PET Imaging of Neovascularization with 68Ga-3PRGD2 for Assessing

Aug 26, 2014 - Antiangiogenic therapy is an effective strategy to inhibit tumor growth. Endostar, as an approved antiangiogenesis agent, inhibits the ...
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PET Imaging of Neovascularization with 68Ga-3PRGD2 for Assessing Tumor Early Response to Endostar Antiangiogenic Therapy Jiyun Shi,†,‡,# Zhongxia Jin,†,§,# Xujie Liu,†,§ Di Fan,†,§ Yi Sun,∥ Huiyun Zhao,†,⊥ Zhaohui Zhu,∥ Zhaofei Liu,†,§ Bing Jia,†,§ and Fan Wang*,†,‡,§ †

Medical Isotopes Research Center, Peking University, Beijing 100191, China Interdisciplinary Laboratory, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China § Department of Radiation Medicine, Basic Medical Sciences, Peking University, Beijing 100191, China ∥ Department of Nuclear Medicine, Peking Union Medical College Hospital, Beijing 100857, China ⊥ Medical and Healthy Analytical Center, Peking University, Beijing 100191, China ‡

ABSTRACT: Antiangiogenic therapy is an effective strategy to inhibit tumor growth. Endostar, as an approved antiangiogenesis agent, inhibits the newborn vascular endothelial cells, causing the decrease of integrin αvβ3 expression. Radiolabeled 3PRGD2, a novel PEGlayted RGD dimer probe (PEG4-E[PEG4-c(RGDfK)]2) showed highly specific targeting capability to integrin αvβ3, which could be used for monitoring the efficacy of Endostar antiangiogenic therapy. In this study, 68Ga-3PRGD2 PET imaging was performed in Endostar treated/untreated Lewis Lung Carcinoma (LLC) mice on days 3, 7, 14, and 21 post-treatment for monitoring the tumor response to Endostar treatment, with the 18F-FDG imaging as control. As a result, 68Ga-3PRGD2 PET reflected the tumor response to Endostar antiangiogenic therapy much earlier (day 3 post-treatment vs day 14 posttreatment) and more accurately than that of 18F-FDG metabolic imaging, which provides new opportunities to develop individualized therapeutic approaches, establish optimized dosages and dose intervals for effective treatment that improve the survival rate of patients. KEYWORDS: antiangiogenic therapy, in vivo monitoring, molecular imaging, radiotracer, endostar

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reduction in tumor size.12,13 Molecular imaging can characterize and quantify the biological processes in living subjects noninvasively from molecular and cellular levels, with the potential for predicting response very early after initiation of therapy.14 Among several molecular imaging modalities, positron emission tomography (PET) is a promising technology that allows noninvasive imaging of tumor angiogenesis at the molecular level.15 18F-FDG is the most commonly used PET imaging agent and has been successfully applied for the diagnosis of many diseases worldwide.16 However, 18F-FDG is not a tumor-specific radiotracer and can lead to a false-positive in various forms of infection, inflammation, and granulomatous disease.16−18 A tripeptide moiety of Arg-Gly-Asp (RGD) has been highlighted for use in angiogenesis imaging, as it specifically binds to integrin αvβ3, which is richly expressed in endothelial cells activated for angiogenesis.19−23 Previously, we have

INTRODUCTION Angiogenesis is essential for the growth and metastasis of solid tumor.1−3 Tumors cannot grow beyond 1−2 mm3 in size without the neovasculature providing oxygen and nutrients.4,5 Inhibition of angiogenesis can ultimately provoke vascular regression, impeding delivery of oxygen and nutrients, and ultimately starving the tumor. Antiangiogenic therapy has been approved by many countries as an effective strategy to inhibit tumor growth, providing a novel treatment approach for cancer patients.6−8 However, lacking an available strategy to noninvasively monitor angiogenesis of tumor in vivo and evaluating the efficacy of antiangiogenic treatment are still an obstacle for the antiangiogenic treatment in clinic. Conventional methods to monitor angiogenesis are inconvenient, time-consuming, and mostly unrepeatable, as it usually requires to invasively obtain tumor tissue for biopsy and visualizing microvessels by immunohistochemical (IHC) staining of endothelial cellspecific markers. Therefore, there is an urgent need to develop a novel and noninvasive method to quickly evaluate the therapeutic response of tumors.9−11 The contrast-enhanced MRI and CT have been used to monitor the antiangiogenesis therapy by reflecting the morphologic changes of tumor. However, the vascular effects of antiangiogenic therapy may occur earlier before there is any © 2014 American Chemical Society

