124I-HuCC49deltaCH2 for TAG-72 antigen-directed positron emission tomography (PET) imaging of LS174T colon adenocarcinoma tumor implants in xenograft mice: preliminary results

Background 18F-fluorodeoxyglucose positron emission tomography (18F-FDG-PET) is widely used in diagnostic cancer imaging. However, the use of 18F-FDG in PET-based imaging is limited by its specificity and sensitivity. In contrast, anti-TAG (tumor associated glycoprotein)-72 monoclonal antibodies are highly specific for binding to a variety of adenocarcinomas, including colorectal cancer. The aim of this preliminary study was to evaluate a complimentary determining region (CDR)-grafted humanized CH2-domain-deleted anti-TAG-72 monoclonal antibody (HuCC49deltaCH2), radiolabeled with iodine-124 (124I), as an antigen-directed and cancer-specific targeting agent for PET-based imaging. Methods HuCC49deltaCH2 was radiolabeled with 124I. Subcutaneous tumor implants of LS174T colon adenocarcinoma cells, which express TAG-72 antigen, were grown on athymic Nu/Nu nude mice as the xenograft model. Intravascular (i.v.) and intraperitoneal (i.p.) administration of 124I-HuCC49deltaCH2 was then evaluated in this xenograft mouse model at various time points from approximately 1 hour to 24 hours after injection using microPET imaging. This was compared to i.v. injection of 18F-FDG in the same xenograft mouse model using microPET imaging at 50 minutes after injection. Results At approximately 1 hour after i.v. injection, 124I-HuCC49deltaCH2 was distributed within the systemic circulation, while at approximately 1 hour after i.p. injection, 124I-HuCC49deltaCH2 was distributed within the peritoneal cavity. At time points from 18 hours to 24 hours after i.v. and i.p. injection, 124I-HuCC49deltaCH2 demonstrated a significantly increased level of specific localization to LS174T tumor implants (p = 0.001) when compared to the 1 hour images. In contrast, approximately 50 minutes after i.v. injection, 18F-FDG failed to demonstrate any increased level of specific localization to a LS174T tumor implant, but showed the propensity toward more nonspecific uptake within the heart, Harderian glands of the bony orbits of the eyes, brown fat of the posterior neck, kidneys, and bladder. Conclusions On microPET imaging, 124I-HuCC49deltaCH2 demonstrates an increased level of specific localization to tumor implants of LS174T colon adenocarcinoma cells in the xenograft mouse model on delayed imaging, while 18F-FDG failed to demonstrate this. The antigen-directed and cancer-specific 124I-radiolabled anti-TAG-72 monoclonal antibody conjugate, 124I-HuCC49deltaCH2, holds future potential for use in human clinical trials for preoperative, intraoperative, and postoperative PET-based imaging strategies, including fused-modality PET-based imaging platforms.

preoperative, intraoperative, and postoperative PET-based imaging strategies, including fused-modality PET-based imaging platforms.

Background
The origin of positron imaging dates back to the early 1950's [1], culminating in the development of positron emission tomography (PET) and its subsequent evolution over the last 40 years [1][2][3][4]. The clinical application of PET-based imaging strategies to the field of oncology has had a significant impact upon the care of cancer patients [5][6][7][8][9][10][11]. Therefore, the development and selection of the most appropriate and specific radiotracer for PET-based imaging is critical to its success in oncology [12][13][14][15]. 18 F-fluorodeoxyglucose ( 18 F-FDG) is currently the most widely used radiotracer for PET-based imaging strategies [16]. In this regard, 18 F-FDG-PET-based imaging is considered state-of-the-art for the diagnostic imaging, staging, and follow-up of a wide variety of malignancies, including colorectal cancer [10,11]. However, there are several intrinsic limitations related to the use of 18 F-FDG-PET imaging that remain a challenge and a concern to those involved in the care of cancer patients [6][7][8][9][16][17][18][19][20][21][22][23][24]. First, false positive results can occur with 18 F-FDG-PET imaging in the presence of any pathologic conditions in which there is a high rate of glucose metabolism, such as inflammatory or infectious processes. Second, false negative results can occur with 18 F-FDG-PET imaging secondary to poor avidity of 18 F-FDG to certain tumor types and secondary to impaired uptake of 18 F-FDG in patients with elevated blood glucose levels. Third, due to system resolution limitations, 18 F-FDG-PET imaging is generally limited in its ability to detect small-volume, early-stage primary disease or to detect microscopic disease within the lymph nodes. Fourth, 18 F-FDG-PET imaging can produce either false positive or false negative results secondary to the normal physiologic accumulation of 18 F-FDG within certain tissues with an elevated level of glucose metabolism (most striking in the brain and heart, and to a lesser degree in the mucosa and smooth muscle of the stomach, small intestine and colon, as well as in liver, spleen, skeletal muscle, thyroid, and brown fat) and secondary to the excretion and accumulation of 18 F-FDG within the urinary tract (kidneys, ureters, and bladder). Overall, these factors have a negative impact on optimizing the specificity and sensitivity of 18 F-FDG-PET for accurate diagnostic cancer imaging [6][7][8][9][16][17][18][19][20][21][22][23][24].
