Investigate Anti-angiogenesis Effect Of Bevacizumab And The Optimal Time Window Of Combined Chemotherapy

Background:Tumor angiogenesis is indispensable for the growth of solid tumors.If without,most of the solid tumors will stop growing and enter into a dormant state.As angiogenesis is critical to tumor growth and metastasis,the drugs targeting tumor angiogenesis will be very promising.In recent years,Zebrafish has been a popular organism for the study of screening and assessing anti-angiogenesis drugs instead of traditional animal model.The primary advantages of zebrafish for novel angiogenesis inhibitors discovery include their high genetic,physiologic,and pharmacologic similarity with humans,as well as the small size,optical transparency,rapiddevelopment,and large numbers of their embryos and larvae,which are the primary system for experimental analys is.Combined,these features define zebrafish as an ideal in vivo model for the systematic identification of novel anti-angiogenesis inhibitors with therapeutic potential.Zebrafish fli1 promoter is able to drive expression of enhanced green fluorescent protein(EGFP)in all blood vessels throughout embryogenesis.These transgenic lines permits detailed analys is of vascular defects associated with anti-angiogenesis drugs.Bevacizumabcompete with VEGF to bind to VEGFR-1 and-2,and then inhibit ECs proliferation and angiogenesis.Several preclinical and clinical studies showed that bevacizumab not only have anti-angiogenes is effect,but also induce vascular normalization.It is hypothesized that appropriate application of anti-angiogenes is agents can make abnormal tumor vascular normalization,and then lead to drug and oxygen transported more effectively to the tumor.The increased permeability of drug to the tumor can enhance the effect of chemotherapies.Similarly,the higher oxygen can enhance the effect of radiotherapy and chemotherapy.Several studies have proposed that vascular normalization induced by anti-angiogenes is therapy was in a time-dependent manner.Within this window,adjuvant chemotherapy showed the best therapeutic effect.Purpose:Inthe first part of this research,we first investigate anti-angionenesis effect of bevacizumab on Zebrafish to provide supplementary evidence for the use of bevacizumab in clinic.And then,in the second part,we focus on identifying a time window for vascular normalization to make best strategies of combined therapy.Methods:In the first part of this study,we used transgenic zebrafish(tgfil:EGFP)as experimental animals.Adult zebrafish were maintained at 28.5?on a 14 h light/10 h dark cycle.Five to six pairs of zebrafish were set up for nature mating every time.On average,200–300 embryos were generated.Embryos were maintained at 28.5?in fish water(0.2%Instant Ocean Salt in deionized water).Bevacizumab solution was injected into the yolk sac.The site for injection was chosen because proteins in the yolk are often taken up by the embryo,therefore,after injection,proteins end up in the circulation of the embryo.The injection was performed as follows:1 mg/ml and 2 mg/ml solution of Bevacizumab in PBS was back-filled into a pulled-glass micropipet.The micropipet was then attached to a micromanipulator.Approximately 40 nanoliters of Bevacizumab solution was injected into the yolk sac for each embryo.To evaluate blood vessels formation,2.5dpf zebrafish were treated with Bevacizumab for a treatment period of 36h.Control embryos were treated with the equivalent amount of PBS solution.The area of the subintestinal vessels(SIV)and the average number of blood vessel segments per eye were quantified with NIS-Elements D3.1 software.Embryos were analyzed with Nikon SMZ 1500Fluorescence microscope and subsequently photographed with digital cameras.Quantitative image analyses were performed using image based morphometric analysis.All data are presented as mean±SEM.Statistical analysis and graphical representation of the data were performed using GraphPad Prism5.0.Statistical significance was performed using a Student's t test or ANOVA as appropriate.Statistical significance is indicated by*,where P<0.05,and***,where P<0.0001.In the second research,we constructed a xenograft mouse model.The study can divided into two parts.First,3-4weeks'nude mice were kept in specific pathogen free(SPF)environment.The A549lung cells(5x10~5?100?l)were inoculated in the right rib of each nude mouse.Nude mice beared NSCLC(Non Small Cell Lung Cancer)xenografted tumor were assessed by vernier caplipers every other day.After 1 week,when the tumors had grown to 150 mm~3,the tumor-bearing nude mice were randomly divided into two groups(n=10 per group)as follows:The control group was intraperitoneally injected with 100?l PBS;the bevacizumab group intraperitoneally injected at a dose of5mg?kg.Starting from the day of treatment(day0),three mice from each group were at euthanized day1?day3?day5?day8.Second,3-4weeks'nude mice were kept in specific pathogen free(SPF)environment.The A549 lung cells(5x10 ~5?100?l)were inoculated in the right rib of each nude mouse.Nude mice beared NSCLC xenografted tumor were assessed by vernier caplipers every other day.After 1 week,when the tumors had grown to 80 mm~3,the tumor-bearing nude mice were randomly divided into six groups(n=5 per group)as follows:The control group(PBS),the bevacizumab(day0)group,the paclitaxel(day0)group,the bevacizumab(day0)+paclitaxel(day0)group,the bevacizumab(day0)+paclitaxel(day3)group,the bevacizumab(day0)+paclitaxel(day5)group,the control group was intraperitoneally injected with 100?l PBS;Bevacizumab was intraperitoneally injected at a dose of 5mg?kg.Paclitaxel was intraperitoneally injected at a dose of 3mg?kg.The drugs were gave every week.All the mice were euthanized after three cycles.Tumor tissue samples from two parts would be weighted and calculated,then fixed by opti-mum cutting temperature compound(OCT)in Liquid nitrogen to prepare frozen sections or fixed by paraformaldehyde to perform Immunohistochemistry.Western blot was performed to analysis the expression level of VEGF in tumor samples.All data are presented as mean±SEM.Statistical analysis and graphical representation of the data were performed using GraphPad Prism 5.0.Statistical significance was performed using a Student's t test or ANOVA as appropriate.Statistical significance is indicated by*,where P<0.05,and***,where P<0.0001.Results:Our research first identify the inhibitory effect of bevacizumab on zebrafish angiogenesis.When the embryos treatment with bevacizumab at the pectoral fin stage(2.5dpf)causes specific vasculature formation defects in the SIVs,The vascular defect in SIV(subintestinal vein)resulted in further complications such as severe pericardial edema after bevacizumab treatment.In contrast,truncal vasculature of the zebrafish show no appreciable phenotypic differences relative to control.Angiogenes is has become an attractive target for drug therapy because of its key role in many diseases,such as cancer,age-related macular degeneration(AMD),Diabetic retinopathy(DR).The results also show that bevacizumab inhibits zebrafish retinal angiogenesis.In the second research,based on the theory of vascular normalization,we verify that combined paclitaxel on day0 and day3 after administration of bevacizumab can showed the best therapeutic effect on subcutaneous tumor.During this time window,the expression of VEGF achieved the least level,as well as the density and derangement of microvascular in tumor.The results may provide a suitable time for the combined chemotherapy with bevacizumab in clinic.