Special Issue: Positron Emission Tomography: State of the Art Received: Revised: Accepted: Published: 3915

April 30, 2014 August 18, 2014 August 26, 2014 August 26, 2014 dx.doi.org/10.1021/mp5003202 | Mol. Pharmaceutics 2014, 11, 3915−3922

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Cell Culture. The U87MG human glioma cell line and Lewis lung carcinoma (LLC) cell line LL/2 (LLC1) were purchased from American Type Culture Collection (Manassas, VA). U87MG cells were cultured in low glucose Dulbecco’s Modified Eagle’s Medium (DMEM) culture medium, and LLC cells were cultured in high glucose DMEM culture medium. Both cell lines were cultured in medium supplemented with 10% (v/v) fetal bovine serum (FBS) at 37 °C in a humidified atmosphere with 5% CO2. Animal Model Establishment. Female C57BL/6J mice (4−6 weeks of age) were purchased from Department of Experimental Animal, Peking University Health Science Center. All animal experiments were performed in accordance with guidelines of Institutional Animal Care and Use Committee (IACUC) of Peking University. LLC mice model was established by subcutaneous injection of 2 × 106 LLC cells into the right rear legs of mice. Once the tumor volume reached 60−70 mm3, the mice were initiated with Endostar treatment (1 week after inoculation of LLC cells). Flow Cytometric Analysis. LLC cells were harvested and trypsinized, then a single cell suspension containing 1 × 106 cells was prepared in a total volume of 100 μL of 1% bovine serum albumin (BSA)/phosphate buffered saline (PBS) (w/v) and immediately stained for 2 h at room temperature with hamster antimouse CD61 (integrin β3) antibody (1:200; Biolegend, San Diego, CA, USA), and then incubated with the FITC-conjugated goat antihamster secondary antibody (Biolegend, San Diego, CA, USA). PBS and mouse IgG were used as controls. Stained cells were analyzed by a FACS Calibur flow cytometer (Becton-Dickinson, Rutherford, NJ, USA). Endostar Treatment Protocol. LLC tumor-bearing C57BL/6J mice with the tumor size of ∼60 mm3 were randomly assigned to two groups (n = 20 mice per group). The therapeutic group was injected with Endostar intraperitoneally at 0.003 mL of Endostar injection/g body weight to give a dose of 8 mg/kg, and 0.9% saline was used as negative control. Endostar and saline were administered daily for 21 days continuously. Tumor dimensions were measured everyday with digital calipers, and the tumor volume was calculated using the formula (volume = 1/2 length × width × width). To assess potential toxicity, body weight was measured daily. When the tumor size exceeded the volume of 1500 mm3 or the body weight lost >20% of original weight, mice were euthanized. PET Imaging Protocol. PET scans and image analyses were performed on days 3, 7, 14, and 21 after Endostar treatment using a microPET R4 rodent model scanner (Siemens Medical Solutions, Malvern, PA) as previously reported.38,39 The microPET studies were performed by tailvein injection of about 5.55 MBq (150 μCi) 18F-FDG or 68Ga3PRGD2 into C57BL/6J mice bearing LLC xenografts under isoflurane anesthesia. Ten minute static PET images were acquired at 1 h time point postinjection (p.i.) (n = 4/group). The images were reconstructed by a two-dimensional orderedsubsets expectation maximum (OSEM) algorithm, and no correction was necessary for attenuation or scatter correction. Regions of interest (ROIs) were drawn over the tumor, kidney, liver, and muscle by using vendor software ASI Pro 5.2.4.0 on decay-corrected whole-body coronal images for each microPET scan. The maximum radioactivity concentrations (accumulation) within an organ were obtained from mean pixel values within the multiple ROI volume and then converted to megabecquerels (MBq) per milliliter per minute using a conversion factor. These values were then divided by the

