Monitoring of biodistribution and persistence of conditionally replicative adenovirus in a murine model of ovarian cancer using capsid-incorporated mCherry and expression of human somatostatin receptor subtype 2 gene.

A significant limiting factor to the human clinical application of conditionally replicative adenovirus (CRAd)-based virotherapy is the inability to noninvasively monitor these agents and their potential persistence. To address this issue, we proposed a novel imaging approach that combines transient expression of the human somatostatin receptor (SSTR) subtype 2 reporter gene with genetic labeling of the viral capsid with mCherry fluorescent protein. To test this dual modality system, we constructed the Ad5/3Δ24pIXcherry/SSTR CRAd and validated its capacity to generate fluorescent and nuclear signals in vitro and following intratumoral injection. Analysis of 64Cu-CB-TE2A-Y3-TATE biodistribution in mice revealed reduced uptake in tumors injected with the imaging CRAd relative to the replication-incompetent, Ad-expressing SSTR2 but significantly greater uptake compared to the negative CRAd control. Optical imaging demonstrated relative correlation of fluorescent signal with virus replication as determined by viral genome quantification in tumors. Positron emission tomography/computed tomography studies demonstrated that we can visualize radioactive uptake in tumors injected with imaging CRAd and the trend for greater uptake by standardized uptake value analysis compared to control CRAd. In the aggregate, the plasticity of our dual imaging approach should provide the technical basis for monitoring CRAd biodistribution and persistence in preclinical studies while offering potential utility for a range of clinical applications.

Triazine-based tool box for developing peptidic PET imaging probes: syntheses, microfluidic radiolabeling, and structure-activity evaluation.

This study was aimed at developing a triazine-based modular platform for targeted PET imaging. We synthesized mono- or bis-cyclo(RGDfK) linked triazine-based conjugates specifically targeting integrin αvß3 receptors. The core molecules could be easily linked to targeting peptide and radiolabeled bifunctional chelator. The spacer core molecule was synthesized in 2 or 3 steps in 64-80% yield, and the following conjugation reactions with cyclo(RGDfK) peptide or bifunctional chelator were accomplished using "click" chemistry or amidation reactions. The DOTA-TZ-Bis-cyclo(RGDfK) 13 conjugate was radiolabeled successfully with (64)Cu(OAc)2 using a microfluidic method, resulting in higher specific activity with above 95% labeling yields compared to conventional radiolabeling (SA ca. 850 vs 600 Ci/mmol). The dimeric cyclo(RGDfK) peptide was found to display significant bivalency effect using I(125)-Echistatin binding assay with IC50 value as 178.5 ± 57.1 nM, which displayed a 3.6-fold enhancement of binding affinity compared to DOTA-TZ-cyclo(RGDfK) 14 conjugate on U87MG human glioblastoma cell. Biodistribution of all four conjugates in female athymic nude mice were evaluated. DOTA-"Click"-cyclo(RGDfK) 15 had the highest tumor uptake among these four at 4 h p.i. with 1.90 ± 0.65%ID/g, while there was no clear bivalency effect for DOTA-TZ-BisRGD in vivo, which needs further experiments to address the unexpected questions.

MicroPET imaging of gene transfer with a somatostatin receptor-based reporter gene and (94m)Tc-Demotate 1.

