1、/molecularpharmaceuticsPET Imaging of Neovascularization with 68Ga-3PRGD2 for AssessingTumor Early Response to Endostarangiogenic TherapyJiyun Shi,# Zhongxia Jin,§,# Xujie Liu,§ Di Fan,§ Yi Sun, Huiyun Zhao, Zhaohui Zhu, Zhaofei Liu,§ Bing Jia,§ and Fan Wang*,

2、67;Medical Isotopes Research Center, Peking University,Interdisciplinary Laboratory, Institute of Biophysics,100191,Academy of Sciences,100101,100191,100857,§Department of Radiation Medicine, Basic Medical Sciences, Peking University,Department of Nuclear Medicine, Peking Union Medical College

3、Hospital,Medical and Healthy Analytical Center, Peking University,100191,reduction in tumor size.12,13 Molecular imaging can characterizeINTRODUCTIONand qufy the biological processes in living subjectsAngiogenesis is essential for the growth and metastasis of solid tumor.13 Tumors cannot grow beyond

4、 12 mm3 in size without the neovasculature providing oxygen and nutrients.4,5noninvasively from molecular and cellular levels, with thepotential for predicting response very early after initiation of therapy.14 Among several molecular imaging modalities, positron emission tomography (PET) is a promi

5、sing technology that allows noninvasive imaging of tumor angio genesis 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 specic radiotracer and can lead to a

6、false positive in various forms of infection, inammation, and granulomatous disease.1618A tripeptide moiety of Arg Gly Asp (RGD) has been highlighted for use in angiogenesis imaging, as it specically binds to integrin v3, which is richly expressed in endothelial cell ivated for angiogenesis.1923 Pre

7、viously, we haveInhibition oiogenesis can ultimately provoke vascularregression, impeding delivery of oxygen and nutrients, and ultimately starving the tumor. angiogenic therapy has been approved by many countries as an eective strategy to inhibittumor growth, providing a novel treatment approach fo

8、r cancer patients.68 However, lacking an available strategy to non invasively monitor angiogenesis of tumor in vivo and evaluating the ecacy of angiogenic treatment are still an obstacle fortheangiogenic treatment in clinic. Conventional methodsto monitor angiogenesis are inconvenient, time consu, a

9、nd mostly unrepeatable, as it usually requires to invasively obtaintumor tissue for biopsy and visualizing microvessels by immunohistochemical (IHC) staining of endothelial cell specic markers. Therefore, there is an urgent need to develop a novel and noninvasive method to quickly evaluate the thera

10、peutic response of tumors.911The contrast enhanced MRI and CT have been used toSpecial Issue: Positron Emission Tomography: State of the ArtReceived: Revised: Accepted: Published:April 30, 2014August 18, 2014August 26, 2014August 26, 2014angiogenesis therapy by reecting themonitor themorphologic cha

11、nges of tumor. However, the vascular eectsofangiogenic therapy may occur earlier before there is any3915© 2014 American Chemical S/10.1021/mp5003202 | Mol. Pharmaceutics 2014, 11, 39153922ArticleABSTRACT:angiogenic therapy is an eective strategy to inhibit tumor growth. Endostar

12、, as an approvedangiogenesis agent, inhibits the newborn vascular endothelial cells, causing the decrease of integrin v3 expression. Radiolabeled 3PRGD2, a novel PEGlayted RGD dimer probe (PEG4 EPEG4 c(RGDfK)2) showed highly specic targeting capability to integrin v3, which could be used for monitor

13、ing the ecacy of Endostar angiogenic 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

14、result, 68Ga 3PRGD2 PET reected the tumor response to Endostar angiogenic therapy much earlier (day 3 post treatment vs day 14 post treatment) and more accurately than that of 18F FDG metabolic imaging,which provides new opportunities to develop individualized therapeutic approaches, establish optim

15、ized dosages and doseintervals for eective treatment that improve the survival rate of patients.KEYWORDS:angiogenic therapy, in vivo monitoring, molecular imaging, radiotracer, endostarCell Culture. The U87MG human glioma cell line and Lewis lung carcinoma (LLC) cell line LL/2 (LLC1) were purchased

16、from American Type Culture Collection (Manassas, VA). U87MG cells were cultured in low glucose Dulbeccos Modied Eagles 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 bovi rum (FB

17、S) at 37 °C in a humidied atmosphere with 5% CO2.Animal M Establishment. Female C57BL/6J mice (46 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 An

18、imal Care and Use Committee (IACUC) of Peking University. LLC mice m was established by subcutaneous injection of 2 × 106 LLC cells into the right rear legs of mice. Once the tumor volume reached 6070 mm3, the mice were initiated with Endostar treatment (1 week after inoculation of LLC cells).p

