Quantitative, noninvasive, in vivo longitudinal monitoring of gene expression in the brain by co-AAV transduction with a PET reporter gene.

In vivo imaging of vector transgene expression would be particularly valuable for repetitive monitoring of therapy in the brain, where invasive tissue sampling is contraindicated. We evaluated adeno-associated virus vector expression of a dopamine-2 receptor (D2R) mutant (D2R80A) by positron emission tomography in the brains of mice and cats. D2R80A is inactivated for intracellular signaling and binds subphysiologic amounts of the radioactive [(18)F]-fallypride analog of dopamine. The [(18)F]-fallypride signal bound to D2R80A in the injection site was normalized to the signal from endogenous D2R in the striatum and showed stable levels of expression within individual animals. A separate adeno-associated virus type 1 vector with identical gene expression control elements, expressing green fluorescent protein or a therapeutic gene, was coinjected with the D2R80A vector at equal doses into specific sites. Both transgenes had similar levels of gene expression by immunohistochemistry, in situ hybridization, and quantitative PCR assays, demonstrating that D2R80A is a faithful surrogate measure for expression of a gene of interest. This dual vector approach allows the D2R80A gene to be used with any therapeutic gene and to be injected into a single site for monitoring while the therapeutic gene can be distributed more widely as needed in each disease.

Development of anti-EGF receptor peptidomimetics (AERP) as tumor imaging agent.

EGFR is over-expressed in several solid tumors including breast, prostate, pancreas, and lung cancers and is correlated to the metastatic potential of the tumor. Anti-EGFR receptor-binding peptidomimetics (AERP) were examined to assess the small molecule's potential use as tumor-specific imaging agents. The aim of this work was to design and characterize the binding specificity of the radiolabeled peptidomimetics to EGFR over-expressing cell lysate and to A431 xenograft tumors. Our newly designed peptidomimetic, AERP, was conjugated to DTPA and labeled with (99m)Tc. The in vivo tumor accumulation of [(99m)Tc] DTPA-AERP-2 was 1.6±0.1%ID/g and tumor to muscle ratio was 5.5. Our studies suggest that this novel peptidomimetic, AERP-2, warrants further development as an EGFR specific tumor-imaging agent.

Early response assessment in prostate carcinoma by ¹8F-fluorothymidine following anticancer therapy with docetaxel using preclinical tumour models.

PURPOSE: The aim of the study was to assess the potential usefulness of 3-deoxy-3-(18)F-fluorothymidine (FLT) as a radiopharmaceutical for imaging the early therapeutic effects of docetaxel (DTX) on tumour proliferation in hormone-refractory prostate cancer (HRPC). METHODS: Cells of the androgen-independent human prostate tumour cell line, 22Rv1, were implanted in athymic male mice. Approximately 3 weeks after cell implantation, the mice were treated with DTX or vehicle. Before and after the treatment, the mice were imaged with a microPET-Focus-F120 scanner (Concorde Microsystems, Knoxville, TN, USA) using FLT and (18)F-fluorodeoxyglucose (FDG). Tracer accumulations in the tumours were then analysed and compared with the proliferation activity and apoptotic index of the tumours. In a separate cell study, 22Rv1 cells were treated with DTX, then incubated with FLT or FDG and examined for their tracer uptake. RESULTS: The microPET imaging showed a significant decrease of FLT uptake in tumours after administration of DTX, while the changes of FDG uptake were minimal. Immunohistochemical analysis of the tumours revealed that the changes of FLT uptake were well correlated with those of proliferation activity but not with the apoptotic index. In vitro studies demonstrated that the significant decrease of FLT uptake in the cells after incubation with DTX correlated with the % S-phase cell fraction, while there were only minimal changes in the prostate-specific antigen concentration of the cell medium and FDG uptake in the cells. CONCLUSION: These results indicate that FLT is a promising tracer for monitoring the early effects of anticancer therapy with DTX in patients with HRPC.

Death and proliferation time course of stem cells transplanted in the myocardium.

