Reproducibility of 18F-FDG microPET studies in mouse tumor xenografts.

(18)F-FDG has been used to image mouse xenograft models with small-animal PET for therapy response. However, the reproducibility of serial scans has not been determined. The purpose of this study was to determine the reproducibility of (18)F-FDG small-animal PET studies. METHODS: Mouse tumor xenografts were formed with B16F10 murine melanoma cells. A 7-min small-animal PET scan was performed 1 h after a 3.7- to 7.4-MBq (18)F-FDG injection via the tail vein. A second small-animal PET scan was performed 6 h later after reinjection of (18)F-FDG. Twenty-five sets of studies were performed. Mean injected dose per gram (%ID/g) values were calculated from tumor regions of interest. The coefficient of variation (COV) from studies performed on the same day was calculated to determine the reproducibility. Activity from the second scans performed after 6 h were adjusted by subtracting the estimated residual activity from the first (18)F-FDG injection. For 7 datasets, an additional scan immediately before the second injection was performed, and residual activity from this additional delayed scan was subtracted from the activity of the second injection. COVs of both subtraction methods were compared. Blood glucose values were measured at the time of injection and used to correct the %ID/g values. RESULTS: The COV for the mean %ID/g between (18)F-FDG small-animal PET scans performed on the same day 6 h apart was 15.4% +/- 12.6%. The delayed scan subtraction method did not produce any significant change in the COV. Blood glucose correction increased the COV. The injected dose, tumor size, and body weight did not appear to contribute to the variability of the scans. CONCLUSION: (18)F-FDG small-animal PET mouse xenograft studies were reproducible with moderately low variability. Therefore, serial small-animal PET studies may be performed with reasonable accuracy to measure tumor response to therapy.     

Characterization of osteolytic, osteoblastic, and mixed lesions in a prostate cancer mouse model using 18F-FDG and 18F-fluoride PET/CT.

The combination of small-animal PET/CT scans and conventional imaging methods may enhance the evaluation of in vivo biologic interactions of murine models in the study of prostate cancer metastasis to bone. METHODS: Small-animal PET/CT scans using (18)F-fluoride ion and (18)F-FDG coregistered with high-resolution small-animal CT scans were used to longitudinally assess the formation of osteoblastic, osteolytic, and mixed lesions formed by human prostate cancer cell lines in a severe combined immunodeficient (SCID) mouse tibial injection model. These scans were correlated with plain radiographs, histomorphometry, and soft-tissue measurements. RESULTS: Small-animal PET/CT scans were able to detect biologic activity of cells that induced an osteoblastic lesion 2 wk earlier than on plain radiographs. Furthermore, both the size and the activity of the lesions detected on PET/CT images significantly increased at each successive time point (P < 0.05). (18)F-FDG lesions strongly correlated with soft-tissue measurements, whereas (18)F-fluoride ion activity correlated with bone volume measured on histomorphometric analysis (P < 0.005). Osteolytic lesions were successfully quantified using small-animal CT, whereas lesion sizes measured on (18)F-FDG PET scans also strongly correlated with soft-tissue tumor burden (P < 0.05). In contrast, for mixed lesions, (18)F-fluoride ion and (18)F-FDG PET/CT scans detected only minimal activity. CONCLUSION: (18)F-FDG and (18)F-fluoride ion PET/CT scans can be useful tools in characterizing pure osteolytic and osteoblastic lesions induced by human prostate cancer cell lines. The value of this technology needs further evaluation to determine whether these studies can be used effectively to detect more subtle responses to different treatment regimens in animal models.     

Pegylated Arg-Gly-Asp peptide: 64Cu labeling and PET imaging of brain tumor alphavbeta3-integrin expression.