A PET-based imaging approach that specifically targets the cancer cell environment would clearly have a significant potential advantage for improving the accuracy of diagnostic cancer imaging over that of the more nonspecific nature of 18 F-FDG. In that regard, tumorassociated glycoprotein-72 (TAG-72) is a mucin-like glycoprotein complex that is overexpressed by many adenocarcinomas, including colorectal, pancreatic, gastric, esophageal, ovarian, endometrial, breast, prostate, and lung [22,[24][25][26][27]. Such overexpression of TAG-72 is noted in up to approximately 90% of these various adenocarcinomas [24]. In xenograft mice bearing subcutaneous tumor implants of the TAG-72-expressing human colon adenocarcinoma cell line, LS174T [27][28][29], anti-TAG-72 monoclonal antibodies have been shown to accumulate up to 18-fold higher in LS174T tumor implants than in normal tissues [25,30,31]. Over the last 25 years, our group at The Ohio State University, as well as others, have evaluated a variety of radioiodine labeled anti-TAG-72 monoclonal antibodies for tumorspecific antigen targeting at the time of surgery for known primary, recurrent, and metastatic disease, as well as for targeting occult disease and affected lymph nodes in colorectal cancer patients [22,24,. Most recently, we have evaluated the complimentary determining region (CDR)-grafted humanized C H 2domain-deleted anti-TAG-72 monoclonal antibody, HuCC49deltaC H 2 [60][61][62][63], radiolabeled with iodine-125 ( 125 I), for intraoperative tumor detection of colorectal cancer in both a preclinical xenograft mouse model and in a human clinical trial [22,24,[57][58][59]. Collectively, our experience with radiolabeled anti-TAG-72 monoclonal antibodies in combination with a handheld gamma detection probe has clearly shown that this technology provides the surgeon with real-time intraoperative information for more precise tumor localization and resection and has demonstrated improved long-term patient survival after surgery [22,24].
Because of the drawbacks of using 125 I as the radioiodine label for anti-TAG-72 monoclonal antibodies, including the extremely long physical half-life of 125 I of approximately 60 days (which generates handling, storage, and disposal issues within the operating room environment and in the surgical pathology department) and the inability of 125 I to allow for diagnostic imaging capabilities, other radionuclides have been sought for use with anti-TAG-72 monoclonal antibodies. One such alternative is iodine-124 ( 124 I) [64]. In this regard, 124 I is a positron emitting radionuclide that has a physical halflife of approximately 4.2 days, for which its positron emitting properties makes it well-suited for PET-based imaging and for which its shorter physical half-life simplifies the handling, storage, and disposal issues. Therefore, the aim of this preliminary study was to evaluate 124 I-HuCC49deltaC H 2 as an antigen-directed and cancer-specific targeting agent for PET-based imaging.

Tissue culture and reagents
Cell culture medium (DMEM), fetal bovine serum (FBS), trypsin, and other tissue culture materials were purchased from Invitrogen (Carlsbad, California).