PURPOSE: Noninvasive positron emission tomography (PET) imaging of reporter gene is combined with quantitative real-time polymerase reverse transcription (RT-PCR) method to study the time course of death and proliferation of stem cells transplanted in the myocardium. METHODS: Male murine embryonic stem cells (ESCs) were stably transfected with a mutant version of herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) reporter gene; 5 x 10(6) such cells were injected into the myocardium of female athymic rats. While the transplanted cells was monitored by in vivo 9-(4-[F-18]fluoro-3-hydroxymethylbutyl)guanine ([F-18]FHBG) PET imaging of the heart, their absolute number was estimated by RT-PCR from hearts harvested at 3-5 h, 24 h, days 4, 7, and 14 after transplantation. RESULTS: (1) Forty percent of injected cells were retained in the heart while majority of injected cells were lost within a few hours after injection. Cell death was peaked at 24 h when 18% of donor cells retained in the heart were dead. (2) The substantial cell loss was reversed by significant proliferation of ESCs. This led to the recovery of cell number to 3.4 million (70% of injected dose) at day 4 and first visual observation of in vivo [F-18] signal in the heart. (3) A robust correlation (R (2) = 0.9) between percent of injected dose per gram of tissue derived from in vivo PET signal and the number of donor cells estimated by RT-PCR was revealed. CONCLUSIONS: The time course of transplanted stem cells surviving in the heart reveals a process of substantial cell loss within 24 h of injection and subsequent recovery of cell number through proliferation. Such proliferation can be noninvasively monitored by reporter gene imaging.

Autoradiographic and small-animal PET comparisons between (18)F-FMISO, (18)F-FDG, (18)F-FLT and the hypoxic selective (64)Cu-ATSM in a rodent model of cancer.

INTRODUCTION: Copper(II)-diacetyl-bis(N(4)-methylthiosemicarbazone), or Cu-ATSM, a hypoxia imaging agent, has been shown to be predictive of response to traditional cancer therapies in patients with a wide range of tumors. It is known that the environment of the tumor results in a myriad of physiological consequences, including hypoxia, alterations in metabolism and proliferation. In an effort to better characterize the relationships between Cu-ATSM and other prominent radiopharmaceuticals, this current study was undertaken to compare the regional distribution of (64)Cu-ATSM with [(18)F]fluoromisonidazole ((18)F-FMISO), [(18)F]fluoro-2-deoxy-d-glucose ((18)F-FDG) and [(18)F]fluorothymidine ((18)F-FLT) in 9L tumors. METHODS: Taking advantage of the different half-life of (18)F (t(1/2)=110 min) in comparison to (64)Cu (t(1/2)=12.7 h), we undertook a dual-tracer autoradiography study in 9L tumors. Four groups were examined: (a) (18)F-FMISO, 2 h postinjection (p.i.) and (64)Cu-ATSM, 10 min p.i.; (b) (18)F-FMISO, 2 h p.i. and (64)Cu-ATSM, 24 h p.i.; (c) (18)F-FDG, 1 h p.i. and (64)Cu-ATSM, 10 min p.i.; and (d) (18)F-FLT, 1 h p.i. and (64)Cu-ATSM, 10 min p.i. Small-animal PET imaging was performed in 9L tumor-bearing rats with imaging on concurrent days comparing (64)Cu-ATSM with (18)F-FMISO and (18)F-FLT. RESULTS: It was shown that the regional distribution of (18)F-FMISO and (64)Cu-ATSM showed an excellent correlation when the (64)Cu-ATSM had been allowed to distribute for either 10 min (R(2)=.84) or 24 h (R(2)=.86). The regional comparisons between (64)Cu-ATSM (10 min) and (18)F-FDG (1 h) resulted in a very poor correlation (R(2)=.08) between the regional uptake of the two agents. The comparison between (18)F-FLT and (64)Cu-ATSM showed a strong relationship (R(2)=.83) between the two tracers. The small-animal PET images for the distribution comparisons between (64)Cu-ATSM and (18)F-FMISO and (18)F-FLT were in agreement with the data generated from the autoradiography studies. CONCLUSIONS: The data show that it is important to remember that a number of different metabolic situations can exist when considering the relationship between regions of high glucose uptake, proliferation and hypoxia.

Comparison of radiolabeled choline and ethanolamine as probe for cancer detection.

