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Experimental Therapeutics |
Mallinckrodt Institute of Radiology [J. S. L., M. J. W.], and the Department of Surgery [J. M. C., T. L. B., J. W. F.], Washington University School of Medicine, St. Louis, Missouri 63110; The Department of Chemistry, Washington University, St. Louis, Missouri 63110 [J. R. G.]; and Biomedical Imaging Research Center, Fukui Medical University, Matsuoka, Fukui 910-1193, Japan [Y. F.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3.2 x 104 cells in 0.5 ml) were injected into the peritoneum through a midline incision. Ten min later, hamsters were randomized to receive 5, 3, or 1 mCi of 64Cu-PTSM through the same midline incision. High-resolution magnetic resonance imaging and microPET were used to monitor tumor volume and morphology after surgery. After 7 weeks, animals were sacrificed, and trocar and midline wounds were harvested for macroscopic and histological analysis. No macroscopic tumor was found in any of the group treated with 5 mCi of 64Cu-PTSM, whereas 96% of the wound sites in the group treated with saline had macroscopic tumor growth (P < 0.001). This study demonstrates the therapeutic potential of 64Cu-PTSM in inhibiting cancer cell implantation and growth at doses well below the maximum tolerated dose, with no signs of toxicity to the hamsters. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Laparoscopic colectomy is the process of resecting portions of the colon, using trocars, video laparoscopy, and carbon dioxide pneumoperitoneum to minimize abdominal wall access. The use of laparoscopic colectomy for curable colon cancer remains controversial because of the documentation of metastasis at the incision sites (10 , 11) . Since 1993, there have been a number of case reports of tumor recurrence at trocar sites following laparoscopic colon resections (12 , 13) . The development of abdominal wall implants may be attributable to bonafide disseminated disease or to tumor cells disseminated by surgical manipulation and the favorable environment of the wound site for cell implantation. At Washington University Medical School, a hamster model of colorectal cancer has been developed that mimics the implantation of cancer cells following invasive surgery (14 , 15) . In this model, injection of disaggregated human GW39 colon cancer cells into the hamster peritoneum represents a model of tumor spillage that may occur during dissection, manipulation, resection, and extraction of tumor during an operation.
The present work is based on the hypothesis that therapeutic doses of 64Cu-PTSM could ablate any loose tumor cells within the abdomen. This idea is based on the evidence obtained in vitro that uptake of Cu-PTSM in single-cell suspensions is rapid and quantitative as described above. The present investigation was performed to test this hypothesis by measuring the ability of 64Cu-PTSM to inhibit the implantation of loose cancer cells in wound sites following laparoscopic surgery.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animal Models.
All animal experiments were conducted in compliance with the Guidelines for the Care and Use of Research Animals established by Washington University’s Animal Studies Committee. Biodistribution data in nontumor-bearing hamsters were obtained by administering 64Cu-PTSM (10 µCi) i.p. to 100-g golden Syrian hamsters (Sasco Inc., Omaha, NE). The hamsters (n = 5 each group) were euthanized at 5, 30, 120, and 240 min postinjection. Selected tissues and organs were harvested, and weighed, and the activity was counted on a gamma counter. The percentage of injected dose per gram and percentage of injected dose per organ for each tissue were calculated. The laparoscopic model was performed according to previously reported methods (14
, 15)
. Briefly, anesthetized golden Syrian hamsters (100 g) had four 5-mm trocars inserted through the anterior abdominal wall; GW39 cells (3.2 x 104 cells in 0.5 ml of PBS) were then injected into the peritoneum through a 2-cm midline incision. Trocars were then removed, and the trocar wounds were closed. Viability of cells was confirmed to be 98% at the time of injection with Trypan vital blue staining.
Radiotherapy Experiments.