UNLABELLED: Gene therapy trials would benefit greatly from the use of noninvasive imaging to determine the location, magnitude, and time course of gene transfer. Somatostatin receptor subtype 2 (SSTR2) has been used as a reporter probe for gamma-camera imaging of gene transfer in animal models. PET has greater sensitivity than gamma-camera imaging and therefore would have an advantage for the imaging of SSTR2 gene transfer. METHODS: An adenovirus (AdHASSTR2) carrying sstr2, which encodes an N-terminal hemagglutinin epitope, was used for evaluating SSTR2 gene transfer. The somatostatin analog Demotate 1 (Tyr(3)-octreotate conjugated to the 1,4,8,11-tetraazaundecane chelator) was used for chelation of the positron emitter (94m)Tc (half-life, 52 min) and targeting to SSTR2. Gene transfer was evaluated in vitro with A-427 non-small cell lung cancer cells after infection with AdHASSTR2 by (94m)Tc-Demotate 1 binding and internalization assays. In vivo biodistribution and microPET studies were conducted with mice bearing A-427 tumor xenografts directly injected with AdHASSTR2 to determine the tumor localization of (94m)Tc-Demotate 1. RESULTS: (94m)Tc-Demotate 1 bound with high affinity and was internalized rapidly into AdHASSTR2-infected A-427 cells. Biodistribution studies showed uptake of (94m)Tc-Demotate 1 in tumors infected with AdHASSTR2 (4.0 percentage injected dose per gram [%ID/g] at 2 h) and background uptake in tumors infected with a control adenovirus (0.8 %ID/g at 2 h). The uptake of (94m)Tc-Demotate 1 in AdHASSTR2-infected tumors was greater than the uptake in all other tissues, except for the kidneys and the SSTR2-positive pancreas. MicroPET imaging showed similar results, with clear uptake of (94m)Tc-Demotate 1 in AdHASSTR2-infected tumors, background uptake in control tumors, and clearance through the kidneys. CONCLUSION: These studies show that the positron-emitting somatostatin analog (94m)Tc-Demotate 1 could be used to determine SSTR2 gene transfer by microPET imaging, a result that will improve the sensitivity of the SSTR2 reporter gene system.

Expression of the proto-oncogene Fos after exposure to radiofrequency radiation relevant to wireless communications.

In this study the expression levels of the proto-oncogene Fos were measured after exposure to radiofrequency (RF) radiation at two relatively high specific absorption rates (SARs) of 5 and 10 W/kg for three types of modulated signals: 847.74 MHz code division multiple access (CDMA), 835.62 MHz frequency division multiple access (FDMA), and 836.55 MHz time division multiple access (TDMA). This work was undertaken to confirm a previous report by Goswami et al. (Radiat. Res. 151, 300-309, 1999) that CDMA and FDMA radiation caused small but statistically significant increases in Fos levels as cells entered plateau phase during exposure. No effects on Myc or Jun levels were observed in that study. Therefore, in the present study, analyses were restricted to Fos expression during the transition from exponential growth to plateau phase. Fos expression was measured using the real-time polymerase chain reaction (RT-PCR) technique. Serum-stimulated C3H 10T(1/2) cells were used as a positive control for Fos expression. Possible influences of final cell number or pH variability on Fos expression were evaluated. Expression of Fos mRNA in C3H 10T(1/2) cells was not significantly different from that found after sham exposure at either SAR level for any signal modulation. Therefore, the results of Goswami et al. could not be confirmed.

Development of a Radiolabeled Irreversible Peptide Ligand for PET Imaging of Vascular Endothelial Growth Factor.

UNLABELLED: Imaging agents based on peptide probes have desirable pharmacokinetic properties provided that they have high affinities for their target in vivo. An approach to improve a peptide ligand's affinity for its target is to make this interaction covalent and irreversible. For this purpose, we evaluated a (64)Cu-labeled affinity peptide tag, (64)Cu-L19K-(5-fluoro-2,4-dinitrobenzene) ((64)Cu-L19K-FDNB), which binds covalently and irreversibly to vascular endothelial growth factor (VEGF) as a PET imaging agent. We compared the in vivo properties of (64)Cu-L19K-FDNB in VEGF-expressing tumor xenografts with its noncovalent binding analogs, (64)Cu-L19K-(2,4-dinitrophenyl) ((64)Cu-L19K-DNP) and (64)Cu-L19K. METHODS: The L19K peptide (GGNECDIARMWEWECFERK-CONH2) was constructed with 1,4,7-triazacyclononane-1,4,7-triacetic acid at the N terminus for radiolabeling with (64)Cu with a polyethylene glycol spacer between peptide and chelate. 1,5-difluoro-2,4-dinitrobenzene was conjugated at the C-terminal lysine for cross-linking to VEGF, resulting in L19K-FDNB. (64)Cu-L19K-FDNB was assayed for covalent binding to VEGF in vitro. As a control, L19K was conjugated to 1-fluoro-2,4-dinitrobenzene, resulting in L19K-DNP. PET imaging and biodistribution studies of (64)Cu-L19K-FDNB, (64)Cu-L19K-DNP, and the native (64)Cu-L19K were compared in HCT-116 xenografts. Blocking studies of (64)Cu-L19K-FDNB was performed with a coinjection of excess unlabeled L19K-FDNB. RESULTS: In vitro binding studies confirmed the covalent and irreversible binding of (64)Cu-L19K-FDNB to VEGF, whereas (64)Cu-L19K-DNP and (64)Cu-L19K did not bind covalently. PET imaging showed higher tumor uptake with (64)Cu-L19K-FDNB than with (64)Cu-L19K-DNP and (64)Cu-L19K, with mean standardized uptake values of 0.62 ± 0.05, 0.18 ± 0.06, and 0.34 ± 0.14, respectively, at 24 h after injection (P < 0.05), and 0.53 ± 0.05, 0.32 ± 0.14, and 0.30 ± 0.09, respectively, at 48 h after injection (P < 0.05). Blocking studies with (64)Cu-L19K-FDNB in the presence of excess unlabeled peptide showed a 53% reduction in tumor uptake at 48 h after injection. CONCLUSION: In this proof-of-concept study, the use of a covalent binding peptide ligand against VEGF improves tracer accumulation at the tumor site in vivo, compared with its noncovalent binding peptide analogs. This technique is a promising tool to enhance the potency of peptide probes as imaging agents.