19、repared a novel RGD dimer probe PEG4 EPEG4 c (RGDfK)2 (3PRGD2), which can specically target integrin v3 with relatively high a , 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 (RG

20、D4).2427 The radiolabeled3PRGD2 has been successfully used for tumor detection inclinic.2831Endostar, a recombinant human endostatin, which wasapproved by theFood and Drug Administration(CFDA) in 2005 fortreatment of lung cancer, was mainlyused as anangiogenic agent for cancer treatment.3234 In this

21、 study, we conjugated 3PRGD2 with DOTA and labeledwith 68Ga.68Gais 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

22、investigate 68Ga DOTA 3PRGD2 (68Ga 3PRGD2) as an integrin v3 targeting imaging agent for monitoring theFlow Cytometric Analysis. LLC cells weecacy of Endostararvested andangiogenic therapy with the compartrypsinized, then a single cell suspension containing 1 × 106 cells was prepared in a total

23、 volume of 100 L of 1% bovine serum albumin (BSA)/phosphate buered saline (PBS) (w/v) and immediately stained for 2 h at room temperature withison of 18F FDG in an animal m.EXPERIMENTAL SECTIONMaterials. All commercially available chemical reagentshamstermouse CD61 (integrin 3)body (1:200;purchased

24、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 carbodiiBiolegend, San Diego, CA, USA), and then incubated with theFITC conjugat

25、ed goathamster secondarybody (Biolegend, San Diego, CA, USA). PBS and mouse IgG were used as controls. Stained cells were analyzed by a FACS Calibur ow cytometer (Becton inson, erford, NJ, USA).Endostar Treatment Protocol. LLC tumor bearing C57BL/6J mice with the tumor size of 60 mm3 weremide (EDC),

26、 N hydroxysulfono succinimide (SNHS), and Chelex 100 resin (50100 mesh) were purchased from Sigma Aldrich (St. Louis, MO). Water and all buers were passed through a Chelex 100 column (1 × 15 cm) before use for DOTA conjugation and radiolabeling to ensure that aqueous buers were metal . The pept

27、ide PEG4 EPEG4 c(RGDfK)2 (3PRGD2) was synthesized by Peptides Interna tional (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

28、the same as that previously reported.37 The Endostar wasrandomly 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.

29、Endostar and saline weministered daily for 21 dayscontinuously. Tumor dimensions were measured everyday withdigital 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

30、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 m scanner (Siemens Medical Solutions, Mal

31、vern, PA) as previously reported.38,39 The microPET studies were performed by tail vein injection of about 5.55 MBq (150 Ci) 18F FDG or 68Ga 3PRGD2 into C57BL/6J mice bearing LLC xenografts under isourane anesthesia. Ten minute static PET images were acquired at 1 h time point postinjection (p.i.) (

32、n = 4/group). The images were reconstructed by a two dimensional orderedpurchased from ShXiansheng Maidejin BiologicalPharmaceutical Co.,.Synthesis of DOTA-3PRGD2. DOTA 3PRGD2 conjugatewas prepared as we have previously described.25 In brief, the DOTA OSu (6 mol, calculated on the basis of N hydroxy

33、sulfonosuccinimide) was added to peptides (2 mol)in 0.1 N NaHCO3 solution (pH 9.0). After being stirred at 25°C overnight, the DOTA conjugate was isolated by semi preparative HPLC. HPLC analysis and mass spectroscopy were used to conrm the identity of the product.68GaRadiolabeling.68Gawas elute

34、d from 68Ge/68Gagenerator in 4 × 1 mL 0.05 M HCl, the 68Ga in the second 1mL vial was directly used for radiolabeling without furthersubsets expectationum (OSEM) algorithm, and nopurication. One milliliter of 370 MBq (10 mCi) of68Gacorrection was necessary for attenuation or scatter correction.

35、solution was added into a lyophilized kit containing NaOAc buer (31.25 mol) 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 toRegions of interest (ROIs) were drawn over the tumor, kidney, liv

36、er, and muscle by using vendor software ASI Pro on decay corrected whole body coronal images for each microPETscan. Theum radioactivity concentrations (accumuRadio HPLC analysis. The product was then formulated in phosphate buered saline (PBS) and passed through a 0.22 m Millipore lter into

37、a sterile multidose vial for in vivo experiments.lation) 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 /10.1021/mp5

38、003202 | Mol. Pharmaceutics 2014, 11, 39153922Molecular PharmaceuticsArticleMolecular PharmaceuticsFigure 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 v3 expression in LLC cel

39、l and tumor tissue by (A) ow cytometrtric analysis, (B,C) Western blot studies, (D) ex vivo LLC tumor tissue immunouorescence staining, and (E) microPET imaging of LLC bearing mice at 1 h after intravenous injection of 68Ga 3PRGD2.Western Blotting. The U87MG human glioma cell lysates, Lewis lung car

