【作者】 杨光杰；

【导师】 韩建奎；

【作者基本信息】 山东大学 ， 影像医学与核医学， 2015， 博士

【摘要】 第一部分’8F标记新型RGD二聚体(18F-RGD2)探针构建及其在SKOV-3卵巢癌荷瘤鼠靶向显像中的应用研究研究背景卵巢癌发病率在全球妇科恶性肿瘤中排第二位,早期症状隐匿,发现时往往处于进展期或者已经扩散。临床研究证据表明卵巢癌对化疗敏感,治疗上一般采取手术切除后行卡铂或紫杉醇辅助化疗。而对于丧失手术机会的病人则直接采取化疗疗法。近年来尽管卵巢癌病人5年生存率有所改善,但死亡率仍居高不下。大量临床试验研究证实,抗肿瘤血管生成治疗对各个分期的卵巢癌病人的预后都具有重要影响。近来研究证实,抗肿瘤血管生成治疗联合放化疗,可以明显增强抗肿瘤疗效,改善预后。因此,目前临床上急需一种无创性影像学检查方法对肿瘤进行早期检测、监测抗血管生成治疗疗效以及筛选适合进行抗血管生成治疗的肿瘤患者。近年研究表明,整合素αvβ3在肿瘤新生血管和某些类型的肿瘤细胞上高表达,而在成熟的血管和正常组织无表达或者极低表达。RGD多肽是整合素avβ3的配体,可以高亲和力与整合素αvβ3特异性结合,因此利用RGD多肽类探针进行整合素αvβ3靶向显像,对早期诊断肿瘤和监测抗肿瘤药物疗效具有重要临床价值。紫杉醇是最重要的一线抗癌药物之一,但是其抗肿瘤机制迄今为止尚未完全阐明。目前认为主要包括细胞周期停滞、诱导凋亡、使微管聚集。此外最新研究证实紫杉醇还有抗肿瘤新生血管生成作用。本论文合成一种新型18F标记聚乙二醇修饰的RGD探针18F-FB-NH-PEG4-E[PEG4-c(RGDfK)]2(记为18F-RGD2),并研究了其在人卵巢癌SKOV-3荷瘤鼠模型中靶向肿瘤显像及监测抗血管生成药物疗效的作用及意义。实验方法1、制备探针18F-RGD2将纯化的18F-SFB溶于DMSO,然后添加NH2-PEG4-E[PEG4-c(RGDfK)]2(RGD2)的DMSO溶液和DIPEA,60℃加热30 mmin,产物用Sep-Pak C-18柱进行纯化,并应用radio-HPLC检测放化纯。2、建立荷瘤鼠模型选取4-6周龄的雌性裸鼠,左肩皮下注射含5×106个SKOV-3卵巢癌细胞的生理盐水溶液,操作在通风的无菌条件下完成,建模后进行常规饲料喂养。肿瘤大小约50 mm3时进行生物学分布研究。3、生物学分布研究及阻断实验荷瘤鼠尾静脉注射1 MBq/0.1 ml 18F-RGD2,30 min、60 min、120 min后分批处死小鼠。采集肿瘤、血液、肌肉、骨、心脏、肝脏、肺、肾脏、肠等重要组织和器官,称重后,用Y计数器进行放射性计数,计算%ID/g。阻断实验：另选SKOV-3荷瘤小鼠,每只尾静脉同时注入1 MBq/0.1 ml剂量的18F-RGD2和过量未标记的E[c(RGDfK)]2,60 min后处死小鼠,进行生物学分布研究。4. MicroPET显像两组SKOV-3荷瘤鼠分别注射3.7 MBq/0.1 ml 18F-RGD2或者5 MBq/0.1 ml 18F-FDG,60 min后,腹腔内注射0.6%戊巴比妥钠,麻醉后行MicroPET扫描,扫描模式为静态采集5 min。5、紫杉醇治疗方案将紫杉醇溶于DMSO后用PBS缓冲液稀释到所需浓度。将实验小鼠分为治疗组和对照组,每组12只。治疗组给予0.1ml的紫杉醇(10mg/kg),对照组给予0.1ml的PBS溶液,每3天给药一次,连续给药6次。6、免疫组织化学分析将生物学分布研究中处死小鼠的肿瘤组织做免疫组织化学分析。将肿瘤组织4%多聚甲醛固定后,制备成石蜡切片,利用抗CD34抗体进行免疫组织化学染色,定量分析肿瘤的微血管密度。实验结果1、成功合成新型18F标记RGD探针18F-RGD2。2、生物学分布研究结果表明18F-RGD2在SKOV-3肿瘤组织明显浓聚,肿瘤靶向性好。3、MicroPET显像证实18F-RGD2在卵巢癌组织明显聚集,18F-RGD2的T/NT(靶/非靶)比值明显大于18F-FGD的T/NT比值。4、抗肿瘤血管生成治疗后,治疗组的肿瘤18F-RGD2摄取低于对照组,注射后60 min降低31.31%±7.18%(P=0.009),注射后120 min降低38.92%±8.31%(P<0.001)。5、免疫组化染色证实SKOV-3肿瘤组织高表达CD34,紫杉醇抗血管生成治疗后肿瘤组织CD34表达下降,微血管密度降低,与生物学分布结果一致。结论18F-RGD2具有较强的整合素αvβ3亲和力,在肿瘤组织明显浓聚,可用于抗肿瘤血管生成疗效监测和评价。第二部分99mTc-3PRGD2监测不同抗肿瘤血管生成药物疗效的差异性研究研究背景近年来,靶向肿瘤新生血管药物治疗取得重大进展,但是如何非侵入性实时监测该类药物的疗效是临床亟待解决的问题。通过标记可与肿瘤细胞特异性靶点相结合的配体,引入机体后,通过影像学设备实时成像,可为临床抗肿瘤治疗提供重要参考。近年来,有关肿瘤特异性靶点研究有多项报道,其中研究最多,也最有潜力的是整合素αvβ3。整合素αvβ3主要表达在肿瘤新生血管和某些肿瘤细胞表面,而在成熟的血管和正常组织无表达或者极低表达。多项研究证实RGD多肽可与整合素αvβ3高效特异性结合,RGD多肽类探针可被用于各种模态的整合素αvβ3靶向显像,进行肿瘤早期诊断和治疗疗效监测。多项研究报道RGD多肽类探针可有效监测肿瘤化疗或抗血管生成治疗疗效。这些研究涉及多种药物治疗和不同类型的肿瘤。一般认为,RGD探针在肿瘤局部的浓聚程度反映肿瘤新生血管密度,是正相关关系,尽管研究证实RGD多肽类探针可有效监测实体肿瘤的抗血管生成药物治疗疗效,而且也已见临床研究报道。但在前期体外细胞学实验中,研究组发现抗血管生成药物夫拉平度(细胞周期依赖性蛋白激酶抑制剂)与紫杉醇不同,在抑制血管内皮细胞增殖的同时,可以上调SKOV-3肿瘤细胞表面的整合素αvβ3表达。这种双重效应可能影响RGD多肽类探针监测抗肿瘤血管生成治疗疗效的有效性和准确性。论文第一部分已经证实18F-RGD2用于卵巢癌SKOV-3荷瘤鼠模型肿瘤靶向显像及监测紫杉醇抗肿瘤血管生成治疗疗效的有效性。在第二部分,课题组尝试通过利用目前比较成熟的放射性探针99mTc-3PRGD2,研究RGD多肽类探针监测不同抗肿瘤血管生成药物治疗疗效的有效性,并探讨其在监测不同抗肿瘤血管生成药物疗效中是否存在差异性及分子机理。研究方法1、制备探针99mTc-3PRGD299mTc-3PRGD2通过HYNIC-3PRGD2药盒制备。将1ml 99mTcO4-溶液(25 mCi)加入到HYNIC-3PRGD2药盒中,震荡,溶解充分后100℃水浴加热20 min,室温冷却5 min后放射性薄层纸层析法检测放化纯。2、建立荷瘤鼠模型选取4-6周龄的雌性裸鼠,左肩皮下注射含5×106个SKOV-3卵巢癌细胞生理盐水溶液,建模操作在通风的无菌条件先完成。建模后进行常规饲料喂养。肿瘤大小约50 mm3时进行生物学分布研究。