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Basic Science Investigations |
1 Division of Diagnostic Imaging, University of Texas M.D. Anderson Cancer Center, Houston, Texas
2 Department of Surgical Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas
3 Department of Gastrointestinal Medical Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Key Words: apoptosis • nuclear imaging • annexin V • paclitaxel • polyethylene glycol
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Owing to its high affinity to phosphatidylserine molecules that are exposed on the surface of apoptotic cells and other dead cells or cell fragments, annexin V has been proposed as a marker for noninvasive imaging of apoptosis (2). Blankenberg et al. (3) labeled annexin V directly with 99mTc through a small-molecular-weight chelator, hydrazinonicotinamide, and tested the ability of 99mTc-labeled annexin V to reveal apoptosis in a murine B-cell lymphoma tumor model. Unfortunately, no data about the intratumoral distribution of 99mTc-labeled annexin V were available with which to ascertain the correlation between apoptosis and tumor uptake of 99mTc-annexin V. Furthermore, owing to the short blood half-life of 99mTc-annexin V (<7 min) (2) and the short half-life of 99mTc radionuclide (6 h), it is difficult to extend the time of imaging using 99mTc-labeled annexin V beyond 6 h.
Recent clinical studies by Belhocine et al. (4) demonstrated for the first time that overall survival and progression-free survival were significantly related to uptake of 99mTc-labeled annexin V in treated lung cancers and lymphomas. As noted by Blankenberg (5) in an accompanying editorial, the timing of annexin V imaging after a given therapy was critical. If peak apoptotic activity after anticancer therapy varies from patient to patient and from treatment to treatment, then it will be necessary to determine for each patient the best time to scan after the start of chemotherapy. It has been suggested that an imaging protocol using multiple separate injections of 99mTc-labeled annexin V and multiple radionuclide scans could be used to assess peak apoptotic activity (5). Such a protocol, however, may be difficult to implement in larger-scale trials.
We hypothesized that long-circulating annexin V may make it possible to capture therapy-induced apoptotic cells over a prolonged period. Thus, we have developed a simple procedure to synthesize polyethylene glycol (PEG)-modified, 111In-labeled annexin V (6). PEG-modified proteins have been shown to exhibit reduced liver uptake and increased blood circulation half-lives, resulting in improved biologic activity (7,8). 111In has a longer physical half-life (2.81 d) than 99mTc. In our previous study, we showed that PEGylated annexin V, 111In-DTPA-PEG-annexin V (where DTPA is diethylenetriaminepentaacetic acid), selectively bound to apoptotic cells in vitro and had increased blood half-life in vivo. The half-life (t1/2, 
) for PEGylated annexin V in nude mice was 4.90 h, whereas that of unPEGylated annexin V was only 0.07 h (6).
In this study, we sought to compare the imaging properties of 111In-labeled PEGylated annexin V and 111In-labeled unPEGylated annexin V in a mammary cancer xenograft.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Synthesis and Radiolabeling of PEGylated Proteins
The PEG precursor containing an NH2-reactive p-SCN group on one end and DTPA on the other end of the PEG chain, SCN-PEG-DTPA, was synthesized in accordance with procedures reported previously (6). To conjugate DTPA-PEG to proteins, aliquots of SCN-PEG-DTPA (15 µL, 0.18 µmol) in dimethylformamide were added to a solution of annexin V (0.40 mL, 0.006 µmol) or ovalbumin (0.52 mL, 0.006 µmol) in 0.1 mol/L phosphate-buffered saline (PBS) solution (pH 8.5), and the mixture was allowed to react at 4°C overnight. Unreacted SCN-PEG-DTPA was removed using an AKTA fast-protein liquid chromatography system (Amersham Pharmacia Biotech) equipped with a Resource Q 1-mL anion-exchange column. The samples were eluted with 20 mmol/L Tris buffer (pH 7.5) and with a linear gradient of 0%–100% 1N NaCl in 15 mL at a flow rate of 4 mL/min. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 12%) was used to analyze the extent of protein PEGylation.
To label DTPA-PEG-annexin V and DTPA-PEG-ovalbumin with 111In, a solution of DTPA-PEG-annexin V or DTPA-PEG-ovalbumin (300 µL, 200 µg/mL) in 20 mmol/L Tris buffer (pH 7.5) was incubated with 29.6 MBq (800 µCi) 111InCl3 for 15 min. Free 111In3+ was removed by gel filtration from a PD-10 column eluted with PBS. The purity of radiolabeled compound was analyzed by radio-gel permeation chromatography (GPC) and by gel electrophoresis. The GPC system consisted of an LDC pump (Laboratory Data Control), a LUDLUM radiometric detector (Measurement Inc.), and an SP 8450 UV/VIS detector (Spectra-Physics). The samples were separated using a Phenomenex Biosep SEC-S3000 7.8 mm x 30 cm column, eluted with PBS containing 0.1% LiBr at a flow rate of 1 mL/min, and detected by radioactivity and ultraviolet (UV) absorbance at 254 nm.