DOTA chelated HuCC49deltaC H 2 antibody was supplied by Dr. Jeffrey Schlom (Laboratory of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD).

Anion Exchange Resin Filters
100-to 200-mesh AG1X8 anion exchange resin (Bio-Rad Labs, Richmond, CA) was washed using sterile, pyrogen free water. The anion exchange resin was aseptically loaded onto a 0.22 μm filter disc (1.1 to 1.5 gram wet resin/filter unit) (Millipore Corporation, Milford, MA). The resin was washed using the following solutions in a sequence: 10 ml sterile, pyrogen-free water; 10 ml sterile 0.1 N NaOH; 10 ml pyrogen free water; 10 ml 0.1 N sodium phosphate buffer (pH 7.4); and finally by 3.3 ml 0.1 N sodium phosphate buffer with 1% HSA.
Labeling process [65,66] 0.50 ml of HuCC49deltaC H 2 antibody (1.5 mg/ml) was added to a 10 ml vial coated with 1 mg of iodogen. Then, 0.8 ml of phosphate buffered Na 124 I (150 MBq/ ml) (IBA Molecular, Dulles, VA) was added to the vial. The reagents were allowed to react for 15 minutes. Free 124 I was removed using an exchange resin filter disc. Then, 1 ml of 5% sucrose with 0.05% Tween 20 in saline was used to elute the labeled antibody. The purified 124 I-HuCC49deltaC H 2 was passed through a 0.22 mm Millipore filter (Millipore Corporation, Milford, MA) for in vivo applications. Radiolabeling efficiency was monitored using thin layer chromatography, which was performed on silica-gel-impregnated glass fiber sheets (Pall Corporation, East Hills, NY). 0.02 M citrate buffer (pH 5.0) was used as the mobile phase.

Xenograft mouse model with human colon adenocarcinoma cells (LS174T)
The human colon adenocarcinoma cells, LS174T, were trypsinized for 2 minutes, collected, and washed with PBS under 1000 rpm × 2 minutes. The washed cells (5×10 6 cells) were resuspended in a mixture of 50 μl of PBS and 50 μl of matrigel medium (Invitrogen, Carlsbad, California) and then injected subcutaneously into the dorsal surface (back) of female athymic Nu/Nu nude mice (National Cancer Institute at Frederick, Frederick, MD) that were 4 to 6 weeks of age. The resultant LS174T tumor implants on the xenograft mice were allowed to grow for approximately two weeks, reaching a tumor implant volume of up to 300 mm 3 . The xenograft mice used in this preliminary study were not pretreated with an oral saturated solution of potassium iodide (SSKI).

I-HuCC49deltaC H 2 and 18 F-FDG injections of the xenograft mice
Two xenograft mice were successfully injected intravenously (i.v.), by way of tail vein injection, with 124 I -HuCC49deltaC H 2, at a dose of 0.6 MBq and 0.75 MBq, respectively. Two additional xenograft mice were successfully injected intraperitoneally (i.p.) with 124 I-HuCC49del-taC H 2, at a dose of 1.4 MBq and 2.5 MBq, respectively. As a control, one xenograft mouse was successfully injected i. v., by way of tail vein injection, with 7.4 MBq of 18 F-FDG. Pre-injection and post-injection blood glucose levels were not monitored in the xenograft mice.