3、抗血管生成治疗方案将紫杉醇和夫拉平度用DMSO溶解,然后用PBS缓冲液稀释到所需浓度。将24只荷瘤鼠随机分为紫杉醇组、夫拉平度组和对照组,每组8只。紫杉醇组每次腹腔内注射0.1 ml紫杉醇溶液(20mg/kg),夫拉平度组每次腹腔内注射0.1m1夫拉平度溶液(5mg/kg),对照组裸鼠每次腹腔内注射0.1 mlPBS缓冲液。每3天注射一次,共注射6次。4、生物学分布研究及阻断实验荷瘤鼠尾静脉注射0.37 MBq/0.1 ml 99mTc-3PRGD2,2 h和4h后分批处死小鼠。采集采集肿瘤、血液、肌肉、骨、心脏、肝脏、肺、肾脏、肠等重要组织和器官,称重后,用γ计数器进行放射性计数,计算%ID/g。另选SKOV-3荷瘤鼠4只,每只尾静脉同时注入0.37 MBq/0.1 ml剂量的99mTc-3PRGD2和过量未标记的E[c(RGDfK)]2,进行阻断实验。5、免疫组织化学分析取生物学分布研究中处死的小鼠肿瘤组织做免疫组织化学分析。将肿瘤组织常规4%多聚甲醛固定后,制备成石蜡切片,进行Ki-67、CD34和整合素αv抗体免疫组织化学染色,定量分析肿瘤增殖程度、微血管密度和整合素αvβ3表达。6、内皮细胞管型形成实验分别用含紫杉醇(100 mM)和夫拉平度(300 mM)的培养液培养人脐静脉血管内皮细胞(HUVECs),37 ℃,5% CO2培养24 h,然后通过内皮细胞管型形成实验检测药物在体外对HUVECs形成血管能力的影响。7、流式细胞术分析整合素avβ3表达分别用含紫杉醇(100 mM)和夫拉平度(300 mM)的培养液培养人脐静脉血管内皮细胞(HUVECs),37 ℃,5% CO2培养24 h,荧光抗体染色后通过流式细胞术测定药物治疗对SKOV-3肿瘤细胞表面整合素avβ3表达的影响。结果1、肿瘤体积测定及Ki-67免疫组化结果显示紫杉醇和夫拉平度治疗都减缓了肿瘤生长,明显抑制SKOV-3细胞增殖,P<0.05。2、CD34免疫组化染色及内皮细胞管型形成实验分别从体内、体外证实了紫杉醇和夫拉平度都明显降低血管生成,P<0.05。3、紫杉醇治疗后肿瘤放射性摄取明显降低(2小时降低39.96%±8.23%,P=0.044；4小时降低35.76%±11.42%,P=0.024),而夫拉平度治疗后肿瘤放射性摄取轻度升高(2小时升高4.42%±0.24%,P=0.898；4小时升高12.2%±1.84%,P=0.702)。4、免疫组化染色显示紫杉醇和夫拉平度抗血管生成治疗后SKOV-3肿瘤整合素αvβ3表达具有差异性：紫杉醇治疗后整合素αvβ3表达降低34.2%±5.9%(P<0.01),而夫拉平度治疗后整合素αvβ3表达轻度升高8.7%±3.2%(P=0.078)。5、流式细胞术分析表明紫杉醇治疗对于SKOV-3细胞表面整合素αvβ3表达无明显影响,而夫拉平度治疗可上调SKOV-3细胞表面整合素αvβ3表达,P<0.05。结论99mTc-3PRGD2在监测不同抗肿瘤血管生成药物治疗中具有明显差异性,因此在应用此类探针评价抗血管生成药物疗效时应特别慎重。论文创新点1、成功制备了新型聚乙二醇修饰的可靶向肿瘤整合素avβ3表达显像的探针18F-FB-NH-PEG4-E[PEG4-c(RGDfK)]2。2、通过MicroPET显像及生物学分布研究证实了“二价”新探针18F-RGD2可用于卵巢癌显像及抗肿瘤新生血管疗效监测,并初步阐明18F-RGD2卵巢癌显像及监测抗肿瘤新生血管药物疗效的分子机制。3、首次发现RGD多肽类探针在监测抗肿瘤血管生成治疗疗效中具有差异性,并初步阐明了其分子机制。更多还原

【Abstract】 Part Ⅰ Preparation of a novel 18F-labeled RGD dimer (18F-RGD2) and targeted imaging in SKOV-3 ovarian bearing miceResearch BackgroundAs the second most common gynecological malignancy worldwide, ovarian cancer is often not found until it is advanced or has spread. It is generally considered sensitive to chemotherapy. Patients should be treated with optimal cytoreductive surgery combined with adjuvant carboplatin-paclitaxel chemotherapy, while some unresectable patients are treated with up-front chemotherapy. Although the 5-year survival for women presenting with advanced ovarian cancer has shown some improvement for the past few years, the mortality rate is still high. A large number of trials has proven that antiangiogenic therapy plays an important role in the treatment of various stages of ovarian cancer. Recently, it is reported that antiangiogenic therapy can enhance the therapeutic efficacy of chemotherapy and radiotherapy, and can improve the prognosis in various carcinomas. Consequently, there is an increasing demand for noninvasive imaging to diagnosis, facilitate early response monitoring and screening of the appropriate patients who will benefit from antiangiogenic treatment with positive effects.It is reported that integrin avp3 is expressed on tumor neovessels as well as on some tumor cells but not on quiescent blood vessels in normal tissue. Due to the high integrin