prepared a novel RGD dimer probe PEG 4 -E[PEG4 -c(RGDfK)]2 (3PRGD2), which can specifically target integrin αvβ3 with relatively high affinity, leading to high tumor uptake and improved in vivo pharmacokinetics as compared with RGD monomer and conventional RGD multimers, including RGD dimer (RGD2) and tetramer (RGD4).24−27 The radiolabeled 3PRGD2 has been successfully used for tumor detection in clinic.28−31 Endostar, a recombinant human endostatin, which was approved by the China Food and Drug Administration (CFDA) in 2005 for treatment of lung cancer, was mainly used as an antiangiogenic agent for cancer treatment.32−34 In this study, we conjugated 3PRGD2 with DOTA and labeled with 68Ga. 68Ga is a 68Ge/68Ga generator producible radioisotope for clinical PET, making it more accessible and less expensive than other PET isotopes, and its short half-life (68 min) can lead to low level of radiation exposure.35,36 Our goal was to investigate 68Ga-DOTA-3PRGD2 (68Ga-3PRGD2) as an integrin αvβ3 targeting imaging agent for monitoring the efficacy of Endostar antiangiogenic therapy with the comparison of 18F-FDG in an animal model.

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EXPERIMENTAL SECTION Materials. All commercially available chemical reagents purchased from J. T. Baker (USA) were of analytical grade. The bifunctional chelator 1,4,7,10-tetraazadodecane- N,N′,N″,N‴tetraacetic acid (DOTA) was purchased from Macrocyclics, Inc. (Dallas, TX). 1-Ethyl-3-[3-(dimethylamino)-propyl] carbodiimide (EDC), N-hydroxysulfono-succinimide (SNHS), and Chelex 100 resin (50−100 mesh) were purchased from Sigma-Aldrich (St. Louis, MO). Water and all buffers were passed through a Chelex 100 column (1 × 15 cm) before use for DOTA conjugation and radiolabeling to ensure that aqueous buffers were metal free. The peptide PEG4-E[PEG4c(RGDfK)]2 (3PRGD2) was synthesized by Peptides International (Louisville, KY). 68GaCl3 solution was obtained from a commercial 68Ge/68Ga generator (ITG Isotope Technologies Garching GmbH, Garching, Germany). The reversed-phase high-performance liquid chromatography (HPLC) system was the same as that previously reported.37 The Endostar was purchased from Shandong Xiansheng Maidejin Biological Pharmaceutical Co., Ltd. Synthesis of DOTA-3PRGD2. DOTA-3PRGD2 conjugate was prepared as we have previously described.25 In brief, the DOTA-OSu (6 μmol, calculated on the basis of Nhydroxysulfonosuccinimide) was added to peptides (2 μmol) in 0.1 N NaHCO3 solution (pH 9.0). After being stirred at 25 °C overnight, the DOTA conjugate was isolated by semipreparative HPLC. HPLC analysis and mass spectroscopy were used to confirm the identity of the product. 68 Ga Radiolabeling. 68Ga was eluted from 68Ge/68Ga generator in 4 × 1 mL 0.05 M HCl, the 68Ga in the second 1 mL vial was directly used for radiolabeling without further purification. One milliliter of 370 MBq (10 mCi) of 68Ga solution was added into a lyophilized kit containing NaOAc buffer (31.25 μmol) and DOTA-3PRGD2 (10 nmol), incubated at 100 °C for 10 min. After cooling down at room temperature for 5 min, the 68Ga-labeled DOTA-3PRGD2 was subjected to Radio-HPLC analysis. The product was then formulated in phosphate-buffered saline (PBS) and passed through a 0.22 μm Millipore filter into a sterile multidose vial for in vivo experiments. 3916

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Figure 1. Chemical structure of 68Ga-3PRGD2 (68Ga-DOTA-3PRGD2). RGD2 represents the dimeric version of c(RGDfK) and 3P represents the 3PEG4.

