Correlating EGFR expression with receptor-binding properties and internalization of 64Cu-DOTA-cetuximab in 5 cervical cancer cell lines.

UNLABELLED: The anti-epidermal growth factor receptor (anti-EGFR) antibody cetuximab is clinically approved for the treatment of EGFR-expressing metastatic colorectal cancer and advanced head and neck cancer. Overexpression of EGFR has also been found in more than 70% of carcinomas of the cervix. The overall goal of this study was to determine whether (64)Cu-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-cetuximab has potential as an agent for measuring EGFR concentration by PET imaging in cervical cancer tumors. METHODS: Cetuximab was conjugated to the bifunctional chelator DOTA and labeled with (64)Cu. EGFR messenger RNA (mRNA) expression was correlated with EGFR densities on the cell surface of 5 different cervical cancer cell lines and with receptor function, measured by internalization of (64)Cu-DOTA-cetuximab. Imaging in tumor-bearing mice with small-animal PET was performed using the highest-expressing cervical cancer cell line. RESULTS: The affinity of (64)Cu-DOTA-cetuximab binding for the EGFR was similar in 4 EGFR-positive lines, varying from 0.1 to 0.7 nM. The mRNA expression corresponded well with EGFR densities and levels of internalization, with responses decreasing in the order of CaSki>ME-180>DoTc2 4510>HeLa>C-33A. Biodistribution and small-animal PET studies with (64)Cu-DOTA-cetuximab in CaSki tumor-bearing nude mice showed relatively high tumor uptake at 24 h after injection (13.2+/-1.2 percentage of injected activity per gram), although there was also significant retention of activity in the blood and liver accumulation. CONCLUSION: (64)Cu-DOTA-cetuximab was successfully used to detect and quantify EGFR expression in cervical cancer tumors, and small-animal PET/CT of EGFR-expressing CaSki tumors suggests potential for PET/CT of EGFR-positive tumors.

Receptor-binding, biodistribution, and metabolism studies of 64Cu-DOTA-cetuximab, a PET-imaging agent for epidermal growth-factor receptor-positive tumors.

The epidermal growth-factor receptor (EGFR) and its ligands have been recognized as critical factors in the pathophysiology of tumorigenesis. Overexpression of the EGFR plays a significant role in the tumor progression of a wide variety of solid human cancers. Therefore, the EGFR represents an attractive target for the design of novel diagnostic and therapeutic agents for cancer. Cetuximab (C225, Erbitux) was the first monoclonal antibody targeted against the ligand-binding site of EGFR approved by the Food and Drug Administration for the treatment of patients with EGFR-expressing, metastatic colorectal carcinoma, although clinical trials showed variability in the response to this treatment. The aim of this study involved using cetuximab to design a positron emission tomography (PET) agent to image the overexpression of EGFR in tumors. Cetuximab was conjugated with the chelator, DOTA, for radiolabeling with the positron-emitter, 64Cu (T(1/2) = 12.7 hours). 64Cu-DOTA-cetuximab showed high binding affinity to EGFR-positive A431 cells (K(D) of 0.28 nM). Both biodistribution and microPET imaging studies with 64Cu-DOTA-cetuximab demonstrated greater uptake at 24 hours postinjection in EGFR-positive A431 tumors (18.49% +/- 6.50% injected dose per gram [ID/g]), compared to EGFR-negative MDA-MB-435 tumors (2.60% +/- 0.35% ID/g). A431 tumor uptake at 24 hours was blocked with unlabeled cetuximab (10.69% +/- 2.72% ID/g), suggesting that the tumor uptake was receptor mediated. Metabolism experiments in vivo showed that 64Cu-DOTA-cetuximab was relatively stable in the blood of tumor-bearing mice; however, there was significant metabolism in the liver and tumors. 64Cu-DOTA-cetuximab is a potential agent for imaging EGFR-positive tumors in humans.

The role of p53 in the trafficking of copper-64 to tumor cell nuclei.

Copper-64 (T(1/2) = 12.7 h; beta(+): 17.8%, beta(-): 41%) has applications in both positron emission tomography (PET) imaging and targeted radiotherapy of cancer. Copper-64 radiopharmaceuticals have shown tumor growth inhibition with a relatively low radiation dose in animal models; however, the mechanism of cytotoxicity has not been fully elucidated. Here, we report an investigation on the potential role of the tumor suppressor protein p53 in trafficking (64)Cu to tumor cell nuclei. Two EGFR expressing human colorectal cell lines (HCT 116 +/+ and HCT 116 -/-) that are positive or negative for p53 expression respectively, were used to compare internalization and nuclear localization of [(64)Cu]copper acetate and of (64)Cu-DOTA-cetuximab, a monoclonal anti-EGFR antibody. [(64)Cu]copper acetate uptake into cells was similar between the two cell lines during a 24 h time course. In contrast, the uptake of [(64)Cu]copper acetate in the nuclei of HCT 116 +/+ cells was significantly higher than in HCT 116 -/- cells (p < 0.0001) at 24 h. There was no difference in receptor binding, receptor-mediated internalization, and efflux of (64)Cu-DOTA-cetuximab between the two HCT 116 cells lines. However, nuclear localization of (64)Cu-DOTA-cetuximab showed increased uptake in the nuclei of HCT 116 +/+ cells as early as 4 h. These data demonstrate that (64)Cu is delivered to tumor cell nuclei in a p53 positive cell line in significantly greater amounts than in p53 negative cells by both non-specific and receptor-mediated uptake mechanisms.