avP3 binding affinity, many RGD probes have been developed for multimodality imaging of integrin expression with the purpose of tumor diagnosis and monitoring tumor treatment response, which is of great clinical significance. Paclitaxel is one of the most important first line anticancer drugs for ovarian cancer. But the mechanisms of its antitumor activity are not entirely understood. Various mechanisms of paclitaxel have been reported including cell cycle arrest, induction of apoptosis and stabilization of microtubules. In addition, the recent studies indicate that inhibition of angiogenesis also plays an important role in its antitumor activity. In this study we prepared a novel 18F-labeled and PEG modified RGD dimer porbe 18F-FB-NH-PEG4-E[PEG4-c(RGDfK)] 2 (denoted as 18F-RGD2). The value of 18F-RGD2 as a probe for targeted imaging and therapy response monitoring in SKOV-3 xenograft-bearing mice also were evaluated.Methods1. Preparation of 18F-RGD2The purified 18F-SFB was redissolved in dimethyl sulfoxide (DMSO), and added to a DMSO solution of RGD2 and N, N-diisopropylethylamine (DIPEA). The reaction mixture was allowed to incubate at 60 ℃ for 30 min. The mixture purified by a Sep-Pak C-18 column, and the radiochemical purity was checked by the radio high-performance liquid chromatography (Radio-HPLC).2. Establishment of animal modelsFemale athymic nu/nu mice at 4 to 6 weeks of age were subcutaneously implanted with 5×106 SKOV-3 cells into the left front flank. All procedures were performed in a laminar flow cabinet using aseptic techniques. The animals were allowed to feed ad libitum. Biodistribution studies were performed when the tumor volume reached about 50 mm3.3. Biodistribution and blocking studiesMice were injected with 1 MBq/0.1ml 18F-RGD2 via tail vein and were humanely sacrificed at 30 min,60 min and 120 min after injection. Tumor, blood, muscle, bone, heart, lung, kidney and intestines were collected, weighed, and counted using a gamma-counter for %ID/g.For the blocking experiment, SKOV-3 xenograft-bearing mice were injected with 0.1 ml of the above solution containing 1 MBq 18F-RGD2 along with excess unlabeled E[c(RGDfK)]2. Mice were sacrificed at 60 min after injection for organ biodistribution using the same procedure above.4. MicroPET imagingTwo groups of animals were injected 3.7 MBq 18F-RGD2 or 5 MBq 18F-FDG, respectively. Mice were anesthetized with intraperitoneal injection of 0.6% sodium pentobarbital. And then five-minute static MicroPET scans were acquired.5. Paclitaxel therapy protocolPaclitaxel were dissolved in dimethylsulfoxide and diluted by PBS to specified concentrations before use. Mice with ovarian cancer were divided into 2 groups randomly (n=12 per group). The treatment group were injected with 0.1 ml of paclitaxel at a dosage of 10 mg/kg body weight, and the control group were injected with 0.1 ml of PBS. Therapies were performed every 3 days for 