The alphav-integrins, cell adhesion molecules that are highly expressed on activated endothelial cells and tumor cells but not on dormant endothelial cells or normal cells, present an attractive target for tumor imaging and therapy. We previously coupled a cyclic Arg-Gly-Asp (RGD) peptide, c(RGDyK), with 1,4,7,10-tetraazacyclododecane-N,N',N',N'-tetraacetic acid (DOTA) and labeled the RGD-DOTA conjugate with 64Cu (half-life, 12.8 h; 19% beta+) for solid tumor targeting, with high tumor-to-background contrast. The rapid tumor washout rate and persistent liver and kidney retention of this tracer prompted us to optimize the tracer for improved pharmacokinetic behavior. In this study, we introduced a polyethylene glycol (PEG; molecular weight, 3,400) moiety between DOTA and RGD and evaluated the 64Cu-DOTA-PEG-RGD tracer for microPET imaging in brain tumor models. METHODS: DOTA was activated in situ and conjugated with RGD-PEG-NH2 under slightly basic conditions. alphavbeta3-Integrin-binding affinity was evaluated with a solid-phase receptor-binding assay in the presence of 125I-echistatin. Female nude mice bearing subcutaneous U87MG glioblastoma xenografts were administered 64Cu-DOTA-PEG-RGD, and the biodistributions of the radiotracer were evaluated from 30 min to 4 h after injection. microPET (20 min of static imaging at 1 h after injection) and then quantitative autoradiography were used for tumor visualization and quantification. The same tracer was also applied to an orthotopic U87MG model for tumor detection. RESULTS: The radiotracer was synthesized with a high specific activity (14,800-29,600 GBq/mmol [400-800 Ci/mmol]). The c(RGDyK)-PEG-DOTA ligand showed intermediate binding affinity for alphavbeta3-integrin (50% inhibitory concentration, 67.5 +/- 7.8 nmol/L [mean +/- SD]). The pegylated RGD peptide demonstrated rapid blood clearance (0.57 +/- 0.15 percentage injected dose [%ID]/g [mean +/- SD] at 30 min after injection and 0.03 +/- 0.02 %ID/g at 4 h after injection). Activity accumulation in the tumor was rapid and high at early time points (2.74 +/- 0.45 %ID/g at 30 min after injection), and some activity washout was seen over time (1.62 +/- 0.18 %ID/g at 4 h after injection). Compared with (64)Cu-DOTA-RGD, this tracer showed improved in vivo kinetics, with significantly reduced liver uptake (0.99 +/- 0.08 %ID/g vs. 1.73 +/- 0.39 %ID/g at 30 min after injection and 0.58 +/- 0.07 %ID/g vs. 2.57 +/- 0.49 %ID/g at 4 h after injection). The pegylated RGD peptide showed higher renal accumulation at early time points (3.51 +/- 0.24 %ID/g vs. 2.18 +/- 0.23 %ID/g at 30 min after infection) but more rapid clearance (1.82 +/- 0.29 %ID/g vs. 2.01 +/- 0.25 %ID/g at 1 h after injection) than 64Cu-DOTA-RGD. The integrin receptor specificity of this radiotracer was demonstrated by blocking of tumor uptake by coinjection with nonradiolabeled c(RGDyK). The high tumor-to-organ ratios for the pegylated RGD peptide tracer (at 1 h after injection: tumor-to-blood ratio, 20; tumor-to-muscle ratio, 12; tumor-to-liver ratio, 2.7; and tumor-to-kidney ratio, 1.2) were confirmed by microPET and autoradiographic imaging in a subcutaneous U87MG tumor model. This tracer was also able to detect an orthotopic brain tumor in a model in which U87MG cells were implanted into the mouse forebrain. Although the magnitude of tumor uptake in the orthotopic xenograft was lower than that in the subcutaneous xenograft, the orthotopic tumor was still visualized with clear contrast from normal brain tissue. CONCLUSION: This study demonstrated the suitability of a PEG moiety for improving the in vivo kinetics of a 64Cu-RGD peptide tracer without compromising the tumor-targeting ability and specificity of the peptide. Systematic investigations of the effects of the size and geometry of PEG on tumor targeting and in vivo kinetics will lead to the development of radiotracers suitable for clinical applications such as visualizing and quantifying alphav-integrin expression by PET. In addition, the same ligand labeled with therapeutic radionuclides may be applicable for integrin-targeted internal radiotherapy.     

Evaluation of the antitumor effects of rilotumumab by PET imaging in a U-87 MG mouse xenograft model

IntroductionDysregulation of the hepatocyte growth factor (HGF)/MET pathway has been implicated in various cancers. Rilotumumab is an investigational, fully human monoclonal antibody that binds and neutralizes HGF. The purpose of this study was to evaluate the efficacy of rilotumumab in a U-87 MG mouse xenograft tumor model using ¹⁸ F-FDG and ¹⁸ F-FLT PET.MethodsU-87 MG tumor-bearing nude mice received rilotumumab or control IgG2. In the dose response study, increasing doses of rilotumumab (10, 30, 100, 300, or 500 μg) were administered, and mice were evaluated with ¹⁸ F-FDG PET at baseline and 7 days post-treatment. In the time course study, 300 μg of rilotumumab twice per week was used for the treatment, and mice were evaluated over 7 days using ¹⁸ F-FDG and ¹⁸ F-FLT PET.ResultsIn the dose response study, rilotumumab at doses of 300 and 500 μg was similarly effective against tumor growth. Treatment with 300 and 500 μg rilotumumab inhibited ¹⁸ F-FDG accumulation with significant decreases of ? 37% and ? 40% in the percent injected dose per gram of tissue (%ID/g), respectively. In the time course study, treatment with 300 μg rilotumumab inhibited ¹⁸ F-FDG and ¹⁸ F-FLT accumulation with a maximum %ID/g of ? 41% and ? 64%, respectively. No apparent differences between the use of either tracer to evaluate rilotumumab efficacy were observed.ConclusionsRilotumumab inhibited ¹⁸ F-FDG and ¹⁸ F-FLT accumulation as early as 2 and 4 days after treatment, respectively, in a mouse tumor model. Further studies to evaluate ¹⁸ F-FDG PET imaging as an early tumor response marker for rilotumumab are warranted. Rilotumumab is currently being tested in patients with MET-positive, advanced gastric and gastroesophageal cancer.     

Multimodality imaging of tumor xenografts and metastases in mice with combined small-animal PET, small-animal CT, and bioluminescence imaging.

Recent developments have established molecular imaging of mouse models with small-animal PET and bioluminescence imaging (BLI) as an important tool in cancer research. One of the disadvantages of these imaging modalities is the lack of anatomic information. We combined small-animal PET and BLI technology with small-animal CT to obtain fusion images with both molecular and anatomic information. METHODS: We used small-animal PET/CT and BLI to detect xenografts of different cell lines and metastases of a melanoma cell line (A375M-3F) that had been transduced with a lentiviral vector containing a trimodality imaging reporter gene encoding a fusion protein with Renilla luciferase, monomeric red fluorescent protein, and a mutant herpes simplex virus type 1 thymidine kinase. RESULTS: Validation studies in mouse xenograft models showed a good coregistration of images from both PET and CT. Melanoma metastases were detected by 18F-FDG PET, 9-[4-(18)F-fluoro-3-(hydroxymethyl)butyl]guanine (18F-FHBG) PET, CT, and BLI and confirmed by ex vivo assays of Renilla luciferase and mutant thymidine kinase expression. 18F-FHBG PET/CT allowed detection and localization of lesions that were not seen on CT because of poor contrast resolution and were not seen on 18F-FDG PET because of higher background uptake relative to 18F-FHBG. CONCLUSION: The combination of 18F-FHBG PET, small-animal CT, and BLI allows a sensitive and improved quantification of tumor burden in mice. This technique is potentially useful for the study of the biologic determinants of metastasis and for the evaluation of novel cancer treatments.     