MicroPET tumor imaging of the xenograft mice
Selection of microPET imaging time points was based on historical data as well as the physical half-lives of 18 F (110 minutes) and 124 I (4.2 days). For 18 F-FDG, the standard accepted injection to scan time for humans and small animals is approximately 60 ± 10 minutes [68][69][70]. For 124 I-HuCC49deltaC H 2 injected xenograft mice, an initial 1 hour time point for baseline microPET imaging was used, as well as a time range of delayed microPET imaging from 18 hours to 24 hours after administration of 124 I-HuCC49deltaC H 2 to allow for distribution, uptake, and clearance. At selected time points (ranging from approximately 1 hour to 24 hours after injection of 124 I-HuCC49deltaC H 2), the xenograft mice were anesthetized with i.p. Ketamine (100 mg/kg)/Xylazine (10 mg/kg) and then scanned on an Inveon microPET scanner (Siemens Medical Solutions, Knoxville, TN). Image acquisition and analysis were performed by using Inveon Acquisition Workplace (Siemens Medical Solutions, Knoxville, TN). Xenograft mice initially underwent a transmission scan with a cobalt-57 source for 402 seconds for attenuation correction and quantification. Xenograft mice then underwent a PET emission scan at approximately 1 hour, 18 hours, and 20 hours after injection of 124 I-HuCC49deltaC H 2 with an acquisition time of 400 seconds and again at approximately 23 hours or 24 hours after injection of 124 I-HuCC49del-taC H 2 with an acquisition time of 800 seconds. For the 18 F-FDG injected xenograft mouse, a PET emission scan was obtained at approximately 50 minutes after injection of 18 F-FDG with an acquisition time of 400 seconds. The energy window of all PET emission scans was set to 350 keV to 650 keV, with a time resolution of 3.4 ns. Each emission acquisition data set was attenuation corrected with the attenuation transmission scan taken of each individual mouse at each designated time point and arranged into sinograms. The resultant sinograms were iteratively reconstructed into three dimensional volumes using an ordered-subset expectation maximization (OSEM) reconstruction algorithm. The transmission acquisition yielded an approximation of body volume and anatomic localization, such that regions of interest could be created to represent portions of the mouse anatomy, specifically, whole body, the LS174T tumor implant, and a designated background area (i.e., left lower quadrant of the abdomen).
The region of interest (ROI), for determination of tumor implant volume, was drawn manually by qualitative assessment to cover the entire tumor implant volume by summation of voxels using the Inveon software (Siemens Medical Solutions, Knoxville, TN) in a manner similar to that previously published by Jensen et al. [70]. In the study by Jensen et al., they compared the accuracy of xenograft measurement by in vivo caliper measurement versus microCT-based and microPETbased measurement and found microCT to be the most accurate measurement method [70]. We used a similar method in conjunction with the transmission image to generate the tumor implant volume. PET activity within the volumetric ROI then yielded the resultant average intensity counts for the tumor implant and for the designated background area. Finally, to generate a quantification measurement value for the activity of 124 I-HuCC49deltaC H 2 and of 18 F-FDG that was imaged on microPET within a given LS174T tumor implant, we utilized the unitless value of the relative ratio of the average intensity counts. This relative ratio of the average intensity counts was determined by dividing the average intensity counts from the tumor implant volume by the average intensity counts of the designated background area. We elected to generate this relative ratio of the average intensity counts as a quantification measurement value due to the fact that mouse body weights and tumor implant weights were not recorded and microCT was not obtained on all of the xenograft mice during the course of the current preliminary study.

Statistical analysis
The software program IBM SPSS® 18 for Windows® (SPSS, Inc., Chicago, Illinois) was used for the data analysis. One-way analysis of variance (ANOVA) was utilized for the comparison of the relative ratio of average intensity counts of the LS174T tumor implants.

Results
After chromatographic purification, 98% of 124 I was bound to the chelated HuCC49deltaC H 2 antibody, as determined by thin layer chromatography. The radioactivity of 124 I-HuCC49deltaC H 2 obtained was 15 MBq/ml.  (Figure 4). This was presumed to be secondary to use of the chelated form of the HuCC49deltaC H 2 antibody. Figure 5 shows the xenograft mouse injected i.v. with 7.4 MBq of 18 F-FDG and imaged by the microPET at approximately 50 minutes after injection. Multiple sites of tumor-nonspecific 18 F-FDG accumulation were noted in the xenograft mouse. 18 F-FDG was noted to avidly accumulate in the heart, the brown fat of the posterior neck, and the Harderian glands within the bony orbits of the eyes, all secondary to the high rate glucose metabolism within these tissues. 18 F-FDG was noted to be rapidly eliminated from kidneys and bladder within 50 minutes after the i.v. injection. Only very minimal localization of 18 F-FDG to the LS174T tumor implant was noted in the xenograft mouse model.