Following laparoscopic surgery as described above, hamsters (n 
7 in each group) were randomized to receive saline or 1, 3, or 5 mCi of 64Cu-PTSM. Radioactivity was given within 10 min of surgery through the sutured midline incision. The hamsters were provided water and hamster chow immediately after the operation and inspected daily for evidence of complications. Seven weeks after surgery and radiotherapy, animals were sacrificed, and trocar and midline wounds were examined for macroscopic tumor. Wound sites without gross tumor were excised, fresh frozen, and examined histologically for the presence of microscopic tumor implants. The tumors were examined histologically ex vivo, using H&E and Mucin staining techniques (15)
. The Diagnostic Services Laboratory in the Department of Comparative Medicine at Washington University School of Medicine performed toxicity analysis on select animals from the 5 mCi-treated animals. The hematology analysis included hemoglobin, WBC counts, RBC counts, platelet counts, hematocrit, and differential WBCs. Liver and kidney analyses included blood urea nitrogen, creatinine, alanine aminotransferase, and aspartate aminotransferase.
Imaging.
High resolution MRI and microPET (Concorde Microsystems; Knoxville, TN) were used to monitor tumor volume and morphology after surgery. Hamsters were imaged weekly in an Oxford Instruments 200/330 (4.7 tesla; 33-cm clear bore) magnet equipped with a 16 cm (i.d.) actively shielded gradient coil (maximum gradient, 18 G/cm; rise time; 400 µs). The magnet and gradient coil were interfaced to a Varian UNITY-INOVA console. Magnetic resonance images were collected weekly following laparoscopic surgery, and tumor growth was measured by manually segmenting individual slices in each image and calculating volumes using Varian’s Image Browser software. To confirm the presence of established GW39 tumors in control animals, 2 weeks after the initial surgery, select animals were injected with tracer amounts of 64Cu-PTSM (200–250 µCi) and imaged on the microPET 2-h postinjection. One week later, these same select hamsters were injected with tracer amounts of 64Cu-TETA-1A3 (Refs. 3
, 7
; 200–250 µCi) and imaged 2-h postinjection. An additional group of control hamsters was also imaged with 18F-FDG on the microPET at 3, 5, and 7 weeks post surgery. In each imaging session, fasted hamsters received injections containing 1 mCi of 18F-FDG and were imaged at 1 h postinjection. A third group of hamsters that underwent the same laparoscopic operation (trocar and midline incision), but did not receive GW39 cells, underwent both 18F-FDG-microPET and 64Cu-PTSM-microPET as controls. Tumor volume from the microPET images was determined using AnalyzeAVW 3.0 (Biomedical Imaging Resource, Rochester, MN).
Statistical Analysis.
To determine statistical significance, a Student’s t test was performed and a 95% confidence level assumed, with P < 0.05 being considered significantly different. 
2 or Fisher’s exact tests were used to compare proportions and frequency data.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. 64Cu-PTSM exits the peritoneum to enter the blood stream within 5 min to reach a maximum blood level at 120 min postinjection. 64Cu-PTSM is then seen to distribute to all of the organs harvested, including the heart, brain, and lung, again reaching maximum uptake values at 120 min postinjection. 64Cu-PTSM is cleared through the liver and kidney. Liver uptake is followed by uptake in the intestines prior to excretion. The uptake observed in the intestines, however, could also be attributed to uptake by the intact serosa of the bowel and intact peritoneum directly following the i.p. injection of radioactivity and not from the normal excretory pathways. It is important to note that the systemic administration of 64Cu-PTSM results in a much more rapid distribution of the tracer, with peak values being reached more rapidly and larger amounts of radioactivity being taken up by the organs examined (8
, 9)
.
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. After treatment with 1 mCi of 64Cu-PTSM, 17.8% inhibition of tumor implantation inhibition was noted, compared with 66.8% inhibition when 3 mCi was administered. In hamsters treated with 5 mCi of 64Cu-PTSM, there was no evidence of macroscopic tumor in any of the animals. Conversely, in the saline control animals (n = 10), all of the wound sites (n = 35) had macroscopic tumor growth (P < 0.001). When examined microscopically, the animals treated with 5 mCi exhibited only 2% tumor implantation in the total possible wound sites. The doses administered to achieve inhibition did not produce any signs of toxicity in the animals: the mean weight of the treated hamsters increased similarly to that of the control hamsters, and they maintained a healthy physical appearance (with no sign of scruffy coat or diarrhea) over the experimental period. There were no significant changes in blood chemistry levels. Animals receiving H2-PTSM or nonradioactive Cu-PTSM in amounts equivalent to those in the radioactive studies had survival rate identical to that of the vehicle controls (data not shown).