OBJECTIVE: Recently [11C] or [18F] labeled choline has been developed as a promising tracer for cancer detection. However choline may not be best agent, as the development of in vivo 1H-decoupled, NOE-enhanced 31P- MRS enabled the resolution of the resonances of phosphorylethanolamine (PEt) and phosphorylcholine (PCho) peaks that normally overlap under the broad phosphomonoester peak (PME). In human non-Hodgkin's lymphoma in vivo, the PEt/PCho ratio was reported as 2.9 +/- 1.0 (n = 11). The objective of this work was to compare radiolabeled choline with ethanolamine as an agent for cancer detection using various cancer cell lines. RESULTS: Cell uptake of [14C] ethanolamine and [14C] N, N'-dimethyl ethanolamine was compared to [14C] choline uptake in a wide variety of different cancer cell lines. Our data clearly demonstrates that, ethanolamine and N, N'-dimethyl ethanolamine showed 2-7 fold more uptake than choline. We showed that proliferating fibroblasts have significantly higher uptake of ethanolamine and N, N'-dimethyl-ethanolamine, than does growth arrested fibroblasts. Time course studies in A172 cells indicate continuous linear uptake of ethanolamine after four hours of tracer exposure, which was halted if tracer containing, media was replaced with regular media after one hour. The androgen stimulated and depleted study with prostate cancer cell lines showed that increased trapping of radiotracers in cells is well correlated with their proliferation status. METHODS: We carried out comparison of radiolabeled choline and ethanolamine by cell uptake study using 8 different cancer cell lines. To evaluate the response of radiotracers towards proliferation we also carried out their cell uptake study of androgen dependent and androgen independent cells in androgen stipulated and deprived media. CONCLUSIONS: Our data demonstrate that ethanolamine and N, N'-dimethyl ethanolamine are taken up by a wide variety of tumor cells significantly better (2-7 fold) than the clinically utilized radiolabeled choline tracer.

18F-fluoroacetate: a potential acetate analog for prostate tumor imaging--in vivo evaluation of 18F-fluoroacetate versus 11C-acetate.

UNLABELLED: PET with (11)C-acetate ((11)C-ACE) has a high sensitivity for detection of prostate cancer and several other cancers that are poorly detected with (18)F-FDG. However, the short half-life (20.4 min) of (11)C limits the general availability of (11)C-ACE. (18)F-Fluoroacetate ((18)F-FAC) is an analog of acetate with a longer radioactive half-life ((18)F = 110 min). This study was undertaken to assess the potential usefulness of (18)F-FAC as a prostate tumor imaging agent. METHODS: We developed an efficient radiosynthesis for (18)F-FAC, which has already been adapted to a commercial synthesizer. Biodistribution studies of (18)F-FAC were compared with (11)C-ACE in normal Sprague-Dawley male rats and CWR22 tumor-bearing nu/nu mice. We also performed a small-animal PET study of (18)F-FAC in CWR22 tumor-bearing nu/nu mice and a whole-body PET study in a baboon to examine defluorination. RESULTS: We obtained (18)F-FAC in a radiochemical yield of 55% +/- 5% (mean +/- SD) in approximately 35 min and with a radiochemical purity of >99%. Rat biodistribution showed extensive defluorination, which was not observed in the baboon PET, as indicated by the standardized uptake values (SUVs) (SUVs of iliac bones and femurs were 0.26 and 0.3 at 1 h and 0.22 and 0.4 at 2 h, respectively). CWR22 tumor-bearing nu/nu mice showed tumor uptake (mean +/- SD) of 0.78 +/- 0.06 %ID/g (injected dose per gram of tissue) for (11)C-ACE versus 4.01 +/- 0.32 %ID/g for (18)F-FAC. For most organs-except blood, muscle, and fat-the tumor-to-organ ratios at 30 min after injection were higher with (18)F-FAC, whereas the tumor-to-heart and tumor-to-prostate ratios were similar. CONCLUSION: All of these data indicate that (18)F-FAC may be a useful alternative to (11)C-ACE tracer for the detection of prostate tumors by PET.

Carbon-11 labeled sigma2 receptor ligands for imaging breast cancer.