A preliminary study of anti-1-amino-3-18F-fluorocyclobutyl-1-carboxylic acid for the detection of prostate cancer.

We evaluated the feasibility of anti-1-amino-3-(18)F-fluorocyclobutyl-1-carboxylic acid (anti-(18)F-FACBC) in diagnosing prostate cancer (PCa), using a rat orthotopic prostate cancer transplantation (OPCT) model. Furthermore, using in vivo experiments, we examined the potential of anti-(18)F-FACBC for differentiating between PCa and inflammation and between PCa and benign prostatic hyperplasia (BPH). METHODS: The OPCT model was developed by transplanting DU145, a human PCa cell line, into the ventral prostate of athymic F344 rats. To develop a dual PCa and inflammation (DPCI) model, MAT-Ly-Lu-B2--a rat PCa cell line--was transplanted subcutaneously into male Copenhagen rats. Streptozotocin was injected into the hind footpad of these rats for inducing popliteal lymphadenitis. For inducing the BPH, normal F344 rats were castrated and injected subcutaneously with testosterone propionate. In biodistribution studies, the rats were injected with anti-(18)F-FACBC or (18)F-FDG and sacrificed at 15 or 60 min after injection. We performed dynamic small-animal PET of the abdominal portion of the OPCT rats for 60 min after the injection of anti-(18)F-FACBC or (18)F-FDG. RESULTS: The biodistribution in the OPCT rats at 60 min after injection showed that the uptake of anti-(18)F-FACBC and (18)F-FDG into the PCa tissue was 1.58 +/- 0.40 %ID/cm(3) (percentage injected dose per cm(3)) and 1.48 +/- 0.90 %ID/cm(3), respectively (P > 0.05). The accumulation of anti-(18)F-FACBC in the urinary bladder at 60 min after injection was 3.09 +/- 1.43 %ID/cm(3), whereas that of (18)F-FDG was 69.31 +/- 16.55 %ID/cm(3) (P < 0.05). Consequently, small-animal imaging with anti-(18)F-FACBC facilitated the visualization of the PCa tissue of the OPCT rats with higher contrast than (18)F-FDG. Furthermore, in comparison with (18)F-FDG, apparently higher ratios of PCa to inflammation and PCa to BPH accumulation of anti-(18)F-FACBC were demonstrated in the animal models. CONCLUSION: FACBC PET is believed to be useful not only for the visualization of human PCa but also for differentiating between PCa and inflammation and between PCa and BHP.     

18F-FDG small-animal PET for monitoring the therapeutic effect of CT-guided radiofrequency ablation on implanted VX2 lung tumors in rabbits.

The primary goals of this study were to investigate the behavior of normal lung tissues after radiofrequency ablation (RFA) and to determine the suitability of 18F-FDG PET, using a dedicated small-animal scanner, for monitoring the early therapeutic effects of RFA on VX2 lung tumors (VX2s) in rabbits. METHODS: Fourteen Japanese white rabbits with normal lungs underwent RFA, followed by 18F-FDG PET at 1 d and at 1, 2, 4, and 8 wk. In addition, 7 rabbits with untreated VX2s underwent 18F-FDG PET, and 13 rabbits with RFA-treated VX2s underwent 18F-FDG PET at 1 d (n = 7) or 1 wk (n = 6) after the treatment. RESULTS: After RFA of normal lungs, ring-shaped accumulations of 18F-FDG, which coincided with inflammation caused by ablation, were observed. The mean early- (40-60 min after injection) and delayed (100-120 min)-phase ablated lesion-to-muscle ratios were, respectively, 2.9 +/- 1.0 and 3.3 +/- 0.8 (1 d), 4.1 +/- 0.6 and 5.2 +/- 0.9 (1 wk), 4.1 +/- 1.0 and 5.3 +/- 1.5 (2 wk), 3.1 +/- 0.5 and 3.6 +/- 1.1 (4 wk), and 1.8 +/- 0.1 and 2.3 +/- 0.1 (8 wk). At 4 and 8 wk, the uptake was less than that at 1 and 2 wk (P < 0.05). VX2s showed mean tumor-to-muscle ratios of 6.6 +/- 2.1 and 8.6 +/- 3.3 at the early and delayed phases, respectively. For ablated tumors, the respective ratios were 0.8 +/- 0.4 and 1.1 +/- 0.7 (1 d) and 1.2 +/- 0.5 and 1.5 +/- 0.7 (1 wk). These values were significantly lower than those for nonablated tumors (P < 0.001). Histopathologic examination confirmed the absence of viable tumors. 18F-FDG accumulation around ablated tumors reflected thermally damaged normal tissues and was significantly lower than that of control VX2s (P < 0.01). CONCLUSION: Our data suggest that 18F-FDG PET is promising for evaluating the therapeutic response of lung malignancies to RFA: Accumulation of 18F-FDG in surrounding normal tissues appears to be time dependent, and the data suggest that, clinically, 18F-FDG PET should be performed 4 wk or more after RFA. Delayed-phase images seem to better distinguish tumor from inflammation than do early-phase images.     

Evaluation of (76)Br-FBAU as a PET reporter probe for HSV1-tk gene expression imaging using mouse models of human glioma.