The SDS-PAGE gel was stained with Coomassie Brilliant Blue for the presence of proteins. For the detection of radioactivity, the dried gel was exposed to a multipurpose storage phosphor screen and analyzed using a Cyclone imager (Packard Instrument Co.).
Synthesis and Radiolabeling of DTPA-Annexin V
DTPA-annexin V was synthesized by reacting annexin V with p-SCN-DTPA in aqueous solution. PD-10 was used to remove unreacted DTPA. DTPA-annexin V was labeled with 111In using the same procedures as described above. The purity of the radiotracer was examined by radio-GPC.
-Imaging with 111In-DTPA-PEG-Annexin V
MDA-MB-468 tumor cells were grown as a monolayer in Dulbecco’s modified Eagle’s medium/F-12 medium supplemented with 10% fetal bovine serum. Female nude mice, 18–22 g (Harlan Sprague–Dawley), were inoculated subcutaneously in the anterior chest wall with 1.5 x 106 MDA-MB-468 cells per site. When the tumors had grown to 200 mm3 in average volume, the mice were assigned to groups of 3 mice each. Groups of mice were given a single intravenous injection of poly(L-glutamic acid)-paclitaxel at a dose of 25 mg equivalent (eq) paclitaxel/kg, a single intravenous injection of poly(L-glutamic acid)-paclitaxel at a dose of 100 mg eq paclitaxel/kg, intraperitoneal injections of C225 at a dose of 1 mg per mouse per injection on day 1 and day 4, combined treatment with poly(L-glutamic acid)-paclitaxel (100 mg eq paclitaxel/kg) and C225 (1 mg per mouse per injection), or no treatment control. On day 1 or day 4 after the initiation of treatment, mice were injected with 111In-DTPA-PEG-annexin V (8 µg eq annexin V per mouse; 1.85 MBq [50 µCi] per mouse) through the tail vein.
Forty-eight hours after injection of the radiotracer (day 3 and day 6 after initiation of treatment), planar -images were acquired in a 64 x 64 matrix for 5 min using a solid-state -camera (model 2020tc; DigiRad) equipped with a medium-energy collimator and Mirage processing software (version 4.322; Segami Corp.). The mice were placed prone on the camera’s parallel-hole collimator after they were anesthetized with an intraperitoneal injection of sodium pentobarbital (35 mg/kg). At the end of the imaging session, the mice were killed and tumors were removed from each animal for radioactivity measurements and autoradiography. Tumor uptake of 111In-DTPA-PEG-annexin V was expressed as percentage of the injected dose per gram of tissue (%ID/g tissue).
Determination of Apoptotic Index
Mice bearing MD-MB-468 tumors were killed at different times after treatments, and the tumors were immediately excised and placed in neutral-buffered formalin. The tissues were then processed and stained with hematoxylin–eosin (H&E) stain. Apoptosis was scored in coded slides by microscopic examination at x400 magnification according to the method of Milas et al. (9). Briefly, 5 fields of nonnecrotic areas were randomly selected in each histologic specimen, and, in each field, the number of apoptotic nuclei per 100 nuclei examined was determined and expressed as a percentage. The values were based on scoring 1,500 nuclei obtained from 3 mice per time point.
Autoradiography
Tumors removed from mice injected with 111In-DTPA-PEG-annexin V or 111In-DTPA-annexin V at the end of the -imaging session (4 d after radiotracer injection) were immediately frozen and processed. Sliced 8-µm sections were fixed in cold acetone, dried, and exposed to a multipurpose storage phosphor screen for 10 d. A Cyclone storage phosphor screen imager (Packard Instrument Co.) was used to detect the distribution of radionuclide activity localized in each section.
TUNEL Assay
DNA fragmentation was analyzed by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay using a commercial kit (Promega) according to the manufacturer’s protocol. Briefly, frozen tissue sections (8 µm) were fixed with 4% methanol-free paraformaldehyde for 10 min at room temperature. The sections were washed and incubated sequentially with equilibration buffer and reaction buffer containing nucleotide mixture and terminal deoxynucleotidyltransferase. To prevent photobleaching, Vectashield mounting medium (Vector Laboratories, Inc.) was used to mount coverslips. Images were recorded using an Olympus fluorescence microscope with a 520-nm filter and an x20 objective lens.