Four conformationally flexible benzamide analogs having a high affinity and outstanding selectivity for sigma(2) versus sigma(1) receptors were synthesized and radiolabeled with carbon-11 by reaction with [(11)C]methyl iodide. The four (11)C-labeled radiotracers were evaluated for their potential to image the proliferative status of breast tumors with positron emission tomography (PET). In vivo studies in female BALB/C mice bearing EMT-6 breast tumors showed that one radiotracer, (2-methoxy-(11)C)-N-(4-(3,4-dihydro-6,7-dimethoxy-isoquinolin-2(1H)-yl)butyl)-5-methylbenzamide ([(11)C]2), had a high tumor uptake and suitable tumor/background ratio for imaging purposes. Blocking studies were consistent with the labeling of sigma(2) receptors in vivo. A study comparing the in vivo properties of [(11)C]2 and (18)F-3'-fluoro-3'-deoxy-L-thymidine ([(18)F]FLT) indicated that [(11)C]2 had either similar (lung, fat) or better (blood, muscle) tumor/organ ratios than [(18)F]FLT in the tissues that are important for breast tumor imaging. Consequently, [(11)C]2 is a potential radiotracer for imaging the proliferative status of breast tumors in vivo with PET.

Quantitation of pulmonary transgene expression with PET imaging.

UNLABELLED: PET imaging represents a promising approach for noninvasive monitoring of reporter gene expression in living subjects. We evaluated the relationship between various methods of quantifying the imaging signal and in vitro assays of the expression of a PET reporter gene (a mutant Herpes simplex virus-1 thymidine kinase (mHSV1-tk); 9-(4-(18)F-fluoro-3-hydroxymethylbutyl)guanine ((18)F-FHBG) was used as the PET reporter probe. METHODS: In 14 rats, pulmonary gene transfer was performed by intratracheal administration of various amounts of an adenovector containing a fusion gene encoding for mHSV1-tk and an enhanced green fluorescent protein. Three days later, the animals were divided into 2 groups. One group (n = 7) did not receive any other interventions. The other group was treated with alpha-naphthylthiourea (ANTU) to increase pulmonary vascular permeability. All rats were injected intravenously with (18)F-FHBG. Two additional rats in both groups received a null adenovector and served as controls. In the normal rats, repetitive blood samples were obtained and PET imaging was performed simultaneously using a dynamic imaging protocol. Rate constants estimating (18)F-FHBG transport (K(1)) or trapping (k(3)) within target cells were generated by compartmental modeling. After euthanasia, pulmonary uptake of (18)F-FHBG was determined using a gamma-counter in all rats, and in vitro assays of transgene expression were performed on lung tissue. RESULTS: In normal rats, pulmonary uptake of (18)F-FHBG increased as thymidine kinase (TK) activity increased only at low levels of mHSV1-tk expression and then plateaued as TK activity continued to increase. Compartmental modeling failed to improve the correlation with in vitro assays of transgene expression. However, a linear relationship was obtained between the pulmonary uptake of (18)F-FHBG and in vitro assays of TK activity in rats treated with ANTU. CONCLUSION: In rodent lungs, (18)F-FHBG uptake appears to be a function of both transport into tissues expressing the transgene as well as the level of transgene expression itself.

Monitoring of therapy in androgen-dependent prostate tumor model by measuring tumor proliferation.