The role of p53 in combination radioimmunotherapy with 64Cu-DOTA-cetuximab and cisplatin in a mouse model of colorectal cancer.

UNLABELLED: Radioimmunotherapy has been successfully used in the treatment of lymphoma but thus far has not demonstrated significant efficacy in humans beyond disease stabilization in solid tumors. Radioimmunotherapy with (64)Cu was highly effective in a hamster model of colorectal cancer, but targeted radiotherapies with this radionuclide have since not shown as much success. It is widely known that mutations in key proteins play a role in the success or failure of cancer therapies. For example, the KRAS mutation is predictive of poor response to anti-epidermal growth factor receptor therapies in colorectal cancer, whereas p53 is frequently mutated in tumors, causing resistance to multiple therapeutic regimens. METHODS: We previously showed that nuclear localization of (64)Cu-labeled DOTA-cetuximab was enhanced in p53 wild-type tumor cells. Here, we examine the role of p53 in the response to radioimmunotherapy with (64)Cu-DOTA-cetuximab in KRAS-mutated HCT116 tumor-bearing mice, with and without cisplatin, which upregulates wild-type p53. RESULTS: Experiments with HCT116 cells that are p53 +/+ (p53 wild-type) and -/- (p53 null) grown in cell culture demonstrated that preincubation with cisplatin increased expression of p53 and subsequently enhanced localization of (64)Cu from (64)Cu-acetate and (64)Cu-DOTA-cetuximab to the tumor cell nuclei. Radioimmunotherapy studies in p53-positive HCT116 tumor-bearing mice, receiving either radioimmunotherapy alone or in combination with cisplatin, showed significantly longer survival in mice receiving unlabeled cetuximab or cisplatin alone or in combination (all, P < 0.01). In contrast, the p53-negative tumor-bearing mice treated with radioimmunotherapy alone or combined with cisplatin showed no survival advantage, compared with control groups (all, P > 0.05). CONCLUSION: Together, these data suggest that (64)Cu specifically delivered to epidermal growth factor receptor-positive tumors by cetuximab can suppress tumor growth despite the KRAS status and present opportunities for personalized clinical treatment strategies in colorectal cancer.

Identification of critical residues involved in ligand binding and G protein signaling in human somatostatin receptor subtype 2.