The utility of 5-(76)Br-bromo-2'-fluoro-2'-deoxyuridine ((76)Br-FBAU), a uracil analog, as a PET reporter probe for use with the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene system for gene expression imaging was evaluated in vivo and in vitro using human and rat glioma cells. METHODS: Human glioma cell lines U87 and U251 were transduced with replication-defective adenovirus constitutively expressing HSV1-tk (Ad.TK) or a control expressing green fluorescent protein (Ad.GFP). These cells were incubated with (76)Br-FBAU for 20-120 min to determine the percentage of total dose uptake. In vitro uptake of equimolar concentrations (1.8 x 10(-8) mol/L) of (76)Br-FBAU and 2'-fluoro-2'-deoxy-5-iodouracil-beta-d-arabinofuranoside ((14)C-FIAU) was also determined in RG2-TK rat glioma cells stably expressing HSV1-tk and in control RG2 cells at 30-120 min. In vivo uptake of (76)Br-FBAU was determined in subcutaneous U87 tumor intratumorally transduced with Ad.TK by ex vivo biodistribution. Uptake in intracranial U87 tumors transduced with Ad.TK expressing HSV1-tk was measured by brain autoradiography. In vivo PET was performed on subcutaneous and intracranial U87 tumors transduced with Ad.TK and on subcutaneous and intracranial stably expressing RG2-TK tumors. RESULTS: U87 and U251 cells transduced with Ad.TK had significantly increased uptake of (76)Br-FBAU compared with cells transduced with Ad.GFP over 20-120 min. In stably expressing cells at 120 min, (14)C-FIAU uptake in RG2-TK tumor cells was 11.3 %ID (percentage injected dose) and in RG2 control cells was 1.7 %ID, and (76)Br-FBAU uptake in RG2-TK tumor cells was 14.2 %ID and in RG2 control cells was 1.5 %ID. Ex vivo biodistribution of subcutaneous U87 tumors transduced with Ad.TK accumulated (76)Br-FBAU significantly more than in the control Ad.GFP transduced tumor and normal tissue, with the lowest uptake in brain. Autoradiography showed localized uptake in intracranial U87 and U251 cells transduced with Ad.TK. PET image analyses of mice with RG2-TK tumors resulted in an increased tumor-to-background ratio of 13 and 26 from 2 to 6 h after injection, respectively, in intracranial tumors. CONCLUSION: (76)Br-FBAU accumulates in glioma cells constitutively expressing HSV1-tk by either adenoviral transduction or in stably expressing cell lines both in vitro and in vivo. (76)Br-FBAU shows promise as a PET reporter probe for use with the HSV1-tk in vivo gene expression imaging system.     

<Superscript>68</Superscript>Ga-labeled multimeric RGD peptides for microPET imaging of integrin α<Subscript>v</Subscript>β<Subscript>3</Subscript> expression

PURPOSE: We and others have reported that (18)F- and (64)Cu-labeled arginine-glycine-aspartate (RGD) peptides allow positron emission tomography (PET) quantification of integrin alpha(v)beta(3) expression in vivo. However, clinical translation of these radiotracers is partially hindered by the necessity of cyclotron facility to produce the PET isotopes. Generator-based PET isotope (68)Ga, with a half-life of 68 min and 89% positron emission, deserves special attention because of its independence of an onsite cyclotron. The goal of this study was to investigate the feasibility of (68)Ga-labeled RGD peptides for tumor imaging. METHODS: Three cyclic RGD peptides, c(RGDyK) (RGD1), E[c(RGDyK)](2) (RGD2), and E{E[c(RGDyK)](2)}(2) (RGD4), were conjugated with macrocyclic chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and labeled with (68)Ga. Integrin affinity and specificity of the peptide conjugates were assessed by cell-based receptor binding assay, and the tumor targeting efficacy of (68)Ga-labeled RGD peptides was evaluated in a subcutaneous U87MG glioblastoma xenograft model. RESULTS: U87MG cell-based receptor binding assay using (125)I-echistatin as radioligand showed that integrin affinity followed the order of NOTA-RGD4 > NOTA-RGD2 > NOTA-RGD1. All three NOTA conjugates allowed nearly quantitative (68)Ga-labeling within 10 min (12-17 MBq/nmol). Quantitative microPET imaging studies showed that (68)Ga-NOTA-RGD4 had the highest tumor uptake but also prominent activity accumulation in the kidneys. (68)Ga-NOTA-RGD2 had higher tumor uptake (e.g., 2.8 +/- 0.1%ID/g at 1 h postinjection) and similar pharmacokinetics (4.4 +/- 0.4 tumor/muscle ratio, 2.0 +/- 0.1 tumor/liver ratio, and 1.1 +/- 0.1 tumor/kidney ratio) compared with (68)Ga-NOTA-RGD1. CONCLUSIONS: The dimeric RGD peptide tracer (68)Ga-NOTA-RGD2 with good tumor uptake and favorable pharmacokinetics warrants further investigation for potential clinical translation to image integrin alpha(v)beta(3).     

In vivo quantitation of glucose metabolism in mice using small-animal PET and a microfluidic device.

The challenge of sampling blood from small animals has hampered the realization of quantitative small-animal PET. Difficulties associated with the conventional blood-sampling procedure need to be overcome to facilitate the full use of this technique in mice. METHODS: We developed an automated blood-sampling device on an integrated microfluidic platform to withdraw small blood samples from mice. We demonstrate the feasibility of performing quantitative small-animal PET studies using (18)F-FDG and input functions derived from the blood samples taken by the new device. (18)F-FDG kinetics in the mouse brain and myocardial tissues were analyzed. RESULTS: The studies showed that small ( approximately 220 nL) blood samples can be taken accurately in volume and precisely in time from the mouse without direct user intervention. The total blood loss in the animal was <0.5% of the body weight, and radiation exposure to the investigators was minimized. Good model fittings to the brain and the myocardial tissue time-activity curves were obtained when the input functions were derived from the 18 serial blood samples. The R(2) values of the curve fittings are >0.90 using a (18)F-FDG 3-compartment model and >0.99 for Patlak analysis. The (18)F-FDG rate constants K(1)(*), k(2)(*), k(3)(*), and k(4)(*), obtained for the 4 mouse brains, were comparable. The cerebral glucose metabolic rates obtained from 4 normoglycemic mice were 21.5 +/- 4.3 mumol/min/100 g (mean +/- SD) under the influence of 1.5% isoflurane. By generating the whole-body parametric images of K(FDG)(*) (mL/min/g), the uptake constant of (18)F-FDG, we obtained similar pixel values as those obtained from the conventional regional analysis using tissue time-activity curves. CONCLUSION: With an automated microfluidic blood-sampling device, our studies showed that quantitative small-animal PET can be performed in mice routinely, reliably, and safely in a small-animal PET facility.     