To generate a quantification measurement value for the activity of 124 I-HuCC49deltaC H 2 and of 18 F-FDG that was imaged on microPET within a given LS174T tumor implant, we utilized the unitless value of the relative ratio of the average intensity counts, as determined by dividing the average intensity counts of the LS174T tumor implant by the average intensity counts of the designated background area. For comparing the localization of 124 I-HuCC49deltaC H 2 within the LS174T tumor implants at approximately 1 hour after i.v. and i.p. injection versus at 18 hours to 24 hours after i.v. and i.p. injection, the mean relative ratio of the average intensity counts was determined to be 0.34 (SD ± 0.29, range implants at differing doses of 124 I-HuCC49deltaC H 2, the mean relative ratio of the average intensity counts was determined to be 2.03 (SD ± 0.14, range 1.93 to 2.13, n = 2) at the lowest dose administered (i.e., 0.6 MBq i.v.) as compared to 3.70 (SD ± 1.23, range 2.84 to 4.57, n = 2) at the highest dose administered (i.e., 2.5 MBq i. p.) (p = 0.195). Finally, for comparing the localization of 124 I-HuCC49deltaC H 2 versus 18 F-FDG within the LS174T tumor implants, the mean relative ratio of the average intensity counts was 2.58 (SD ± 0.99, range 1.57 to 4.57, n = 8) for 124 I-HuCC49deltaC H 2 at 18, 20, 23, and 24 hours after i.v. and i.p. injection as compared to 1.05 (n = 1) for 18 F-FDG at approximately 50 minutes after i.v. injection (p = 0.188). Although this demonstrates that there was 2.46 times greater localization of 124 I-HuCC49deltaC H 2 within the LS174T tumor implants as compared to 18 F-FDG, this particular p-value did not reach statistical significance, and this is likely attributable to the statistic restraints of comparing

Discussion
In the current preliminary report, 124 I-HuCC49deltaC H 2 demonstrated a significantly increased level of specific localization to LS174T tumor implants as compared to background tissues (p = 0.001) in the xenograft mouse model at 18 hours to 24 hours after injection as compared to at approximately 1 hour after injection. In contrast, in the same xenograft mouse model, 18 F-FDG failed to demonstrate any increased level of specific localization to a LS174T tumor implant as compared to background tissues at approximately 50 minutes after injection. These findings, although based on a limited number of xenograft mice, re-enforce the recognized limitations of an 18 F-FDG-based PET imaging strategy as compared to an antigen-directed and cancer-specific 124 I-HuCC49deltaC H 2-based PET imaging strategy.
In the current preliminary report, both i.v. and i.p. administration of 124 I-HuCC49deltaC H 2 resulted in specific localization on microPET imaging to the LS174T tumor implants in the xenograft mouse model at 18 and 23 hours and at 20 and 24 hours after injection, respectively, validating the use of both injection routes for use in preclinical animal studies evaluating 124 I-HuCC49del-taC H 2. Therefore, the end result of the transport of 124 I-HuCC49deltaC H 2 from the peritoneal cavity to the LS174T tumor implants after i.p. administration was similar to the transport of 124 I-HuCC49deltaC H 2 from the systemic circulation to LS174T tumor implants after i.v. administration. These results with 124 I-HuCC49del-taC H 2 are consistent with previous studies which have demonstrated the efficacy of i.p. administered anti-TAG-72 monoclonal antibodies in patients with colorectal cancer [71,72].
Overall, these preliminary results in the LS174T colon adenocarcinoma xenograft mouse model are very encouraging and lay the ground work for further investigations into the use of this antigen-directed and cancerspecific 124 I-radiolabeled anti-TAG-72 monoclonal antibody conjugate in human clinical trials related to preoperative, intraoperative, and postoperative PET-based imaging strategies [73]. Such an approach that utilizes PET-based imaging in conjunction with 124 I-HuCC49del-taC H 2 is clinically feasible and could potentially have a significant impact upon the current management of colorectal cancer, as well as upon other TAG-72 antigenexpressing adenocarcinomas.