G protein signaling through human somatostatin receptor subtype 2 (SSTR2) is well known, but the amino acids involved in stimulation of intracellular responses upon ligand binding have not been characterized. We constructed a series of point mutants in SSTR2 at amino acid positions 89, 139, and 140 in attempts to disrupt G protein signaling upon ligand binding. The aspartic acid changes at position 89 to either Ala, Leu, or Arg generated mutant receptors with varying expression profiles and a complete inability to bind somatostatin-14 (SST). Mutations to Asp 139 and Arg 140 also led to varying expression profiles with some mutants maintaining their affinity for SST. Mutation of Arg 140 to Ala resulted in a mutated receptor that had a B(max) and dissociation constant (K(d)) similar to wild-type receptor but was still coupled to the G protein as determined in both a cAMP assay and a calcium-release assay. In contrast, mutation of Asp 139 to Asn resulted in a mutated receptor with B(max) and K(d) values that were similar to wild type but was uncoupled from G protein-mediated cAMP signaling, but not calcium release. Thus, we identified mutations in SSTR2 that result in either receptor expression levels that are similar to wild type but is completely ablated for ligand binding or a receptor that maintains affinity for SST and is uncoupled from G protein-mediated cAMP signaling.

Evaluation of copper-64-labeled somatostatin agonists and antagonist in SSTr2-transfected cell lines that are positive and negative for p53: implications for cancer therapy.

OBJECTIVES: Radiolabeled somatostatin analogs have become important agents for molecular imaging and targeted radiotherapy of somatostatin receptor-positive tumors. Here we determine the effect of the tumor suppressor protein, p53, on trafficking (64)Cu to tumor cell nuclei from DOTA vs. CB-TE2A-conjugated agonist Y3-TATE and the antagonist (64)Cu-CB-TE2A-sst2-ANT in cell lines that are positive or negative for p53. METHODS: Receptor binding, internalization, cyclic adenosine monophosphate (cAMP) and nuclear localization studies were performed with the somatostatin receptor subtype 2 (SSTr2) agonists, (64)Cu-CB-TE2A-Y3-TATE and (64)Cu-DOTA-Y3-TATE vs. antagonist, (64)Cu-CB-TE2A-sst2-ANT, in SSTr2-transfected p53 +/+ and -/- HCT116 colorectal carcinoma cells. RESULTS: The antagonist, (64)Cu-CB-TE2A-sst2-ANT, bound 8-9-fold more SSTr2 binding sites than did the (64)Cu-labeled agonists. (64)Cu-CB-TE2A-Y3-TATE was more efficiently internalized than (64)Cu-DOTA-Y3-TATE, while (64)Cu-CB-TE2A-sst2-ANT showed lower yet significant levels of internalization. CB-TE2A-Y3-TATE acted as a full agonist, inhibiting cAMP production, whereas CB-TE2A-sst2-ANT showed no inhibition of cAMP production. The (64)Cu from agonists (64)Cu-DOTA-Y3-TATE and (64)Cu-CB-TE2A-Y3-TATE showed greater nuclear localization at 24 h in p53 +/+ vs. -/- cells; however, there was no difference in the levels of (64)Cu from the antagonist based on p53 status. Surprisingly, the DOTA and CB-TE2A-conjugated agonists showed similar nuclear localization in the p53 +/+ and -/- cells, suggesting no difference in (64)Cu release from these chelators in the HCT116 cell lines. CONCLUSION: Based on these in vitro data, the agonist (64)Cu-CB-TE2A-Y3-TATE demonstrates the most promise as an agent for targeted radiotherapy in p53 positive, SSTr2-positive tumors.

Multimodality imaging of gene transfer with a receptor-based reporter gene.