Synthesis and preclinical evaluation of a folic acid derivative labeled with 18F for PET imaging of folate receptor-positive tumors.

Folic acid was linked regioselectively through its alpha- and gamma-carboxyl groups to 4-fluorobenzylamine (FBA), and the alpha- and gamma-FBA-folate regioisomers were evaluated for their ability to bind to folate receptor-positive cells. The 18F-labeled alpha/gamma-FBA-folate counterpart was examined for in vivo tumor targeting efficiency in nude mice bearing folate receptor-positive tumor cells. METHODS: 18F-alpha/gamma-FBA-folate was prepared in a 4-step reaction sequence starting from folic acid. The relative binding affinities of the alpha- and gamma-FBA-folates to the folate receptor with respect to parent folic acid were determined in cultured KB-31 cells (nasopharyngeal epidermal carcinoma cell line) overexpressing the folate receptor using 3H-folic acid. Tumor accumulation of the 18F-labeled alpha/gamma-FBA-folate and 18F-FDG was analyzed in vivo by high-resolution PET. Biodistribution and PET studies were performed under baseline and blockage conditions. RESULTS: The radiochemical yield of the coupling step ranged from 15% to 44%, and the maximum specific radioactivity was 24 GBq/micromol. The in vitro binding affinities of the alpha- and gamma-isomers and folic acid were 71, 62, and 41 nmol/L, respectively. PET revealed heterogeneous uptake of the radioligand, with the highest activity concentrations found in the tumor rim. In contrast, 18F-FDG uptake in a nude mouse bearing KB-31 folate receptor-positive tumors was negligible. Radioligand uptake in tumors at 125 min after injection amounted to 6.56% of the injected dose per gram of tissue (%ID/g) in control animals, whereas radioactivity accumulation in the tumors of folic acid-treated animals was significantly reduced by more than 80%-to 1.07 %ID/g (P = 0.001). CONCLUSION: This new 18F-labeled folic acid derivative is a promising tool for PET imaging of folate receptor-positive tumors.     

Dual-function probe for PET and near-infrared fluorescence imaging of tumor vasculature.

To date, the in vivo imaging of quantum dots (QDs) has been mostly qualitative or semiquantitative. The development of a dual-function PET/near-infrared fluorescence (NIRF) probe can allow for accurate assessment of the pharmacokinetics and tumor-targeting efficacy of QDs. METHODS: A QD with an amine-functionalized surface was modified with RGD peptides and 1,4,7,10-tetraazacyclodocecane-N,N',N',N'-tetraacetic acid (DOTA) chelators for integrin alpha(v)beta(3)-targeted PET/NIRF imaging. A cell-binding assay and fluorescence cell staining were performed with U87MG human glioblastoma cells (integrin alpha(v)beta(3)-positive). PET/NIRF imaging, tissue homogenate fluorescence measurement, and immunofluorescence staining were performed with U87MG tumor-bearing mice to quantify the probe uptake in the tumor and major organs. RESULTS: There are about 90 RGD peptides per QD particle, and DOTA-QD-RGD exhibited integrin alpha(v)beta(3)-specific binding in cell cultures. The U87MG tumor uptake of (64)Cu-labeled DOTA-QD was less than 1 percentage injected dose per gram (%ID/g), significantly lower than that of (64)Cu-labeled DOTA-QD-RGD (2.2 +/- 0.3 [mean +/- SD] and 4.0 +/- 1.0 %ID/g at 5 and 18 h after injection, respectively; n = 3). Taking into account all measurements, the liver-, spleen-, and kidney-to-muscle ratios for (64)Cu-labeled DOTA-QD-RGD were about 100:1, 40:1, and 1:1, respectively. On the basis of the PET results, the U87MG tumor-to-muscle ratios for DOTA-QD-RGD and DOTA-QD were about 4:1 and 1:1, respectively. Excellent linear correlation was obtained between the results measured by in vivo PET imaging and those measured by ex vivo NIRF imaging and tissue homogenate fluorescence (r(2) = 0.93). Histologic examination revealed that DOTA-QD-RGD targets primarily the tumor vasculature through an RGD-integrin alpha(v)beta(3) interaction, with little extravasation. CONCLUSION: We quantitatively evaluated the tumor-targeting efficacy of a dual-function QD-based probe with PET and NIRF imaging. This dual-function probe has significantly reduced potential toxicity and overcomes the tissue penetration limitation of optical imaging, allowing for quantitative targeted imaging in deep tissue.     

PET imaging of colorectal cancer in xenograft-bearing mice by use of an 18F-labeled T84.66 anti-carcinoembryonic antigen diabody.