Biodistribution Studies
Mice bearing MDA-MB-468 tumors were assigned to groups of 3 mice each. Mice in each of the 3 treated groups were given a single intravenous injection of poly(L-glutamic acid)-paclitaxel at a dose of 100 mg eq paclitaxel/kg. Mice in each of the 3 control groups were not treated. Four days after drug treatment, mice in control groups and treated groups were injected with 111In-DTPA-PEG-annexin V (4 µg eq annexin V per mouse; 1.48 MBq [40 µCi] per mouse), 111In-DTPA-PEG-ovalbumin (4 µg eq ovalbumin per mouse; 1.48 MBq [40 µCi] per mouse), or 111In-DTPA-annexin V (4 µg eq annexin V per mouse; 1.11 MBq [30 µCi] per mouse). No cold carriers were added to the radiotracers. -Images were acquired in a 64 x 64 matrix for 15 min at 4 and 48 h after the injection of each radiotracer using a Siemens m.Cam -camera equipped with a medium-energy, low-penetration collimator and ICON software version 9.5. Mice were prepared for the imaging sessions as described before. At the end of the imaging session, the mice were killed, and various tissues were removed from each animal for radioactivity measurements. The data were expressed as %ID/g tissue.
Data Analysis
Statistical comparisons between untreated controls and each treated group for each radiotracer were calculated using the Student t test. P < 0.05 was considered to be statistically significant. The Pearson correlation coefficient was used to assess the correlation between the average tumor uptake of 111In-DTPA-PEG-annexin V and the average apoptotic index. Five treatment groups, each consisting of 3 mice, were used to derive the correlation curve.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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DTPA-annexin V was synthesized from annexin V and p-SCN-DTPA. The radiochemical yield and radiochemical purity of 111In-DTPA-annexin V were 74% and >98%, respectively. The specific activity was 296 kBq (8 µCi/µg).
-Imaging with 111In-DTPA-PEG-Annexin V
To validate the use of 111In-DTPA-PEG-annexin V for the noninvasive imaging of tumor apoptosis, we first evaluated whether 111In-DTPA-PEG-annexin V could capture apoptotic responses of MDA-MB-468 tumors to poly(L-glutamic acid)-paclitaxel and C225 treatments. -Images acquired 48 h after radiotracer injection clearly showed increased radioactivity in the gastrointestinal tract as well as in the tumors of mice treated with either poly(L-glutamic acid)-paclitaxel at a dose of 100 mg eq paclitaxel/kg or combined poly(L-glutamic acid)-paclitaxel and C225 therapy given 4 d before radiotracer injection compared with the radioactivity in tumors of untreated mice (Figs. 2A–2C). Treatment with poly(L-glutamic acid)-paclitaxel at a dose of 25 mg eq paclitaxel/kg did not result in apparent enhancement in tumor uptake of 111In-DTPA-PEG-annexin V (data not shown). Similarly, treatment with C225 alone did not cause detectable change in tumor uptake of the radiotracer (Fig. 2D). These observations were confirmed by scintillation well counting. Treatments with poly(L-glutamic acid)-paclitaxel at a dose of 100 mg eq paclitaxel/kg poly(L-glutamic acid)-paclitaxel or combined poly(L-glutamic acid)-paclitaxel and C225 increased the tumor uptake of 111In-DTPA-PEG-annexin V by 75% (P = 0.001) and 60% (P = 0.029), respectively, when the radiotracer was injected 4 d after the initiation of drug treatment. On the other hand, treatment with C225 alone significantly decreased the tumor uptake of 111In-DTPA-PEG-annexin V by 24% (P = 0.028) (Table 1). No differences were found in tumor uptake of 111In-DTPA-PEG-annexin V between control mice and mice treated with poly(L-glutamic acid)-paclitaxel or C225 when the radiotracer was injected 1 d after the initiation of drug treatment (Table 1).
