| 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. |
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