Figure 2. Validation of the integrin αvβ3 expression in LLC cell and tumor tissue by (A) flow cytometrtric analysis, (B,C) Western blot studies, (D) ex vivo LLC tumor tissue immunofluorescence staining, and (E) microPET imaging of LLC-bearing mice at 1 h after intravenous injection of 68Ga3PRGD2.

Western Blotting. The U87MG human glioma cell lysates, Lewis lung carcinoma (LLC) cell lysates, and the frozen LLC tumor tissues without Endostar treatment or with Endostar treatment on days 3, 7, 14, and 21 were homogenized and extracted using RIPA tissue protein extraction buffer. Protein concentration was determined using a microBCA protein assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA) and adjusted to equivalent values using lysis buffer. After SDSPAGE and transfer, the polyvinylidene fluoride (PVDF) membrane (Invitrogen Corp.; Carlsbad, CA, USA) was blocked with 8% nonfat milk blocking buffer, and then incubated overnight at 4 °C with ITCβ3 (integrin β3) polyclonal primary antibody (1:500; Proteintech, Chicago, IL, USA) followed by incubating at room temperature for 2 h with horseradish peroxidase-conjugated goat antirabbit secondary antibodies. βActin was used as the loading control, and the bands were

administered activity to obtain (assuming a tissue density of 1 g/mL) an image-ROI-derived percent injected dose per gram (ID%/g). Immunofluorescent Staining. The integrin αvβ3 expression patterns of LLC tumor tissues without Endostar treatment or with Endostar treatment on days 3, 7, 14, and 21 were analyzed by fluorescence staining and Western blot. The fluorescence staining study was carried out as previously described.40 Frozen tumor tissue sections were incubated with hamster antimouse CD61 (integrin β3) antibody (1:200; Biolegend, San Diego, CA, USA) and rat antimouse CD31 antibody (1:200; BD Biosciences, San Jose, CA), and then visualized by TRITC-conjugated goat antirat secondary antibody (1:200; CW Biotech, Beijing, China) and FITCconjugated goat antihamster secondary antibody (1:200; Biolegend, San Diego, CA, USA) under the microscope (Carl Zeiss Axiovert 200 M, Carl Zeiss, Thornwood, NY, USA). 3917

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detected using enhanced chemiluminescence (ECL) (Millipore, USA). Statistical Analysis. Quantitative data were expressed as the mean ± SD. Mean values were compared using one-way analysis of variance (ANOVA) and Student’s t test. P values < 0.05 were considered statistically significant.

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RESULTS DOTA-3PRGD2 Conjugation and 68Ga Radiolabeling. DOTA-3PRGD2 (Figure 1) was prepared by direct conjugation of 3PRGD2 peptide with DOTA-OSu. The HPLC purity of DOTA-3PRGD2 was >95%. The labeling was done within 30 min, with a decay-corrected yield ranging from 90% to 95%. The radiochemical purity of 68Ga labeled DOTA-3PRGD2 (named 68Ga-3PRGD2) was >98%. Integrin αvβ3 Expression Validation. The integrin αvβ3 expression in LLC cells and tumor tissue has been evaluated by flow cytometric analysis and Western blot studies. The results of fluorescence-activated cell sorter (FACS) showed that negative rate of LLC cells stained with antimouse integrin β3 (CD61) was 99.96%, almost the same as that of PBS (99.98%) and mouse IgG negative control (99.97%) (Figure 2A). The Western blot study confirmed the finding of the FACS.41,42 As shown in Figure 2B,C, integrin αvβ3 is almost not expressed on LLC cells, but highly expressed in LLC tumor tissues, with U87MG human glioma cells as the positive control. Immunofluorescence staining of LLC tissues was performed. The vascular density and integrin αvβ3 expression in the LLC xenografts were evaluated by ex vivo immunofluorescence staining using anti-CD31 and antimouse integrin β3 antibodies, respectively. As shown in Figure 2D, LLC tissues have abundant vasculatures as indicated by strong CD31 staining. Overlaid staining of CD31 and mouse β3 in the LLC tissues showed that most of CD31 positive areas were also mouse β3positive, indicating that LLC tissues highly express integrin β3 due to its rich vasculatures. Western blot study of LLC tissues also validated the expression of integrin β3 in LLC tissues (Figure 2C). The representative microPET image of LLC-bearing mice at 1 h after intravenous injection of 68Ga-3PRGD2 is shown in Figure 2E. LLC tumor was clearly visualized after injection of the tracer, with high contrast to contralateral background. Antiangiogenic Therapy. The antiangiogenic therapy with Endostar was carried out in LLC tumor-bearing mice. No significant tumor growth inhibition was observed in treatment group as compared with the control group before day 7 posttreatment (p > 0.05, Figure 3). At the end of the therapeutic study (day 21 post-treatment), the tumor growth of the control group was rapid with the tumor size reaching over 1700 mm3, but 270 mm3 in the Endostar group, demonstrating the tumor growth inhibition effect of Endostar. During antiangiogenic therapy, Endostar treatment showed very limited toxicity according to body weight changing rate (data was not shown).43 Monitoring the Efficacy of Antiangiogenic Therapy by microPET. For monitoring the efficacy of antiangiogenic therapy, microPET imaging was performed by using 18F-FDG or 68Ga-3PRGD2 on days 3, 7, 14, and 21 post-treatment, respectively (Figure 4). The whole-body microPET images and tumor uptake (%ID/g) calculated from PET images are presented in Figure 4. The tumor uptake values (%ID/g) of 18 F-FDG in the treatment group were 20.17 ± 3.66, 18.82 ± 4.36, 6.78 ± 4.10, and 11.15 ± 6.59, while the tumor uptake