UNLABELLED: 3'-Deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) has been recently described as a radiopharmaceutical for measuring cellular proliferation using PET imaging. Evaluation of tumor proliferative activity by PET using (18)F-FLT could be a procedure to assess the viability of tumor, such as histologic grade, clinical stage, and prognosis as well as the early effects of cancer therapy. This study was undertaken to determine whether (18)F-FLT is useful in the detection of prostate cancer as well as monitoring therapeutic effects in a human tumor model. METHODS: The androgen-dependent human prostate tumor, CWR22, was implanted into athymic mice. This well-established model of prostate cancer was used in all studies. To determine the optimal imaging times for (18)F-FLT, a biodistribution was performed in CWR22 mice. (18)F-FLT (740 kBq [20 micro Ci]) was administered via the tail vein and uptake was determined in selected tissues at 5 min, 20 min, and 1, 2, and 4 h after injection (n = 5, each time point). Androgen ablation studies were conducted in the CWR22 model with either diethylstilbestrol (DES) or surgical castration. Animals received DES every 2 d for 3 wk. The effectiveness of therapy was monitored using (18)F-FLT microPET as baseline, during treatment, and after treatment. Tracer accumulation in the tumor was then analyzed by comparing tumor-to-muscle ratios derived from reconstructed microPET data. RESULTS: At 2 h after injection, the (18)F-FLT uptake in tumor was 0.69 +/- 0.14 percentage injected dose per gram of tissue, showing the highest activity of all organs measured. The microPET study with dynamic imaging showed that (18)F-FLT uptake in blood reached its plateau within 1 min and was rapidly cleared, whereas (18)F-FLT uptake in tumor reached its plateau in 30 min and remained up to 60 min. microPET using (18)F-FLT successfully imaged the implanted CWR22 tumor in the mice at both 1 and 2 h after injection. There was a marked reduction of (18)F-FLT uptake in tumor after castration or DES treatment; however, there were no differences in (18)F-FLT uptake in the tumor in the control group. These changes of (18)F-FLT uptake in tumor parallel the changes of actual tumor measurement. CONCLUSION: These results indicate that (18)F-FLT is a useful tracer for detection of prostate cancer in an animal model. (18)F-FLT has the potential for monitoring the therapeutic effect of androgen ablation therapy in prostate cancer.

Imaging pulmonary gene expression with positron emission tomography.

We evaluated positron emission tomographic imaging of pulmonary transgene expression, using an enhanced mutant herpes simplex virus-1 thymidine kinase as the reporter gene, in the lungs of normal rats. Sixteen rats were studied 3 days after an intratracheal administration of 5 x 10(9) to 1 x 10(11) viral particles of a replication-incompetent adenovirus containing a fusion gene of the mutant kinase and green fluorescent protein. Three rats infected with adenovirus containing no insert (null vector) served as control subjects. Images were obtained 1 hour after an intravenous injection of 9-(4-[18F]-fluoro-3-hydroxymethylbutyl)guanine, an imaging substrate for the viral kinase. After euthanasia, tissue radioactivity was determined in a gamma counter, and thymidine kinase activity and green fluorescent protein levels were measured in lung tissue samples. Imaging and gamma counting radioactivity measurements were strongly and linearly correlated (r2 = 0.96, p < 0.001). Imaging detected thymidine kinase expression above background (null vector) in 15 of 16 rats, even at low viral doses that produced little to no measurable green fluorescent protein expression. Lung 9-(4-[18F]-fluoro-3-hydroxymethylbutyl)guanine uptake (as assessed by imaging) correlated with in vitro assays of both kinase activity (r(2) = 0.48, p < 0.001) and fluorescent protein (r(2) = 0.46, p < 0.001). We conclude that positron emission tomographic imaging is a sensitive and quantitative method for detecting pulmonary reporter gene expression noninvasively.

Repetitive imaging of reporter gene expression in the lung.

Positron emission tomographic imaging is emerging as a powerful technology to monitor reporter transgene expression in the lungs and other organs. However, little information is available about its usefulness for studying gene expression over time. Therefore, we infected 20 rats with a replication-deficient adenovirus containing a fusion gene encoding for a mutant Herpes simplex virus type-1 thymidine kinase and an enhanced green fluorescent protein. Five additional rats were infected with a control virus. Pulmonary gene transfer was performed via intratracheal administration of vector using a surfactant-based method. Imaging was performed 4-6 hr, and 4, 7, and 10 days after gene transfer, using 9-(4-[18F]-fluoro-3-hydroxymethylbutyl)guanine, an imaging substrate for the mutant kinase. Lung tracer uptake assessed with imaging was moderately but significantly increased 4-6 hr after gene transfer, was maximal after 4 days, and was no longer detectable by 10 days. The temporal pattern of transgene expression measured ex vivo with in vitro assays of thymidine kinase activity and green fluorescent protein was similar to imaging. In conclusion, positron emission tomography is a reliable new tool to evaluate the onset and duration of reporter gene expression noninvasively in the lungs of intact animals.