Despite the promising results of our current preliminary report that clearly show that the 124 I-radiolabled anti-TAG-72 monoclonal antibody conjugate, 124 I-HuCC49deltaC H 2, shows high degree of specific localization to TAG-72 antigen expressing tumor implants in the xenograft mouse model, there are several shortcomings of our current experimental study design which led to non-optimization of our reported results and that will need to be further addressed in future experiments. These shortcomings are the small sample size, the lack of thyroid block by oral administration of SSKI, the use of the chelated form of the HuCC49deltaC H 2 antibody, and the anesthetic and time constraints at the time of these preliminary experiments that did not allow for obtaining fused microPET/CT imaging of all the xenograft mice studied.
First, as is shown in Figures 1, 2, 3, and 4, significant thyroid uptake was seen on microPET imaging at the time points of 18 hours and 23 hours after i.v. injection and at the time points of 20 hours and 24 hours after i. p. injection of 124 I-HuCC49deltaC H 2. It has long been well-known in the nuclear medicine literature that if the thyroid is not blocked by the oral administration of SSKI, then resultant thyroid uptake of circulating radioactive iodine will freely occur [74][75][76]. This has been previously experimentally evaluated with radioiodine labeled anti-TAG-72 monoclonal antibodies [77]. As such, in the current animal experiments, the lack of thyroid blockade resulted in significant thyroid uptake of free 124 I as the unbound 124 I-HuCC49deltaC H 2 was metabolized in the body and before the free circulating 124 I was excreted into the urine. Therefore, pretreatment of the xenograft mice with oral administration of SSKI to minimize thyroid uptake of free 124 I would have resulted in more optimal microPET imaging, thus better illustrating our take-home message of specific localization of 124 I-HuCC49deltaC H 2 to LS174T tumor implants by minimizing the degree of thyroid localization of free 124 I. This shortcoming was an oversight on our part and will be subsequently re-addressed in future xenograft mouse model experiments in which the xenograft mice are pretreated with oral SSKI.
Second, nonspecific liver uptake of 124 I-HuCC49del-taC H 2 was seen on microPET imaging. As best illustrated in Figure Figure 2). A similar pattern of accumulation within the liver has been previously reported for various chelated radiolabeled CC49 monoclonal antibodies [78], as well as for a single-chain Fv version of the radiolabeled CC49 monoclonal antibody [79]. It has been suggested that the high accumulation of these radiolabeled monoclonal antibody in the liver is likely due to the metabolism of the chelated form of the antibody within the liver [78]. Clearance and metabolism of IgG antibodies occurs predominantly through the reticuloendothelial system (RES), primarily in the liver and spleen, which both contain Kupffer cells [78,79]. Furthermore, IgG antibodies are bound and internalized by asialoglycoprotein receptors in the liver cells, increasing the retention of IgG antibodies within the liver. Therefore, it is our contention that the nonspecific liver uptake of 124 I-HuCC49deltaC H 2 seen on microPET imaging is explainable by our use of chelated form of the HuCC49deltaC H 2 antibody. It should be noted that our inadvertent use of the chelated form of the HuCC49del-taC H 2 antibody was not recognized until after analysis of the microPET imaging, as is best exemplified at the time points of 20 hours and 24 hours after i.p. administration of 2.5 MBq of 124 I-HuCC49deltaC H 2. Therefore, use of the non-chelated form of the HuCC49deltaC H 2 antibody would have potentially eliminated the nonspecific liver uptake of 124 I-HuCC49deltaC H 2, thus better illustrating our take-home message of specific localization of 124 I-HuCC49deltaC H 2 to LS174T tumor implants. This shortcoming was an oversight on our part and will be subsequently re-addressed in future xenograft mouse model experiments in which the non-chelated form of the HuCC49deltaC H 2 antibody is utilized.