Figure 3. Tumor growth profiles of control group and Endostar treatment group (9 mice per group). C57BL/6J mice bearing s.c. LLC tumors were treated with Endostar (8 mg/kg/day) via intraperitoneal injection. Saline-treated animals served as controls. Arrows represent the schedule of PET imaging (*p < 0.05; ***p < 0.001).

values in control group were 21.87 ± 5.40, 22.00 ± 3.15, 18.78 ± 3.26, and 24.56 ± 6.31 on days 3, 7, 14, and 21 posttreatment, respectively (Figure 4A,B). Compared with the control group, no significant difference of 18F-FDG uptake was observed in Endostar treated tumors until day 14 (n = 8, p < 0.01) and day 21 post-treatment (n = 6, p < 0.05). The tumor uptake values (%ID/g) of 68Ga-3PRGD2 in the treatment group were 2.93 ± 0.79, 3.99 ± 0.54, 2.81 ± 0.62, and 0.97 ± 0.57, while the tumor uptake values in the control group were 4.92 ± 1.21, 6.08 ± 1.00, 5.76 ± 1.24, and 4.95 ± 1.19 on days 3, 7, 14, and 21 post-treatment (Figure 4C,D), respectively. Different from 18F-FDG microPET imaging, tumor uptake values of 68Ga-3PRGD2 in treatment group were significantly lower than that of the control group, starting from day 3 (n = 8, p < 0.05), day 7 (n = 8, p < 0.05) to day 14 (n = 8, p < 0.01) and day 21 post-treatment (n = 6, p < 0.01), indicating that the tumor growth inhibition of Endostar treatment could be reflected as early as day 3 post-treatment with 68Ga-3PRGD2 PET imaging, which is much earlier than 18 F-FDG PET imaging. Ex Vivo Tumor Tissue Analyses for Assessing Response to Antiangiogenic Therapy. In order to further verify the 68Ga-3PRGD2 PET imaging reflecting the efficacy of Endostar antiangiogenic therapy, the integrin αvβ3 expressions of LLC tumor tissues in both control and treated groups were evaluated on days 3, 7, 14, and 21 post-treatment by immunofluorescence staining of CD31 and CD61 in ex vivo LLC tumor tissues. As indicated by CD31 staining (Figure 5A,B), the angiogenesis in Endostar-treated LLC tumor tissues was found significantly inhibited as early as 3 d post-treatment, as compared with that of the control group, and the inhibition lasted to the end of the 3-week treatment. Significant changes of vascular morphology were observed between the control and treatment groups at all the observed time points. As shown in merged fluorescence signals, almost all the CD61 signals in LLC tumor tissue are colocalized with the CD31 signals, demonstrating that the integrin β3 expression in LLC tumor tissue is almost on the tumor vascular vessels (Figure 5A). Furthermore, the correlations between 68Ga-3PRGD2 tumor uptake and vascular density (CD31) in control and treatment 3918