16 days (6 injections).6. Immunohistochemical analysisImmunohistochemical analysis of the SKOV-3 tumor tissues harvested from the mice was performed one day after the biodistribution studies. After being fixed in 4% paraformaldehyde routinely, paraffin-embedded tissue sections were prepared. MVD was evaluated and quantified by immunohistochemical analysis with antibodies to the endothelial marker CD34.Results1. A novel 18F-labeled RGD dimer probe (18F-RGD2) was prepared successfully.2. Biodistribution study demonstrated 18F-RGD2 accumulated highly in SKOV-3 tumor and had a good targeting.3. MicroPET imaging results demonstrated high contrast visualization of SKOV-3 tumors. And tumor to background ratio (T/NT) of 18F-RGD2 uptake was significantly higher than that of 18F-FDG.4. Studies on antiangiogenic therapy demonstrated percentage of injected dose per gram of tissue (%ID/g) tumor uptake of 18F-RGD2 was obviously decreased in treatment group than control group, especially at 60 min (by 31.31% ± 7.18%, P= 0.009) and 120 min (by 38.92% ± 8.31%, P< 0.001) after injection of 18F-RGD2.5. Immunohistochemical analysis demonstrated CD34 was strongly expressed in SKOV-3 tumors. Paclitaxel therapy decreased CD34 expression and MVD in SKOV-3 tumors, which was in accord with biodistribution study.Conclusions18F-RGD2, with specific affinity and high contrast visualization of SKOV-3 tumors, is a promising tracer for tumor imaging and antiangiogenesis therapy monitoring.Part Ⅱ Diversity of 99mTc-3PRGD2 in monitoring antiangiogenic response to different drugsResearch Background In recent years, there is a great improvement in antiangiogenic drugs for targeted therapy of tumors. And how to monitor the therapy response noninvasively is a big challenge. One promising approach is to identify molecular markers of angiogenesis by conjugating a specific ligand recognizing overexpressed receptors in angiogenic tumors to imaging probes. In this area, one of the most potential and best studied targets is the integrin αvβ3. Integrin αvβ3 is expressed on tumor neovessels as well as on some tumor cells but not on quiescent blood vessels in normal tissue. Due to the high integrin αvβ3 binding affinity, many RGD peptides probes have been developed for multimodality imaging of integrin expression with the purpose of tumor diagnosis and tumor treatment response monitoring.There have been several studies using suitably labeled RGD peptides tracers to monitor chemotherapy or antiangiogenesis therapy efficacy. Different drugs were employed for different types of tumors in previous studies. And all the above researches including some clinical studies concluded that RGD radiotracers had a good ability in noninvasive monitoring antiangiogenic response in the solid tumors. It is generally thought that tumor uptake of RGD radiotracers reflects and correlates with tumor angiogenesis. However, in preliminary experiment we found