In this study, we investigated the 18F-labeled anti-carcinoembryonic antigen (CEA) T84.66 diabody, a genetically engineered noncovalent dimer of single-chain variable fragments, for small-animal PET imaging of CEA expression in xenograft-bearing mice. METHODS: 18F labeling of the anti-CEA T84.66 diabody (molecular mass, 55 kDa) was achieved with N-succinimidyl-4-18F-fluorobenzoate (18F-SFB). The biodistribution of the 18F-fluorobenzyl-T84.66 diabody (18F-FB-T84.66 diabody) was evaluated in athymic nude mice bearing subcutaneous LS 174T human colon carcinoma and C6 rat glioma tumors. Serial small-animal PET imaging studies were performed to further evaluate in vivo targeting efficacy and pharmacokinetics. RESULTS: Radiolabeling required 35 +/- 5 (mean +/- SD) min starting from 18F-SFB, and the tracer 18F-FB-T84.66 diabody was synthesized with a specific activity of 1.83 +/- 1.71 TBq/mmol. The decay-corrected radiochemical yield was 1.40% +/- 0.16% (n = 4), and the radiochemical purity was greater than 98%. The radioimmunoreactivity was 57.1% +/- 2.0%. The 18F-FB-T84.66 diabody showed rapid and high tumor uptake and fast clearance from the circulation in the LS 174T xenograft model, as evidenced by both small-animal PET imaging and biodistribution studies. High-contrast small-animal PET images were obtained as early as 1 h after injection of the 18F-FB-T84.66 diabody, and only a background level of activity accumulation was found in CEA-negative C6 tumors. The tracer exhibited predominantly renal clearance, with some activity in the liver and spleen at early time points. CONCLUSION: The 18F-labeled diabody represents a new class of tumor-specific probes for PET that are based on targeting cell surface antigen expression. The 18F-FB-T84.66 diabody can be used for high-contrast small-animal PET imaging of CEA-positive tumor xenografts. It may be translated to the clinic for PET of CEA-positive malignancies.     

Evaluation of 3'-deoxy-3'-18F-fluorothymidine for monitoring tumor response to radiotherapy and photodynamic therapy in mice.

3'-Deoxy-3'-18F-fluorothymidine (18F-FLT) has been suggested as a new PET tracer for imaging tumor proliferation. We investigated the use of 18F-FLT to monitor the response of tumors to radiotherapy and photodynamic therapy (PDT) in mice. METHODS: C3H/He mice bearing an SCCVII tumor were treated with single-dose x-ray irradiation of 20 Gy. Tumor uptake was examined for 18F-FLT, 3H-thymidine (3H-Thd), 18F-FDG, and 14C-deoxyglucose (14C-DG) at 6 h, 12 h, 24 h, 3 d, and 7 d after radiotherapy. BALB/c nu/nu mice bearing a HeLa tumor were treated with PDT. Tumor uptake was examined for the 4 tracers at 24 h after PDT. Expression of proliferating cell nuclear antigen (PCNA) was determined in untreated and treated tumors. RESULTS: In the biodistribution study, considerable uptake of 18F-FLT was observed in both tumor types. Tumor volumes decreased to 39.3% +/- 22.4% at 7 d after radiotherapy. The PCNA labeling index was reduced in x-ray-irradiated tumors (control, 53.2% +/- 8.7%; 6 h, 38.5% +/- 5.3%; 24 h after radiotherapy, 36.8% +/- 5.3%). 18F-FLT uptake in tumor expressed as the percentage of the injected dose per gram of tumor (%ID/g) decreased significantly at 6 h and remained low until 3 d after radiotherapy (control, 9.7 +/- 1.2 %ID/g; 6 h, 5.9 +/- 0.4 %ID/g; 24 h, 6.1 +/- 1.3 %ID/g; 3 d after radiotherapy, 6.4 +/- 1.1 %ID/g). 18F-FDG uptake tended to gradually decrease but a significant decrease was found only at 3 d (control, 12.1 +/- 2.7 %ID/g; 6 h, 13.3 +/- 2.3 %ID/g; 24 h, 8.6 +/- 1.8 %ID/g; 3 d after radiotherapy, 6.9 +/- 1.2 %ID/g). PDT resulted in a reduction of the PCNA labeling index (control, 82.0% +/- 8.6%; 24 h after PDT, 13.5% +/- 12.7%). Tumor uptake of 18F-FLT decreased (control, 11.1 +/- 1.3 %ID/g; 24 h after PDT, 4.0 +/- 2.2 %ID/g), whereas 18F-FDG uptake did not decrease significantly after PDT (control, 3.5 +/- 0.6 %ID/g; 24 h after PDT, 2.3 +/- 1.1 %ID/g). Changes in the uptake of 18F-FLT and 18F-FDG were similar to those of 3H-Thd and 14C-DG, respectively. CONCLUSION: In our model system, changes in 18F-FLT uptake after radiotherapy and PDT were correlated with those of 3H-Thd and the PCNA labeling index. The decrease in 18F-FLT uptake after treatments was more rapid or pronounced than that of 18F-FDG. Therefore, 18F-FLT may be a feasible PET tracer for monitoring response to therapy in oncology.     

Volumetric analysis of 18F-FDG PET in glioblastoma multiforme: prognostic information and possible role in definition of target volumes in radiation dose escalation.

The use of (18)F-FDG PET for brain tumors has been shown to be accurate in identifying areas of active disease. Radiation dose escalation in the treatment of glioblastoma multiforme (GBM) may lead to improved disease control. On the basis of these premises, we initiated a pilot study to investigate the use of (18)F-FDG PET for the guidance of radiation dose escalation in the treatment of GBM. METHODS: Patients were considered eligible to participate in the study if they had a diagnosis of GBM, were at least 18 y old, and had a score of at least 60 on the Karnofsky Scale. Patients were treated with standard conformal fractionated radiotherapy (1.8 Gy per fraction, to 59.4 Gy), with volumes defined by MRI. At a dose of 45-50.4 Gy, patients underwent (18)F-FDG PET for boost target delineation. Final noncoplanar fields (3-4) were designed to treat the volume of abnormal (18)F-FDG uptake plus a 0.5-cm margin for an additional 20 Gy (2 Gy per fraction), to a total dose of 79.4 Gy. If no abnormal (18)F-FDG uptake was observed, treatment was stopped after the conventional course of 59.4 Gy. Age, Karnofsky score, MRI-based volumes, and (18)F-FDG PET volume were analyzed as prognostic variables for time to tumor progression (TTP) and overall survival. (18)F-FDG PET volumes and MRI-based volumes were compared to assess concordance. RESULTS: For the 27 patients who could be evaluated, median actuarial TTP was 43 wk, and median actuarial survival was 70 wk. On univariate analysis, (18)F-FDG PET, T1-weighted MRI gadolinium enhancement (excluding nonenhancing resection cavity), and T2-weighted MRI volumes were significantly predictive of TTP. On multivariate analysis, only (18)F-FDG PET volume retained significance for predicting TTP. Similar results were obtained on analysis of these variables as prognostic factors for survival. When (18)F-FDG PET-based volumes were compared with MRI-based volumes, a difference of at least 25% was detected in all patients, with all but 2 having smaller (18)F-FDG PET volumes. Of patients in whom (18)F-FDG uptake was initially present but treatment subsequently failed, 83% demonstrated the first tumor progression within the region of abnormal (18)F-FDG uptake. CONCLUSION: In comparison with MRI, (18)F-FDG PET defined unique volumes for radiation dose escalation in the treatment of GBM. (18)F-FDG PET volumes were predictive of survival and time to tumor progression in the treatment of patients with GBM.     