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Figure 4. Representative PET images of 18F-FDG (A) and 68Ga-3PRGD2 (B) in untreated (control) and Endostar-treated LLC tumor bearing mice at days 3, 7, 14, and 21 post-treatment with saline or Endostar (8 mg/kg/day) via i.p. injection (n = 4). Bar charts show ROIs tumor uptake (%ID/g) for 18F-FDG (C) and 68Ga-3PRGD2 (D) in control and treatment groups at days 3, 7, 14, and 21 post-treatment with saline or Endostar (8 mg/kg/ day) via i.p. injection (arrows indicate the presence of the tumor).

Figure 5. (A) Immunofluorescence double staining of CD31 and CD61 in ex vivo LLC tumor tissue of control and Endostar treated LLC tumor bearing mice at days 3, 7, 14, and 21 post-treatment with saline or Endostar (8 mg/kg/day) via i.p. injection. (B) Bar charts show percentage of vascular density (CD31) in ex vivo LLC tumor tissue of control and treatment groups at days 3, 7, 14, and 21 post-treatment with saline or Endostar (8 mg/kg/day) via i.p. injection. (C) Correlative analysis between 68Ga-3PRGD2 tumor uptake and vascular density (CD31) in LLC tumor tissue of control and treatment groups at days 3, 7, 14, and 21 post-treatment with saline or Endostar (8 mg/kg/day) via i.p. injection.

value (represents integrin β3 expression) in control and treatment groups are shown in Figure 6C, which have linear relationships with r2 = 0.7547 and r2 = 0.9642, respectively.

groups are shown in Figure 5C, which have linear relationships with r2 = 0.6902 and r2 = 0.8223, respectively. Western blot study was carried out accordingly to evaluate the integrin β3 expression in LLC tumor tissues, which confirmed the fluorescence staining result. As shown in Figure 6A,B, the mouse integrin β3 expression in treated group is significantly lower than that of control group on day 3 posttreatment, and this difference was observed at every time point. The correlations between 68Ga-3PRGD2 tumor uptake and gray

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DISCUSSION Lung cancer is the most common cancer worldwide and is a leading cause of cancer-related deaths in many countries.44−47 Antiangiogenic therapy is a novel and effective systemic treatment strategy for clinical cancer treatment.11,48 Antiangio3919

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Figure 6. (A) Western blot of ex vivo LLC tumor tissue in control and Endostar-treated LLC tumor bearing mice at days 3, 7, 14, and 21 posttreatment with saline or Endostar (8 mg/kg/day) via i.p. injection. (B) Bar charts show percentage of integrin density (gray value) in ex vivo LLC tumor tissue of control and treatment groups at days 3, 7, 14, and 21 post-treatment with saline or Endostar (8 mg/kg/day) via i.p. injection. (C) Correlative analysis between 68Ga-3PRGD2 tumor uptake and integrin density (gray value) in LLC tumor tissue of control and treatment groups at days 3, 7, 14, and 21 post-treatment with saline or Endostar (8 mg/kg/day) via i.p. injection.

