In the current preliminary report, 124I-HuCC49deltaCH2 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, 18F-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 18F-FDG-based PET imaging strategy as compared to an antigen-directed and cancer-specific 124I-HuCC49deltaCH2-based PET imaging strategy.
In the current preliminary report, both i.v. and i.p. administration of 124
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
2. Therefore, the end result of the transport of 124
2 from the peritoneal cavity to the LS174T tumor implants after i.p. administration was similar to the transport of 124
2 from the systemic circulation to LS174T tumor implants after i.v. administration. These results with 124
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
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 cancer-specific 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
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 antigen-expressing adenocarcinomas.
Despite the promising results of our current preliminary report that clearly show that the 124I-radiolabled anti-TAG-72 monoclonal antibody conjugate, 124I-HuCC49deltaCH2, 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 HuCC49deltaCH2 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 , , , and , 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
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
]. 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
2 was metabolized in the body and before the free circulating 124
I was excreted into the urine. Therefore, pre-treatment 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
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
2 was seen on microPET imaging. As best illustrated in Figure , significant nonspecific liver uptake was most pronounced at the time points of 20 hours and 24 hours after i.p. administration of the higher dose (2.5 MBq) of 124
2. This nonspecific liver uptake was less intense on microPET imaging at the time points of 20 hours and 24 hours after i.p. administration of a lower dose (1.4 MBq) of 124
2 (Figure ) and was minimally present on microPET imaging at the time points of 18 hours and 23 hours after i.v. administration of either dose (0.6 MBq or 0.75 MBq) of 124
2 (Figure and Figure ). 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
]. 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
2 seen on microPET imaging is explainable by our use of chelated form of the HuCC49deltaCH
2 antibody. It should be noted that our inadvertent use of the chelated form of the HuCC49deltaCH
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
2. Therefore, use of the non-chelated form of the HuCC49deltaCH
2 antibody would have potentially eliminated the nonspecific liver uptake of 124
2, thus better illustrating our take-home message of specific localization of 124
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 HuCC49deltaCH
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
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
]. 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
]. 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
2. Therefore, the previously described metabolic effects resulting from only a short-duration 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
]. 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.