UNLABELLED: Gene therapy trials have traditionally used tumor and tissue biopsies for assessing the efficacy of gene transfer. Noninvasive imaging techniques offer a distinct advantage over tissue biopsies in that the magnitude and duration of gene transfer can be monitored repeatedly. Human somatostatin receptor subtype 2 (SSTR2) has been used for the nuclear imaging of gene transfer. To extend this concept, we have developed a somatostatin receptor-enhanced green fluorescent protein fusion construct (SSTR2-EGFP) for nuclear and fluorescent multimodality imaging. METHODS: An adenovirus containing SSTR2-EGFP (AdSSTR2-EGFP) was constructed and evaluated in vitro and in vivo. SCC-9 human squamous cell carcinoma cells were infected with AdEGFP, AdSSTR2, or AdSSTR2-EGFP for in vitro evaluation by saturation binding, internalization, and fluorescence spectroscopy assays. In vivo biodistribution and nano-SPECT imaging studies were conducted with mice bearing SCC-9 tumor xenografts directly injected with AdSSTR2-EGFP or AdSSTR2 to determine the tumor localization of (111)In-diethylenetriaminepentaacetic acid (DTPA)-Tyr3-octreotate. Fluorescence imaging was conducted in vivo with mice receiving intratumoral injections of AdSSTR2, AdSSTR2-EGFP, or AdEGFP as well as ex vivo with tissues extracted from mice. RESULTS: The similarity between AdSSTR2-EGFP and wild-type AdSSTR2 was demonstrated in vitro by the saturation binding and internalization assays, and the fluorescence emission spectra of cells infected with AdSSTR2-EGFP was almost identical to the spectra of cells infected with wild-type AdEGFP. Biodistribution studies demonstrated that the tumor uptake of (111)In-DTPA-Tyr3-octreotate was not significantly different (P > 0.05) when tumors (n = 5) were injected with AdSSTR2 or AdSSTR2-EGFP but was significantly greater than the uptake in control tumors. Fluorescence was observed in tumors injected with AdSSTR2-EGFP and AdEGFP in vivo and ex vivo but not in tumors injected with AdSSTR2. Although fluorescence was observed, there were discrepancies between in vivo imaging and ex vivo imaging as well as between nuclear imaging and fluorescent imaging. CONCLUSION: These studies showed that the SSTR2-EGFP fusion construct can be used for in vivo nuclear and optical imaging of gene transfer.

MicroPET imaging of breast cancer using radiolabeled bombesin analogs targeting the gastrin-releasing peptide receptor.

Mammography is a well-established method for detecting primary breast cancer; however, it has some limitations that may be overcome using nuclear imaging methods. Current radiopharmaceuticals have limited sensitivity for detecting small primary lesions and it has been suggested that novel radiopharmaceuticals are necessary for detection of primary breast cancer, as well as for detecting metastases and recurrence, or for monitoring therapy. The gastrin-releasing peptide receptor (GRPR) is a seven-transmembrane G-protein coupled receptor that is overexpressed on primary breast cancer and lymph node metastases. Bombesin (BN) is a tetradecapeptide that binds with high affinity to GRPR and can be radiolabeled with the positron-emitter, copper-64 ((64)Cu) for imaging with positron-emission tomography (PET). The goal of this study was to evaluate BN analogs that could be radiolabeled with (64)Cu for PET imaging of breast cancer. A series of BN analogs containing 4, 5, 6, 8, and 12- carbon linkers were evaluated with regard to their binding and internalization into T-47D human breast cancer cells. The (64)Cu-labeled analogs were then evaluated in mice bearing T-47D xenografts by tissue biodistribution and microPET imaging. These studies showed that all of the analogs had IC(50) values <100 nM and were all internalized into T-47D cells. Biodistribution studies showed that the BN analog with the 8-carbon linker not only had the highest tumor uptake but also had high normal tissue uptake in the liver. The analogs containing the 6- or 8-carbon linkers demonstrated good tumor uptake as determined by microPET imaging. Overall, this study shows the feasibility of using positron-labeled BN analogs for PET detection of GRPR-expressing breast cancer.

In vitro and in vivo evaluation of 64Cu-labeled DOTA-linker-bombesin(7-14) analogues containing different amino acid linker moieties.

The gastrin-releasing peptide receptor (GRPR) is overexpressed on a variety of tumor types and has been targeted with radiolabeled peptides for detection and therapy of these cancers. Analogues of the 14 amino acid bombesin (BN) peptide have been radiolabeled with both gamma- and positron-emitting radionuclides for detection of GRPR-expressing tumors. We have previously evaluated BN analogues radiolabeled with the positron-emitter, copper-64 (64Cu), that contained various aliphatic linkers placed between the BN peptide and the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator. These studies showed that the analogues could be used for positron-emission tomographic (PET) imaging of GRPR-positive tumors in mice but clinical translation would be hindered by significant uptake in background tissues. Therefore, the purpose of this study was to determine if the use of amino acid linkers placed between the DOTA chelate and the BN peptide would reduce nontarget tissue uptake, while maintaining good prostate tumor uptake. The linkers studied utilized three amino acid combinations of glycine (G), serine (S), or glutamic acid (E). In vitro assays in PC-3 cells showed that the glutamic acid-containing linkers had poor binding and internalization, while the other analogues had IC50 values <100 nM and good internalization. In vivo, these same analogues demonstrated tumor-specific uptake and good imaging characteristics that were comparable to, or better than the previously reported 64Cu-labeled DOTA-BN analogues. Overall, this study shows that BN analogues containing amino acid linkers can be used for the PET imaging of GRPR-expressing prostate cancer and that these linkers lead to lower background tissue uptake.