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. The images demonstrate that the regional uptake of both agents in the hamster’s abdomen are in very good agreement, despite the different specificities of the agents. The size, number, and location of tumors visualized in these microPET studies matched well with subsequent histological analysis performed 7 weeks postsurgery (i.e., in each animal imaged, the regional uptake of the 64Cu-labeled agents as visualized by microPET corresponded to abnormal mass observed by MRI, which was subsequently confirmed by histology to be GW39 tumor). The uptake of 18F-FDG in the abdomen of hamsters as monitored by microPET was localized in tumors (Fig. 1B)
as demonstrated by examining the magnetic resonance images and, subsequently, the animals at the time of sacrifice (Table 3)
. On examination of the 18F-FDG coronal images, the visualized tumors were located at the points of trocar placement and midline incision, confirming the growth of tumor at the points of surgical manipulation (Fig. 1C)
. In the control animals that underwent surgery but were not given GW39 cells, no detectable activity had accumulated in the incision sites with 18F-FDG-microPET or 64Cu-PTSM-microPET at the same imaging time points.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cu-PTSM belongs to a group of compounds that can be classed as either nonhypoxia-selective (e.g., Cu-PTSM) or hypoxia selective (e.g., Cu-ATSM; Refs. 9 , 18, 19, 20, 21, 22, 23, 24 ). 62Cu-PTSM has been evaluated clinically as a radiopharmaceutical for myocardial and cerebral perfusion imaging with PET (23) . The rapid uptake and kinetics demonstrated by 64Cu-PTSM in cultured cell suspensions (9) make this an ideal candidate for the studies described herein. In the present study, 64Cu-PTSM was investigated as a potential radiotherapy agent for the ablation of loose cells created by surgical manipulation or in disseminated disease. Because of the nonspecific uptake mechanisms of the agent, it is reasonable to assume that 64Cu-PTSM would not only discriminate between injected viable cells but also between separated tumor cells, viable cells such as i.p. macrophages, or cells that have been liberated from a primary tumor.
The biodistribution data presented in this report demonstrate that although 64Cu-PTSM does exit from the intact peritoneum, it does so at a much slower rate than that following systemic administration of the agent (9) . The levels of radioactivity in the tissues examined after administration of 64Cu-PTSM are relatively low, suggesting retention of the radioactivity in the peritoneum, which allows easy accessibility of the agent to the loose tumor cells introduced earlier into the cavity in this animal model.
The therapy results clearly demonstrated the ability of 64Cu-PTSM to inhibit the implantation and formation of tumors. The delivery of the radiotherapy agent was performed 10 min after the inoculation of cells, a time point that may not be clinically realistic; thus, additional studies may be required to determine the most efficient time of administration postsurgery. Following the administration of 5 mCi of 64Cu-PTSM, there was no macroscopic evidence of tumor at any wound site after 7 weeks. Furthermore, only 2% of the possible sites showed the presence of microscopic tumors. This inhibition was achieved in a dose-dependent manner that did not produce overt signs of toxicity in the animals. Therefore, the toxicity data indicate that the MTD for this compound was not achieved and that larger quantities of radioactivity could be administered safely. In the 64Cu-ATSM study with solid GW39 tumors, animals that received 10 mCi of 64Cu-ATSM i.v. displayed a transient depression in WBC counts, platelets, and liver enzyme levels, but no significant changes in the total protein, hemoglobin, RBC, and kidney enzyme levels (8) . It is therefore not surprising that the administration of 5 mCi of 64Cu-PTSM i.p. produced significantly lower toxicity than the systemic administration of 10 mCi of 64Cu-ATSM. Previous studies showed that targeted radiotherapy with 131I-labeled MAb 1A3 (1 mCi) was well tolerated in the hamster and showed a 47% inhibition of tumor growth in the GW39 laparoscopic model.4 64Cu compares well with the 131I data: 5 mCi of the nonspecific 64Cu-PTSM produced 98% inhibition of tumor growth. Prior to human use, we will determine the MTD of 64Cu-PTSM in an animal model in which peritoneal or retroperitoneal dissection procedures may yield a more realistic MTD value.