Effects of anesthetic agents and fasting duration on 18F-FDG biodistribution and insulin levels in tumor-bearing mice.

Small-animal PET has opened the way for imaging (18)F-FDG uptake in murine tumor models, but the need for anesthesia raises concern over its potential influence on (18)F-FDG kinetics. We thus investigated such effects on cultured cells and on tumor-bearing mice after short- and long-term fasting. METHODS: Lewis lung carcinoma (LLC) cells and cardiomyoblasts were treated for 2 h with a 100 micromol/L concentration of xylazine, ketamine, xylazine plus ketamine (Xy/Ke), or pentobarbital and were measured for (18)F-FDG uptake. LLC tumor-bearing C57BL6 mice that had been kept fasting for either 4 or 20 h were injected with Xy/Ke, pentobarbital, or saline and were administered 1.8 MBq of (18)F-FDG 15 min later. Biodistribution studies and plasma glucose and insulin assays were performed 45 min after injection. Separate anesthetized and control mice underwent (18)F-FDG PET. RESULTS: (18)F-FDG uptake in LLC cells was unaffected by anesthetic agents, whereas xylazine and ketamine caused a small increase of uptake in cardiomyoblasts. In mice kept fasting 4 h, Xy/Ke induced a marked elevation of (18)F-FDG activity (percentage injected dose [%ID]) in blood (6.8 +/- 0.9%ID/g vs. 1.1 +/- 0.6%ID/g) and kidneys while decreasing myocardial uptake (2.3 +/- 1.3%ID/g vs. 4.7 +/- 1.8%ID/g). Target-to-blood ratios were significantly reduced. Pentobarbital caused a moderate increase in blood activity (2.5 +/- 0.8%ID/g), decreased myocardial uptake (2.8 +/- 0.5%ID/g), and reduced target-to-blood ratios. PET images of mice kept fasting 4 h were consistent with the biodistribution data. Insulin levels were lower with Xy/Ke and higher with pentobarbital. In mice kept fasting 20 h, Xy/Ke and pentobarbital increased blood (18)F-FDG activity (5.5 +/- 2.2 and 4.9 +/- 0.9%ID/g vs. 2.4 +/- 0.3%ID/g) and reduced target-to-blood ratios, but these changes were substantially attenuated, compared with those in mice kept fasting 4 h. In addition, insulin levels were low and unaffected by anesthesia. CONCLUSION: Xy/Ke anesthesia markedly elevates blood (18)F-FDG activity and reduces tumor uptake ratios through inhibition of insulin release in mice kept fasting 4 h, whereas pentobarbital induces a similar but less severe response through insulin resistance. These metabolic effects, however, are substantially attenuated after 20 h of fasting. Hence both the choice of anesthetic and the duration of fasting have important effects on (18)F-FDG kinetics and PET images of tumor-bearing mice and should be considered when such studies are performed.     

Tetraphenylphosphonium as a novel molecular probe for imaging tumors.

Mitochondrial membrane potential (DeltaPsim)-dependent enhanced uptake of phosphonium salts, including (3)H-tetraphenylphosphonium ((3)H-TPP), in tumor cells, suggests the potential use of phosphonium salts as tracers for tumor imaging. In this study, we characterize the tumor accumulation of (3)H-TPP and compare it with (18)F-FDG in cell culture and in xenograft, metastatic, and inflammation models in living animals. METHODS: (3)H-TPP and (3)H-FDG accumulation was compared in cell culture with a variety of cell lines in different glucose concentrations. Normal biodistribution and tumor uptake were assessed using nude mice with or without subcutaneous xenograft tumors (C6). To compare the accumulation of (3)H-TPP and (18)F-FDG in a metastatic tumor, severe combined immunodeficiency mice were tail-vein injected with human melanoma cell lines (A375-FL). To characterize the accumulation of (3)H-TPP and (18)F-FDG in inflammation, an inflammatory reaction was induced by subcutaneous injection of Complete Freund's Adjuvant in the left hind paw of Sprague-Dawley rat. RESULTS: The DeltaPsim data from a separate study and the current (3)H-TPP uptake data showed good correlation (r(2) = 0.82, P < 0.05). (3)H-TPP accumulation was significantly greater than that of (3)H-FDG for glucose >/=100 mg/dL. The biodistribution study of (3)H-TPP showed low uptake in most tissues but high accumulation in the heart and kidneys. (3)H-TPP accumulation in xenograft or metastatic tumors was comparable with that of (18)F-FDG, whereas (3)H-TPP accumulation in inflammatory tissues was markedly lower than that of (18)F-FDG. CONCLUSION: The sensitive tumor accumulation of (3)H-TPP with less propensity for inflammatory regions warrants further investigation of radiolabeled phosphonium analogs for tumor imaging in living subjects.     