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.

64Cu-azabicyclo[3.2.2]nonane thiosemicarbazone complexes: radiopharmaceuticals for PET of topoisomerase II expression in tumors.

UNLABELLED: Topoisomerase II (Topo-II) is an essential enzyme in the DNA replication process and is the primary cellular target for many of the most widely used and effective anticancer agents. It has been reported that thiosemicarbazones (TSCs) are potent antitumor agents that inhibit Topo-II. The aim of this study was to investigate the relationship between the in vitro and in vivo behavior of novel (64)Cu-TSC complexes and the expression of Topo-II activity. METHODS: Four (4)N-azabicyclo[3.2.2]nonane TSC derivatives (EPH142, EPH143, EPH144, and EPH270) were successfully radiolabeled with (64)Cu, to form lipophilic cations of the general formula [(64)Cu(L)]Cl, and the partition coefficient (logP) values were determined. One agent [(64)Cu-EPH270](+) was observed in vitro in cultured cell studies. The kinetics of 2 compounds, [(64)Cu-EPH144](+) and [(64)Cu-EPH270](+), were examined in mice bearing L1210 tumors and small-animal PET was conducted in mice bearing L1210 and PC-3 tumors, which expressed high and low levels of Topo-II, respectively. All data were compared with the activity and levels of Topo-II, as determined by a commercially available assay kit and western blot analysis. RESULTS: The 4 complexes were radiolabeled by incubation of (64)CuCl(2) with the ligand in ethanolic solution. The complexes were isolated in high radiochemical purity, as determined by radio-thin-layer chromatography and radio-high-performance liquid chromatography. The compounds were shown to be lipophilic with logP values ranging from 1.34 to 1.92. In biodistribution studies, good L1210 tumor uptake was noted ([(64)Cu-EPH144](+) at 1 h, 4.70 %ID/g [percentage injected dose per gram]; 4 h, 8.80 %ID/g; 24 h, 6.64 %ID/g; and [(64)Cu-EPH270](+) at 1 h, 2.58 %ID/g; 4 h, 6.00 %ID/g; 24 h, 4.80 %ID/g). Small-animal PET of animals with L1210 tumors (high Topo-II expressing) showed excellent tumor accumulation compared with that of animals with PC-3 tumors (low Topo-II expressing), and the L1210 tumor uptake was significantly reduced by coadministration of a Topo-II poison. CONCLUSION: Here we describe the characterization of a new class of copper-radiolabeled TSC analogs. We demonstrate that the accumulation of the (64)Cu-compounds is related to the expression levels of Topo-II in tumor tissue.

Subcellular localization of radiolabeled somatostatin analogues: implications for targeted radiotherapy of cancer.

Copper-64 (T(1/2) = 12.7 h; beta(+), 17.4%; beta(-), 39%) has been used both in positron emission tomography imaging and in radiotherapy. Copper-64 radiopharmaceuticals have shown tumor growth inhibition with a relatively low radiation dose in animal models; however, the mechanism of cytotoxicity has not been fully elucidated. These studies incorporate the use of somatostatin receptor-positive AR42J rat pancreatic tumor cells in vitro to understand the cell killing mechanism of (64)Cu by focusing on subcellular distribution of the somatostatin analogues (64)Cu-labeled 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid-octreotide ((64)Cu-TETA-OC) and (111)In-labeled diethylenetriaminepentaacetic acid-octreotide ((111)In-DTPA-OC). Cell uptake and organelle isolation studies were conducted on (64)Cu-TETA-OC and (111)In-DTPA-OC. Nuclear localization of (64)Cu and (111)In from (64)Cu-TETA-OC and (111)In-DTPA-OC, respectively, increased over time, with 19.5 +/- 1.4% and 6.0 +/- 1.0% in the cell nucleus at 24 h, respectively. In pulse-chase experiments, in which (64)Cu-TETA-OC was incubated with AR42J cells for 4 h, it was found that the nuclear localization of (64)Cu increased significantly over the next 20 h (from 9.8 +/- 1.0% to 26.3 +/- 5.4%). In a control pulse-chase experiment, levels of (64)Cu from [(64)Cu]cupric acetate decreased from 4 to 24 h postadministration (20.6 +/- 8.7 to 5.4 +/- 1.9), suggesting that the redistribution mechanism, or the kinetics of (64)Cu from (64)Cu-TETA-OC is different from that for (64)Cu from [(64)Cu]cupric acetate. The amount of (64)Cu from (64)Cu-TETA-OC also increased in the mitochondria over time, with 21.1 +/- 3.6% in the mitochondria at 24 h postadministration. These results suggest that localization of substantial quantities of (64)Cu to the cell nucleus and mitochondria may contribute to cell killing with (64)Cu radiopharmaceuticals.