detection,5 fields of each slide at 400×magnification were selected and 200 cells of each filed was counted,and apoptosis index?AI?was defined by the total number of TUNEL positive cells with brown stained nuclei out of all 1000 cells above.Some of the tumor sample were processed and were examined and photographed under a transmission electron microscope under 400×magnification.Results1.Tumor blood perfusionBefore the treatment,all tumors were well perfused and PI and AUC in all groups showed no significant difference?p>0.05?.One day after the treatment,PI and AUC in the Endostar+MEUS group showed a reduce in PI?95.80±10.21?and AUC?5526.29±445.96?compared to that of the other three groups?p<0.001?.Both the Endostar group?PI:115.76±8.06,AUC:6244.41±485.67?and the MEUS group?PI:110.58±8.54,AUC:6197.28±445.36?showed significantly lower PI and AUC compared to the Sham US group?PI:125.43±5.97,AUC:6783.76±215.21?.Four days after the last treatment,the PI?83.50±7.97?and AUC?4485.49±466.03?values of the Endostar+MEUS group still remained the lowest among all groups?p<0.001?.Compared to the Sham US group?PI:110.83±8.95,AUC:5901.06±424.49?,PI?97.53±6.80?and AUC?5103.24±452.65?in the Endostar group were both significantly lower,while not lower in the MEUS group?PI:103.44±6.33,AUC:5890.84±326.89?.2.Tumor volumeTumors showed a homogenous growth and no significant difference was demonstrated in tumor volumes among all the groups before treatment?p>0.05?.One day after the treatment,tumor volume?all units in cm³?in the Endostar+MEUS treatment group?2.01±0.71?was significantly lower compared to other three groups?p<0.001?.The Endostar group?5.03±1.61?and the MEUS group?4.99±1.50?both had significant smaller tumor volumes than the Sham US group?7.61±1.35?.Four days after the treatment,the effect of tumor growth inhibition was still observed in the combined treatment group?5.34±1.65?.Compared to the Sham US group?15.79±3.02?,the tumors of the Endostar group?11.13±2.02?appeared to be smaller,while no volume difference was found between the MEUS group?15.05±2.26?and the Sham US group.A tumor growth curve was plotted with tumor volumes measured for seven consecutive days for every group.A remarkable tumor suppression was seen in the Endostar+MEUS treatment group during the seven days.A significant tumor growth inhibition could be observed within the first three days of the MEUS group and the last three days of the Endostar group.3.Microvessel densityMicrovessels were identified as brown staining areas.Significantly decreased levels of both VEGFA?20.67±2.68?,CD31?17.22±3.25?and CD34?20.49±4.44?were detected in the Endostar+MEUS treatment group.The Endostar group demonstrated a lower level of VEGFA?27.16±2.66?,while CEUS group showed decreased expressions of both CD31?29.09±5.66?and CD34?28.80±4.62?.4.ApoptosisBoth Endostar+MEUS treatment group and MEUS group showed a higher apoptosis index than Sham US group,while there was no significant difference between the Endostar group and the Sham US group.5.Electron microscopyThe TEM imaging revealed integrate structures of endothelial cells of tumor blood vessels in the Sham US group.The Endostar+MEUS treatment group and MEUS group showed structural discontinuity of microvessels characterized by irregular surface of vascular endothelial cells and enlarged intercellular spaces,karyopyknosis,incomplete nuclear membrane and mitochondrial vacuoles in different sizes in the cytoplasm were frequently observed.ConclusionsMEUS combining with Endostar could achieve a synergistic anti-tumor effect by disrupting tumor vasculature and antagonizing angiogenesis,providing a novel method for a comprehensive tumor therapy.