The efficient therapeutic kill noted in this study can best be explained by the fact that in subcellular fractionation studies, a significant portion of the 64Cu-PTSM was delivered to the cell nucleus following uptake (>20% after 24 h; Refs. 25
, 26
). 64Cu emits a 0.58-MeV ß- particle (40%), a 0.66-MeV ß+ particle (19%), and a 
of 1.34 MeV (0.5%), giving a mean range of penetrating radiation of <1 mm in tissue. During decay by electron capture, the copper radionuclide emits highly radiotoxic Auger electrons with high linear energy transfer that have a tissue penetration of 0.02–10 µm, the approximate cell nucleus diameter. Therefore, the Auger electrons would be very toxic if the DNA of the cell is within range (27)
. Because of their short range and relatively larger linear energy transfer, low-energy Auger electrons potentially are more radiotoxic than the higher energy positron or ß- particles. Additionally, 64Cu has a maximum recoil energy resulting from the nuclear transmutation of the copper ion (from ß- = 7.6 eV; from ß+ = 9.15 eV; Ref. 28
) to its highly charged daughter nucleus, which may also increase the cell-killing ability. Copper ions have also been implicated in the maintenance of the nuclear matrix and in DNA folding (29)
. It is also known that the treatment of isolated nuclei with low levels of Cu(II) causes nuclear matrix-associated DNA binding and DNA-protein cross-linking as well as DNA double-strand breaks following irradiation (30)
. The combination of these characteristics may help explain 64Cu toxicity. It is important to note that because low-energy Auger electrons deposit their energy in a very small volume and the 64Cu is likely very close to DNA, conventional macroscopic dose calculations would most likely underestimate the energy imparted and thus the absorbed dose. A microdosimetric approach to future tumor/tumor cell dose calculations that accounts for all Auger electrons could raise estimates and relate more closely to the observed tumor cell kill.
The uptake of 64Cu-PTSM and 64Cu-TETA-MAb-1A3 in the abdomens of hamsters as monitored by microPET was latter confirmed by histology to localize in established GW39 tumors. This is of particular importance when attempting to monitor the biokinetics of the 64Cu agents and for calculating the absorbed doses delivered by a therapeutic dose of 64Cu-labeled radiopharmaceuticals, as were previously shown possible in studies with solid GW39 tumor (8)
. It is, however, important to note that the biokinetics of 64Cu-PTSM are dependent on blood flow. Despite the fact the edges of the tumors are likely to be well vascularized, areas of decreased blood perfusion in a metastasis would have decreased tracer retention, perhaps leading to inaccurate estimation of tumor volume with microPET. Moreover, for imaging purposes in this particular study, the use of 64Cu agents to monitor tumor growth and response may not be appropriate: repeated administration of small amounts of 64Cu for microPET imaging may lead to tumor regression, leading to an inaccurate assessment of therapeutic efficacy of the drug under investigation. Therefore, in this study, the use of the nontherapeutic 18F-FDG was explored to monitor the biochemical responses of the tumors to treatment. Historically, the assessment of tumor geometry and volume has been by the use of caliper measurements. Not only is this mode of measurement limited by the tumor’s irregular shape, it does not yield any physiological information during radiotherapy experiments. In the present study, MRI showed the presence GW39 tumors in trocar sites in the abdomen of animals and allowed for more accurate determination of tumor volume compared with the microPET results (Table 3)
. The use of microPET for volume determination, although accurate for small tumor masses, displayed large discrepancies with larger tumor volumes, presumably as a result of extensive tumor necrosis not visualized by 18F-FDG-microPET (Table 3)
. The use of MRI for volume determination allows for the inclusion of all tumor tissue, including necrotic, that may not otherwise be visualized with the radiopharmaceuticals used in this study.