Characterization of somatostatin receptor subtype 2 expression in stably transfected A-427 human cancer cells.

Although radiolabeled somatostatin analogs have become highly prevalent in the diagnosis and treatment of somatostatin receptor subtype (sst)-positive tumors, there are relatively few options with respect to sst-positive tumor cell lines and animal models. It would be highly beneficial, particularly for therapeutic purposes, to have several clones of one human sst2-positive cell line that express a range of sst2 concentrations for evaluating the dose response and intracellular processing of radiolabeled somatostatin analogs. The human non-small cell lung cancer line A-427 was stably transfected with a hemagglutinin-tagged human sst2. Expression of the receptor was evaluated in vitro using flow cytometry, saturation binding analysis, internalization assays, and quantitative polymerase chain reaction. The receptor expression was also validated in an in vivo mouse model in biodistribution and micro-positron emission tomography (microPET) studies using the somatostatin analog octreotide (OC), which was linked to the (64)Cu chelator 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), or (64)Cu-TETA-OC. Stable clones were isolated, and four clones (2, 4, 5, and 7) were chosen for further examination. In vitro assays showed that clone 4 had no expression of sst2, whereas the others had various levels in the order of 7 > 2 > 5. Biodistribution studies with (64)Cu-TETA-OC showed the same rank order, with tumor uptake of the clones ranging from 0.8 to 6.5% injected dose/g. These studies showed that there was a strong correlation among the in vitro assays and between the in vitro assays and the biodistribution. MicroPET confirmed significant uptake of (64)Cu-TETA-OC in clone 7 and background uptake in clone 4. These studies show that clones of a human cell line can be produced expressing various levels of sst2 that should be useful for the future evaluation of radiolabeled somatostatin analogs.

Molecular imaging of gastrin-releasing peptide receptor-positive tumors in mice using 64Cu- and 86Y-DOTA-(Pro1,Tyr4)-bombesin(1-14).

Bombesin is a tetradecapeptide neurohormone that binds to gastrin-releasing peptide receptors (GRPR). GRPRs have been found in a variety of cancers including invasive breast and prostate tumors. The peptide MP2346 (DOTA-(Pro(1),Tyr(4))-bombesin(1-14)) was designed to bind to these GRP receptors. This study was undertaken to evaluate radiolabeled MP2346 as a positron emission tomography (PET) imaging agent. MP2346 was radiolabeled, in high radiochemical purity, with the positron-emitting nuclides (64)Cu (t(1/2) = 12.7 h, beta+ = 19.3%, E(avg) = 278 keV) and (86)Y (t(1/2) = 14.7 h, beta+ = 33%, E(avg) = 664 keV). (64)Cu-MP2346 and (86)Y-MP2346 were studied in vitro for cellular internalization by GRPR-expressing PC-3 (human prostate adenocarcinoma) cells. Both (64)Cu- and (86)Y-MP2346 were studied in vivo for tissue distribution in nude mice with PC-3 tumors. Biodistribution in PC3 tumor-bearing mice demonstrated higher tumor uptake, but lower liver retention, in animals injected with (86)Y-MP2346 compared to (64)Cu-MP2346. Receptor-mediated uptake was confirmed by a significant reduction in uptake in the PC-3 tumor and other receptor-rich tissues by coinjection of a blockade. Small animal PET/CT imaging was carried out in mice bearing PC-3 tumors and rats bearing AR42J tumors. It was possible to delineate PC-3 tumors in vivo with (64)Cu-MP2346, but superior (86)Y-MP2346-PET images were obtained due to lower uptake in clearance organs and lower background activity. The (86)Y analogue demonstrated excellent PET image quality in models of prostate cancer for the delineation of the GRPR-rich tumors and warrants further investigation.