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Histologic analysis of H&E-stained tumor tissues revealed that poly(L-glutamic acid)-paclitaxel and combined poly(L-glutamic acid)-paclitaxel and C225 significantly increased the percentage of apoptotic cells at 4 d after drug injection. The apoptotic index at that time was 1.67% ± 0.31% in untreated tumors, 7.6% ± 0.72% in poly(L-glutamic acid)-paclitaxel–treated tumors (P < 0.001), and 11.07% ± 1.81% in tumors treated with both poly(L-glutamic acid)-paclitaxel and C225 (P < 0.001) (Table 1). Treatment with C225 alone had no effect on the apoptotic index (P = 0.48). Importantly, there was a significant correlation between tumor uptake of 111In-DTPA-PEG-annexin V expressed as %ID/g and apoptotic index determined histologically (r = 0.87; P = 0.02) (Fig. 3).
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-Imaging with 111In-DTPA-annexin V failed to reveal tumors in either untreated mice or mice treated with poly(L-glutamic acid)-paclitaxel (Fig. 6, left). At 4 h after radiotracer injection, 111In-DTPA-annexin V was primarily taken up by the kidney (Table 2), and there was little blood-pool activity. As in the case of 111In-DTPA-PEG-annexin V (Fig. 2B is representative image), mice injected with 111In-DTPA-PEG-ovalbumin had visible background blood-pool activity at 48 h after radiotracer injection (Fig. 6, right). Tumors were visualized with 111In-DTPA-PEG-ovalbumin in both untreated and treated mice. However, changes in tumor uptake in untreated and poly(L-glutamic acid)-paclitaxel–treated mice could not be discerned with 111In-DTPA-PEG-ovalbumin, whereas increased activity in tumors of poly(L-glutamic acid)-paclitaxel–treated mice was clearly visualized with 111In-DTPA-PEG-annexin V (Fig. 2).
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The distribution pattern of unPEGylated 111In-DTPA-annexin V was different from that of the PEGylated proteins (Table 2). 111In-DTPA-annexin V did show increased tumor uptake after poly(L-glutamic acid)-paclitaxel treatment. However, the uptake value for 111In-DTPA-annexin V (1.23 %ID/g) was much lower than that of 111In-DTPA-PEG-annexin V (15.99 %ID/g) after poly(L-glutamic acid)-paclitaxel treatment. Similarly, the tumor-to-muscle ratio for 111In-DTPA-annexin V (3.62-fold) was much lower than that for 111In-DTPA-PEG-annexin V (13.2-fold) (Table 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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In this study, we evaluated the in vivo imaging properties of 111In-labeled, PEGylated annexin V in mice bearing human mammary MDA-MB-468 tumors. Tumor apoptosis was induced by treatments with a water-soluble taxane, poly(L-glutamic acid)-paclitaxel, and with monoclonal antibody C225. Poly(L-glutamic acid)-paclitaxel is a polymeric paclitaxel conjugate that has demonstrated significant antitumor activity with reduced systemic toxicity in preclinical and clinical studies (11). The conjugate is currently being evaluated in several clinical phase I–III trials. C225 is a human–mouse chimeric monoclonal antibody that targets the EGFRs (12). C225 inhibits EGFR tyrosine kinase activity, proliferation, and tumor-induced angiogenesis in a variety of cultured and xenografted human cancer cells that express high levels of EGFR (12–14). Exposure of MDA-MB-468 cells to C225 inhibits cell proliferation but does not induce caspase-8 activation and apoptosis (15). Recent reports indicate that C225 modulates host responses and enhances the efficacy of paclitaxel (16).
We found that treatment with poly(L-glutamic acid)-paclitaxel or combined poly(L-glutamic acid)-paclitaxel and C225 significantly increased the uptake of 111In-DTPA-PEG-annexin V in tumors (Fig. 2; Table 1). The specificity of the detection was confirmed by strong correlation between the tumor uptake of 111In-DTPA-PEG-annexin V and the apoptosis index determined histologically (Fig. 3) and by colocalization of the radioactivity associated with 111In-DTPA-PEG-annexin V and tumor apoptosis revealed by TUNEL staining (Fig. 5).