40、cinoma (LLC) cell lysates, and the frozen LLC tumor tissues without Endostar treatment or with Endostaradministered activity to obtain (assua tissue density of 1g/mL) an image ROI derived percent injected dose per gram (ID%/g).Immunouorescent Staining. The integrin v3 expres sion patterns of LLC tum

41、or tissues without Endostar treatment or with Endostar treatment on days 3, 7, 14, and 21 were analyzed by uorescence staining and Western blot. Thetreatment on days 3, 7, 14, and 21 weomogenized andextracted using RIPA tissue protein extraction buer. Proteinconcentration was determined using a micr

42、oBCA protein assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA) and adjusted to equivalent values using lysis buer. After SDS PAGE and transfer, the polyvinylidene uoride (PVDF) membrane (Invitrogen Corp.; Carlsbad, CA, USA) was blocked with 8% nonfat milk blocking buer, and then incubated ov

43、ernight at 4 °C with ITC3 (integrin 3) polyclonal primary body (1:500; Proteintech, Chicago, IL, USA) followed by incubating at room temperature for 2 h with horseradishuorescence staining study was carried out as previously described.40 Frozen tumor tissue sections were incubated with hamster

44、mouse CD61 (integrin 3) body (1:200; Biolegend, San Diego, CA, USA) and rat mouse CD31body (1:200; BD Biosciences, San visualized by TRITC conjugated goatJose, CA), and then rat secondary) and FITCbody (1:200; CW Biotech,conjugated goathamster secondarybody (1:200;rabbit secondarybodies. peroxidase

45、conjugated goatBiolegend, San Diego, CA, USA) under the microscope (Carl Zeiss Axiovert 200 M, Carl Zeiss, Thornwood, NY, USA).Actin was used as the loading control, and the bands /10.1021/mp5003202 | Mol. Pharmaceutics 2014, 11, 39153922ArticleMolecular Pharmaceuticsdetected using

46、 enhanced chemiluminescence (ECL) (Millipore, USA).Statistical Analysis. Qutative data were expressed asthe mean ± SD. Mean values were compared using one wayanalysis of variance (ANOVA) ands t test. P values <0.05 were considered statistically signicant.RESULTS68GaDOTA-3PRGD2 Conjugation an

47、dRadiolabeling.DOTA 3PRGD2 (Figure 1) was prepared by direct conjugationof 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%.68GaThe radiochemical purity oflabeled DOTA 3PRGD2(named 68Ga

48、 3PRGD2) was >98%.Integrinv3Expression Validation. The integrin v3Figure 3. Tumor growth proles 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 se

49、rved as controls. Arrows represent the schedule of PET imaging (*p < 0.05; *p < 0.001).expression in LLC cells and tumor tissue has been evaluated byow cytometric analysis and Western blot studies. The resultsof uorescence activated cell sorter (FACS) showed that negative rate of LLC cells sta

50、ined with mouse integrin 3 (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 conrmed the nding of the FACS.41,42 As shown in Figure 2B,C, integrin v3 is almost not expressed on LLC cells, but highly expressed in LLC

51、 tumor tissues, with U87MG human glioma cells as the positive control.Immunouorescence staining of LLC tissues was performed. The vascular density and integrin v3 expression in the LLC xenografts were evaluated by ex vivo immunouorescencevalues in control group were 21.87 ± 5.40, 22.00 ± 3

52、.15, 18.78± 3.26, and 24.56 ± 6.31 on days 3, 7, 14, and 21 post treatment, respectively (Figure 4A,B). Compared with the control group, no signicant dierence 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 <

53、; 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,

54、7, 14, and 21 post treatment (Figure 4C,D), respectively. Dierent from 18F FDG microPET imaging, tumor uptake values of 68Ga 3PRGD2 in treatment group were signicantly 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 <

55、0.01) and day 21 post treatment (n = 6, p < 0.01), indicating that the tumor growth inhibition of Endostar treatment could be reected as early as day 3 post treatment with 68Ga 3PRGD2 PET imaging, which is much earlier than 18F FDG PET imaging.Ex Vivo Tumor Tissue Analyses for Assessing Response

56、to angiogenic Therapy. In order to further verify the 68Ga 3PRGD2 PET imaging reecting the ecacy of Endostar angiogenic therapy, the integrin v3 expressions of LLC tumor tissues in both control and treated groups were evaluated on days 3, 7, 14, and 21 post treatment by immunouorescence staining of

57、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 signicantly 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 we

58、ek treatment. Signicant changes of vascular morphology were observed between the control and treatment groups at all the observed time points. As shown in merged uorescence signals, almost all the CD61 signals in LLC tumor tissue are colocalized with the CD31 signals,demonstrating that the integrin 3 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 treatmentstainin