previous studies have showed that 3PRGD2, as a PEGylated RGD dimer, can achieve high binding affinity to integrin αvβ3/ αvβ5. Here, the 3PRGD2 was radiolabeled with PET nuclide 68 Ga to perform microPET imaging. As shown in Figure 2E, the LLC xenograft can be clearly visualized, indicating that 68Ga3PRGD2 PET can be used for neovasculature imaging in LLC xenograft and can reveal the changing of neovasculature directly. For monitoring the response to Endostar treatment, PET imaging with 68Ga-3PRGD2 and 18F-FDG was performed in treated and untreated LLC tumor bearing mice on days 3, 7, 14, and 21 post-treatment, respectively. As shown in Figure 4A,C, Endostar can lead to 18F-FDG PET imaging detectable for tumor metabolic changes on day 14 post-treatment. However, the changes in tumor uptake of 68Ga-3PRGD2 that responded to antiangiogenic therapy were observed as soon as day 3 posttreatment (Figure 4B,C), which is much earlier than 18F-FDG metabolic response. This is because the Endostar inhibited the newborn vascular endothelial cells, causing the decrease of integrin expression, which led to the decrease of integrintargeted imaging probe uptake in tumor. This finding was confirmed by immunofluorescence staining and Western blot experimental results. Since the fluorescence density values of CD31 and gray values of CD61 Western blot showed a linear correlation with tumor %ID/g values of 68Ga-3PRGD2 (Figures 5C and 6C), suggesting that 68Ga-3PRGD2 imaging can reflect the changes of neovascular density and integrin expression. With this molecular imaging tool, the tumor response to antiangiogenic therapy can be assessed at a very early stage of treatment, much earlier than 18F-FDG metabolic imaging as well as the anatomical structure change by monitoring the tumor volume, which shows very important clinical significance.

genic drugs, unlike cytotoxic drugs, may not induce immediate tumor-cell killing. There may be a period of significant tumor progress before response. Thus, it is necessary to develop an option to monitor the efficacy of antiangiogenic therapy.49 Various methods have been used for clinic to evaluate the efficacy of antiangiogenic therapy. Microvessel density (MVD) count is the most commonly used method and has been used as the golden standard. However, it is an invasive method needing tumor tissue for biopsy, which is nonappreciable and inconvenient for clinical use. PET is a noninvasive and quantitative molecular imaging modality that enables it to noninvasively assess responses to antiangiogenic therapy at molecular level. 18F-FDG PET is commonly performed in clinic for cancer staging and follow-up, which reflects the metabolism in tumor area.50 Herbst et al. reported that the 18F-FDG PET showed complex but generally antiangiogenic agent dosedependent changes in both tumor blood flow and metabolism; however, dose dependence was not evidenced by tumor biopsies.51 Since the antiangiogenic therapy mainly inhibits endothelial cells on neovasculature, monitoring the sensitive and specific biomarkers of endothelial cells may directly reflect the antiangiogenic effects.52 Angiogenesis-induced integrin expression on endothelial cells is a very attractive target for angiogenesis imaging. RGD peptide-based imaging tracers allow specific imaging of integrin αvβ3 and αvβ5, which could serve as a tool to assess antiangiogenic treatment.53−59 Since the integrin αvβ3/αvβ5 is highly expressed on both neovasculature endothelial cells and some tumor cells,23 an integrin αvβ3/αvβ5-negative tumor cell line (LL/2 (LLC1)) was chosen to establish the animal model for the assessment of antiangiogenic treatment.60,61 As shown in Figure 2, integrin αvβ3 is highly expressed only on the neovasculature in LLC xenograft, not on the LLC tumor cell themselves, making the LLC tumor model ideal for RGD-based tracers to assess the efficacy of antiangiogenic treatment. Our 3920

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CONCLUSIONS Ga-3PRGD2, as an integrin-targeted specific probe, can be used for monitoring the efficacy of antiangiogenic therapy. Compared with 18F-FDG metabolic imaging, 68Ga-3PRGD2 PET reflects the tumor response to antiangiogenic therapy much earlier and more accurately, which provides new opportunities to develop individualized therapeutic approaches and establish optimized dosages and dose intervals for effective treatment that improve the survival rate of patients. 68

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AUTHOR INFORMATION

Corresponding Author

*(F.W.) Tel: 86-10-82802871. Fax: 86-10-82801145. E-mail: [email protected]. Author Contributions #

(J.S. and Z.J.) These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported, in part, by the “973” program (2013CB733802 and 2011CB707703), Outstanding Youth Fund (81125011), National Natural Science Foundation of China (NSFC) projects (81201127, 30930030, 81000625, 81222019, 81028009, and 81321003), grants from the Ministry of Science and Technology of China (2012ZX09102301-018, 2011YQ03011407, and 2012BAK25B03-16), and grants from the Ministry of Education of China (31300 and BMU20110263).

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