The advantage of PEGylated annexin V over unPEGylated annexin V was demonstrated by comparing images obtained with 111In-DTPA-PEG-annexin V and 111In-DTPA-annexin V. Whereas no tumors were detectable before or after poly(L-glutamic acid)-paclitaxel treatment at 4 h after 111In-DTPA-annexin V injection (Fig. 6), enhanced tumor uptake of 111In-DTPA-PEG-annexin V was clearly visualized in poly(L-glutamic acid)-paclitaxel–treated mice 48 h after radiotracer injection (Fig. 2). Imaging with DTPA-annexin V at 48 h after radiotracer injection also failed to delineate tumors in mice treated with poly(L-glutamic acid)-paclitaxel (data not shown). The fraction of apoptotic cells in MDA-MB-468 tumors after poly(L-glutamic acid)-paclitaxel treatment was quite low, 7.6%–11.1% (Table 1), indicating that 111In-DTPA-PEG-annexin V is sensitive in the detection of apoptotic cells. It is plausible that increased exposure to 111In-DTPA-PEG-annexin V may have contributed to the enhanced detection sensitivity compared with 111In-DTPA-annexin V. Alternatively, the increased blood half-life of 111In-DTPA-PEG-annexin V could have allowed the radiotracer to diffuse deeper into the tumor mass than 111In-DTPA-annexin V could. This is supported by comparing autoradiographs of tumors from mice injected with 111In-DTPA-PEG-annexin V and 111In-DTPA-annexin V (Fig. 4). Poly(L-glutamic acid)-paclitaxel treatment resulted in strong radioactivity in the central zone in addition to the peripheral zone of tumors from mice injected with 111In-DTPA-PEG-annexin V; in contrast, only weak radioactivity was found in the periphery of tumors from mice treated with poly(L-glutamic acid)-paclitaxel and injected with 111In-DTPA-annexin V (Fig. 4). For both radiolabeled annexin V radiotracers, a potential limitation is on its utility in abdominal imaging. The high accumulation of the radiotracer in the liver and kidney might preclude the use of these agents for abdominal imaging.
To evaluate whether the uptake of PEGylated annexin V was related to nonspecific uptake of macromolecules owing to the enhanced permeability and retention (EPR) effect (11), we compared tumor uptake of 111In-DTPA-PEG-annexin V with tumor uptake of the nonspecific PEGylated protein, 111In-DTPA-PEG-ovalbumin. Ovalbumin has a molecular weight of 43 kDa, which is close to that of annexin V (33 kDa). -Imaging of MDA-MB-468 tumor-bearing mice with 111In-DTPA-PEG-ovalbumin showed the presence of tumors before and after poly(L-glutamic acid)-paclitaxel treatment, although it is difficult to ascertain whether poly(L-glutamic acid)-paclitaxel treatment led to enhanced tumor uptake of the radiotracer (Fig. 6). Scintillation well counting showed that both 111In-DTPA-PEG-annexin V and 111In-DTPA-PEG-ovalbumin had high blood-pool activity at 48 h after radiotracer injection (Table 2). Treatment with poly(L-glutamic acid)-paclitaxel resulted in a 96.7% increase in the tumor uptake of 111In-DTPA-PEG-annexin V and a 55.6% increase in the tumor uptake of 111In-DTPA-PEG-ovalbumin, suggesting that a fraction of the increased tumor uptake of 111In-DTPA-PEG-annexin V after poly(L-glutamic acid)-paclitaxel treatment was due to the nonspecific EPR effect of macromolecules (Table 2). Importantly, only 111In-DTPA-PEG-annexin V (P < 0.005) but not 111In-DTPA-PEG-ovalbumin showed a significant increase in the tumor-to-blood ratio and the tumor-to-muscle ratio between the poly(L-glutamic acid)-paclitaxel–treated group and the nontreated control group (Table 2).
It is interesting to note that C225 treatment alone resulted in a 33% reduction in the tumor uptake of 111In-DTPA-PEG-annexin V 4 d after the initiation of the treatment (Table 1). Similarly, combined poly(L-glutamic acid)-paclitaxel and C225 therapy result in less tumor uptake of 111In-DTPA-PEG-annexin V than poly(L-glutamic acid)-paclitaxel treatment alone. These results are consistent with the finding that C225 is not a potent inducer of apoptosis (15,16). Inoue et al. (16) found that in bladder tumor xenografts, C225 but not paclitaxel significantly reduced microvessel density counts. It is possible that the reduced tumor uptake of 111In-DTPA-PEG-annexin V in mice treated with C225 may reflect reduced blood vessel permeability, which is secondary to downregulation of EGFR, vascular endothelial growth factor receptor, and the expression of other angiogenic factors (16).
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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ACKNOWLEDGMENTS |
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-imaging data and Dr. Tarik Belhocine for helpful discussion. We also thank Stephanie Deming for help in editing the manuscript. This research was supported in part by National Institutes of Health (NIH) grant U54 CA090810 and by the John S. Dunn Foundation. Nuclear magnetic resonance studies and animal experiments were supported by NIH Cancer Center support grant CA16672. |
FOOTNOTES |
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For correspondence or reprints contact: Chun Li, PhD, Division of Diagnostic Imaging, Box 59, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030.
E-mail: cli{at}di.mdacc.tmc.edu
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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