Third, at the time of this preliminary animal experiment, due to limitations in the type of anesthetic available (i.e., only i.p. Ketamine/Xylazine was available and inhalation isoflurane anesthesia was not available), due to the time constraints necessary for repetitive scanning in both a microPET and a microCT format, and due to the limited number of xenograft mice available, fused microPET/CT imaging was only obtained on one of the five xenograft mice. Therefore, while all five xenograft mice were imaged by the dedicated microPET scanner, only one xenograft mouse (i.v. injection of 124 I-HuCC49deltaC H 2 at a dose of 0.6 MBq) was also imaged with the microCT scanner at the time point of 24 hours after i.v. injection, thus allowing for reconstruction of fused microPET/CT images. In this particular case of fused microPET/CT imaging, the microCT images demonstrated relatively good correlation of anatomy with the transmission images and assisted in the accurate determination of tumor implant volume from the transmission scan. It is evident within the molecular imaging literature that fused-modality PET-based imaging is superior to PET alone-based imaging, both for the PET/CT platform and for the PET/MRI platform [73,[80][81][82][83]. These fused imaging platforms can provide both molecular/functional information and structural information that can more accurately and more precisely localize various disease processes. It is our intention to subsequently re-address this shortcoming in future xenograft mouse model experiments by utilizing a fused microPET/CT imaging platform in all of the xenograft mice.
As a last notable point of discussion, some may contend that the lack of specific localization of 18 F-FDG to the LS174T tumor implant as compared to the background tissues was the specific result of the type of anesthetic used for the Nu/Nu nude mice in the current preliminary study (i.e., i.p. Ketamine/Xylazine instead of inhalation isoflurane anesthesia). It has been previously reported that C57BL/6 mice injected with 18 F-FDG and having received Ketamine/Xylazine anesthesia demonstrate increased blood glucose levels, as well as increased 18 F-FDG activity within multiple normal tissues, such as in muscle, lung, liver, kidney, and blood, as compared to C57BL/6 mice injected with 18 F-FDG that received no anesthesia [84,85]. It has been suggested by some authors that these metabolic effects are mediated through the inhibition of insulin release, and that such effects are most prominent in mice kept fasting for only 4 hours, but are substantially attenuated by 20 hours of fasting [84]. In our preliminary animal experiments, the xenograft mice were kept without food for approximately 14 hours prior to the injection of 18 F-FDG and 124 I-HuCC49deltaC H 2. Therefore, the previously described metabolic effects resulting from only a shortduration fast should have been minimized. Furthermore, these same authors reported that Ketamine/Xylazine anesthesia did not significantly alter 18 F-FDG activity within Lewis lung carcinoma (LLC) subcutaneous tumor implants on C57BL/6 mice as compared to the same scenerio with no anesthesia [84]. In contrast to Ketamine/Xylazine, low dose (0.5%) inhalation isoflurane anesthesia has been reported to resulted in no significant increase in 18 F-FDG activity within normal tissues (i.e., muscle, lung, liver, and kidney) of C57BL/6 mice as compared to the same scenerio with no anesthesia [84,85]. These findings indirectly suggest that the use of low dose (0.5%) inhalation isoflurane anesthesia for the Nu/Nu nude mice in our current preliminary study could have potentially provided a means to eliminate any negative impact of the choice of anesthetic on the absolute level of 18 F-FDG activity within the LS174T tumor implant and the various normal tissues. Based upon these findings, it is our plan to use inhalation isoflurane anesthesia in our future proposed animal studies in order to minimize the occurence of any such issues.

Conclusions
On microPET imaging, 124 I-HuCC49deltaC H 2 demonstrates an increased level of specific localization to tumor implants of LS174T colon adenocarcinoma cells as compared to background tissues in the xenograft mouse model, while 18 F-FDG failed to demonstrate this same finding. Clearly, a PET-based imaging approach that utilizes 124 I-HuCC49deltaC H 2 is feasible and could potentially have a significant impact upon the current management of colorectal cancer and other TAG-72 antigen-expressing adenocarcinomas. This antigen-directed and cancer-specific 124 I-radiolabled anti-TAG-72 monoclonal antibody conjugate holds future potential for use in human clinical trials for preoperative, intraoperative, and postoperative PET-based imaging strategies, including fused-modality PET-based imaging platforms.