that flavopiridol, a "special" antiangiogenic agent, CDK (cyclin-dependent kinase) inhibitor, can additionally up-regulate the integrin αvβ3 expression on tumor cells, while inhibiting tumor angiogenesis in ovarian cancer animal models. The dual-effect of antiangiogenic drugs may affect the ability to evaluate antiangiogenic response when using RGD radiotracers.In part I, we have validated that 18F-RGD2 was effective in monitoring antiangiogenic response to paclitaxel in SKOV-3 tumor bearing mice. In part Ⅱ, we will further test the value of RGD radiotracers in monitoring antiangiogenesis to different drugs using a well studied RGD radiotracer,99mTc-3PRGD2, and test whether there is diversity. The mechanism of the diversity also will be studied.Methods1. Preparation of 99mTc-3PRGD299mTc-3PRGD2 was prepared by a lyophilized kit formulation. For 99mTc radiolabeling, to the kit vial was added 1 ml of 99mTcO4 solution (25 mCi) in salin. After brief vortexing, the vial was heated at 100 ℃ for 20 min. After radiolabeling, the vial was allowed to stand at room temperature for 5 min. A sample of the resulting solution was analyzed by radio instant thin layer chromatography (Radio-ITLC).2. Establishment of Animal modelsFemale athymic nu/nu mice at 4 to 6 weeks of age were subcutaneously implanted with 5×106 SKOV-3 cells in 0.1 ml of saline into the left front flank. All procedures were performed in a laminar flow cabinet using aseptic techniques. The animals were allowed to feed ad libitum. Biodistribution studies were performed when the tumor volume reached about 50 mm3.3. Antiangiogenic therapy protocolsPaclitaxel and flavopiridol were dissolved in dimethylsulfoxide and diluted by PBS to specified concentrations before use. Twenty-four mice with ovarian cancer were divided into 3 groups randomly (n= 8 per group). The paclitaxel-treated group were injected i.p. with 0.1 ml of paclitaxel at a dosage of 20 mg/kg body weight. The flavopiridol-treated group were injected i.p. with 0.1 ml of flavopiridol at a dosage of 5 mg/kg body weight. The control group were injected i.p. with 0.1 ml of PBS. Therapies were performed every 3 days for 16 days (6 injections).4. Biodistribution and blocking studiesMice were injected with 0.37 MBq/0.lml 99mTc-3PRGD2 via tail vein and were humanely sacrificed at 2 h and 4 h after injection. Tumor, blood, muscle, bone, heart, lung, kidney and intestines were collected, weighed, and counted using a gamma-counter for%ID/g.For the blocking experiment, SKOV-3 xenograft-bearing mice were injected with 0.1 ml of the above solution containing 0.37 MBq 99mTc-3PRGD2 along with excess unlabeled E[c(RGDfK)]2. Mice were sacrificed at 60 min after injection for organ biodistribution using the same procedure above.5. Immunohistochemical analysisImmunohistochemical analysis of the SKOV-3 tumor tissues harvested from the mice was performed one day after the biodistribution studies. After