MRI and microPET experiments using 18F-FDG, demonstrated that the abdominal tumor could be easily detected and that growth could be monitored (Fig. 1B)
. Furthermore, 18F-FDG imaging confirmed the growth of tumors at the sites of trocar placement and midline incision (Fig. 1C)
. The use of histology further confirmed the presence of tumors, but most importantly identified the presence of microscopic tumors that could not be delineated with the imaging techniques. MicroPET images in conjunction with MRI imaging yielded information and data not normally available with the use of caliper measurements. These results indicate that microPET and MRI can help determine overall treatment effectiveness and monitor therapeutic response and, as such, form a powerful combination of imaging modalities that will find broad application in the characterization of disease states and the development of therapeutics.
Radiotherapeutic effectiveness depends on radioligand delivery to and subsequent accumulation within the cell. Unlike many other radiopharmaceuticals that rely on the high accessibility and up-regulation of antigens, 64Cu-PTSM is a nonreceptor-based agent. Therapeutic quantities of 64Cu-PTSM inhibited GW39 implantation in wound sites with no acute toxicity. In the laparoscopic study, the administration of 64Cu-PTSM into the abdomen produced rapid uptake and ablation of loose tumor cells, resulting in significant reduction of tumor implantation at wound sites. Additional work is required to study the effects of different levels of tumor burden, timing effects, and the quantification of normal tissue dosimetry. This was a proof-of-principle study to examine the use of 64Cu-PTSM treatment as an adjuvant therapy for the inhibition of tumor cell implantation following surgery and not for the treatment of existing primary tumors. Future work will include investigating whether the rapid uptake kinetics of 64Cu-PTSM could also allow this agent to be used for the treatment of accessible tumors by direct intratumoral delivery or for treatment of cancer in the peritoneum, e.g., ovarian tumors, ascites, or carcinomatoses in colorectal cancer (e.g., as a means of reducing local and pelvic recurrences).
In conclusion, addition of 64Cu to the radiotherapy arsenal is both useful and innovative because it allows accurate monitoring of drug distribution and biokinetics through concurrent PET imaging. This study has demonstrated the therapeutic potential of 64Cu-PTSM in inhibiting cancer cell attachment to incision sites and growth of metastasis following laparoscopy surgery. 64Cu-PTSM was shown to inhibit cancer cell implantation and growth at a dose that resulted in no overt signs of toxicity to the hamsters. We have also shown that MRI and PET provided powerful imaging modalities for monitoring tumor growth and development following therapy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by a grant from the United States Department of Energy (Grant DE-FG02-87ER60512). The production of copper radionuclides at Washington University is supported by a grant from the National Cancer Institute (Grant 1 R24 CA86307), and the small animal imaging at Washington University is supported by United States NIH Grant 5 R24 CA83060. 
2 To whom requests for reprints should be addressed, at Mallinckrodt Institute of Radiology, Washington University School of Medicine, 510 S. Kingshighway Boulevard, Campus Box 8225, Saint Louis, MO 63110. Phone: (314) 362-8435; Fax: (314) 362-8399; E-mail: welchm{at}mir.wustl.edu
3 The abbreviations used are: 64Cu-PTSM, 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone); 64Cu-ATSM, 64Cu-diacetyl-bis(N4-methylthiosemicarbazone); MRI, magnetic resonance imaging; 18F-FDG, [18F]fluoro-2-deoxyglucose; MAb, monoclonal antibody; PET, positron emission tomography; MTD, maximum tolerated dose.
4 J. M. Connett et al., Radioimmunotherapy of colon cancer in a laparoscopic model using I-131-MAb-1A3, manuscript in preparation.
Received 7/17/01. Accepted 11/ 5/01.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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