Early tumor response to Hsp90 therapy using HER2 PET: comparison with 18F-FDG PET.

We compared (68)Ga-DOTA-F(ab')(2)-herceptin (DOTA is 1,4,7,10-tetraazacyclododecane-N,N',N',N'-tetraacetic acid [HER2 PET]) and (18)F-FDG PET for imaging of tumor response to the heat shock protein 90 (Hsp90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17AAG). METHODS: Mice bearing BT474 breast tumor xenografts were scanned with (18)F-FDG PET and HER2 PET before and after 17AAG treatment and then biweekly for up to 3 wk. RESULTS: Within 24 h after treatment, a significant decrease in HER2 was measured by HER2 PET, whereas (18)F-FDG PET uptake, a measure of glycolysis, was unchanged. Marked growth inhibition occurred in treated tumors but became evident only by 11 d after treatment. Thus, Her2 downregulation occurs independently of changes in glycolysis after 17AAG therapy, and Her2 reduction more accurately predicts subsequent tumor growth inhibition. CONCLUSION: HER2 PET is an earlier predictor of tumor response to 17AAG therapy than (18)F-FDG PET.     

MicroPET imaging of brain tumor angiogenesis with <Superscript>18</Superscript>F-labeled PEGylated RGD peptide

We have previously labeled cyclic RGD peptide c(RGDyK) with fluorine-18 through conjugation labeling via a prosthetic 4-[¹⁸F]fluorobenzoyl moiety and applied this [¹⁸F]FB-RGD radiotracer for _v-integrin expression imaging in different preclinical tumor models with good tumor-to-background contrast. However, the unfavorable hepatobiliary excretion and rapid tumor washout rate of this tracer limit its potential clinical applications. The aims of this study were to modify the [¹⁸F]FB-RGD tracer by inserting a heterobifunctional poly(ethylene glycol) (PEG, M.W. =3,400) between the ¹⁸F radiolabel and the RGD moiety and to test this [¹⁸F]FB-PEG-RGD tracer for brain tumor targeting and in vivo kinetics. [¹⁸F]FB-PEG-RGD was prepared by coupling the RGD-PEG conjugate with N-succinimidyl 4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB) under slightly basic conditions (pH=8.5). The radiochemical yield was about 20–30% based on the active ester [¹⁸F]SFB, and specific activity was over 100 GBq/mol. This tracer had fast blood clearance, rapid and high tumor uptake in the subcutaneous U87MG glioblastoma model (5.2±0.5%ID/g at 30 min p.i.). Moderately rapid tumor washout was observed, with the activity accumulation decreased to 2.2±0.4%ID/g at 4 h p.i. MicroPET and autoradiography imaging showed a very high tumor-to-background ratio and limited activity accumulation in the liver, kidneys and intestinal tracts. U87MG tumor implanted into the mouse forebrain was well visualized with [¹⁸F]FB-PEG-RGD. Although uptake in the orthotopic tumor was significantly lower (P<0.01) than in the subcutaneous tumor, the maximum tumor-to-brain ratio still reached 5.0±0.6 due to low normal brain background. The results of H&E staining post mortem agreed with the anatomical information obtained from non-invasive microPET imaging. In conclusion, PEGylation suitably modifies the physiological behavior of the RGD peptide. [¹⁸F]FB-PEG-RGD gave improved tumor retention and in vivo kinetics compared with [¹⁸F]FB-RGD.     

In vivo VEGF imaging with radiolabeled bevacizumab in a human ovarian tumor xenograft.

Vascular endothelial growth factor (VEGF), released by tumor cells, is an important growth factor in tumor angiogenesis. The humanized monoclonal antibody bevacizumab blocks VEGF-induced tumor angiogenesis by binding, thereby neutralizing VEGF. Our aim was to develop radiolabeled bevacizumab for noninvasive in vivo VEGF visualization and quantification with the single gamma-emitting isotope 111In and the PET isotope 89Zr. METHODS: Labeling, stability, and binding studies were performed. Nude mice with a human SKOV-3 ovarian tumor xenograft were injected with 89Zr-bevacizumab, 111In-bevacizumab, or human 89Zr-IgG. Human 89Zr-IgG served as an aspecific control antibody. Small-animal PET and microCT studies were obtained at 24, 72, and 168 h after injection of 89Zr-bevacizumab and 89Zr-IgG (3.5 +/- 0.5 MBq, 100 +/- 6 microg, 0.2 mL [mean +/- SD]). Small-animal PET and microCT images were fused to calculate tumor uptake and compared with ex vivo biodistribution at 168 h after injection. 89Zr- and 111In-bevacizumab ex vivo biodistribution was compared at 24, 72, and 168 h after injection (2.0 +/- 0.5 MBq each, 100 +/- 4 microg in total, 0.2 mL). RESULTS: Labeling efficiencies, radiochemical purity, stability, and binding properties were optimal for the radioimmunoconjugates. Small-animal PET showed uptake in well-perfused organs at 24 h and clear tumor localization from 72 h onward. Tumor uptake determined by quantification of small-animal PET images was higher for 89Zr-bevacizumab-namely, 7.38 +/- 2.06 %ID/g compared with 3.39 +/- 1.16 %ID/g (percentage injected dose per gram) for human 89Zr-IgG (P = 0.011) at 168 h and equivalent to ex vivo biodistribution studies. Tracer uptake in other organs was seen primarily in liver and spleen. 89Zr- and 111In-bevacizumab biodistribution was comparable. CONCLUSION: Radiolabeled bevacizumab showed higher uptake compared with radiolabeled human IgG in a human SKOV-3 ovarian tumor xenograft. Noninvasive quantitative small-animal PET was similar to invasive ex vivo biodistribution. Radiolabeled bevacizumab is a new tracer for noninvasive in vivo imaging of VEGF in the tumor microenvironment.