being fixed in 4% paraformaldehyde routinely, paraffin-embedded tissue sections were prepared. MVD, proliferation and integrin αvβ3 expression were evaluated and quantified by immunohistochemical staining with antibodies to CD34, Ki-67 and integrin av.6. Endothelial cell tube formation assayHUVECs were pretreated by paclitaxel (100 nM) or flavopiridol (300 nM) at 37 ℃, 5% CO2 for 24 h. The effects of drugs therapy on angiogenesis were evaluated in vitro by endothelial cell tube formation assay.7. Flow cytometric analysisSKOV-3 cells were pretreated by paclitaxel (100 nM) or flavopiridol (300 nM) at 37 ℃,5% CO2 for 24 h. After being incubated with FITC labeled integrin αvβ3 antibody, integrin αvβ3 expression on SKOV-3 cells were measured by flow cytometric analysis.Results1. Both paclitaxel and flavopiridol therapy could apparently inhibit tumor growth and proliferation, P<0.05.2. Antiangiogenic effects to paclitaxel and flavopiridol therapy were validated in vivo and in vitro by CD34 immunohistochemical staining and endothelial cell tube formation assay.3. Compared with the control group,%ID/g tumor uptake of 99mTc-3PRGD2 showed a significant decrease at 2 hours (by 39.96% ± 8.23%, P= 0.044) and at 4 hours (by 35.76% ± 11.42%, P= 0.024) post injection in the paclitaxel-treated group, but a slight increase of tumor uptake in the flavopiridol-treated group at 2 hours (by 4.42% ± 0.24%, P= 0.898) and at 4 hours (by 12.2% ± 1.84%, P= 0.702).4. Compared with the control group, integrin αv expression was different in the paclitaxel-treated group and the flavopiridol-treated group. A significant decrease of av expression was observed in the paclitaxel-treated group (by 34.2% ± 5.9%, P< 0.01), while a slight increase in the flavopiridol-treated group (by 8.7% ± 3.2%, P= 0.078).5. Flavopiridol significantly increased integrin αvβ3 expression on SKOV-3 cells compared with that of untreated SKOV-3 cells (P<0.05), while there was no significant difference in integrin αvβ3 expression between paclitaxel-treated SKOV-3 cells and untreated SKOV-3 cells.ConclusionsThere is diversity in evaluating antiangiogenic response when using 99mTc-3PRGD2, which may be an important reminder in future clinical applications of RGD radiotracers as a strategy for antiangiogenesis therapy response monitoring.Points of Innovation1. Prepared a novel, PEG modified integrin αvβ3-targeted probe 18F-FB-NH-PEG4-E[PEG4-c(RGDfK)]2 for tumor imaging.2. Proved the effectiveness of F-RGD2 dimer in imaging and monitoring paclitaxel therapy response in ovarian cancer xenograft-bearing mice by MicroPET imaging and biodistribution studies, and studied the molecular mechanism of RGD radiotracers in tumor imaging and antiangiogenic therapy monitoring preliminarily.3. Firstly proved there is diversity of RGD radiotracers in monitoring antiangiogenic therapy response, and studied the underlying molecular mechanism preliminarily.更多还原