Copper-64-pyruvaldehyde-bis(N⁴-methylthiosemicarbazone) for the Prevention of Tumor Growth at Wound Sites following Laparoscopic Surgery: Monitoring Therapy Response with microPET and Magnetic Resonance Imaging¹

Copper-64 [t_1/2 = 12.8 h; β⁺ = 0.655 MeV (19%); β⁻ = 0.573 MeV (40%)] is a cyclotron-produced positron-emitting isotope (1, 2) that has utility in both diagnostic medicine (3, 4) and as a therapeutic radionuclide (5, 6, 7, 8). The technology developed for the production of ⁶⁴Cu (Newton Scientific, Inc., Cambridge, MA) has been obtained by institutions in the United States, Europe, Japan, and Canada. ⁶⁴Cu-PTSM³ belongs to a class of neutral, lipophilic complexes that have demonstrated rapid diffusion into cells. In experiments using cultured single-cell suspensions of EMT6 mammary carcinoma cells, 80% of ⁶⁴Cu-PTSM added was retained within the cells after only 1 min (9). Recently we showed that the hypoxia-selective ⁶⁴Cu-ATSM, an analogue of Cu-PTSM, has promise as an agent for radiotherapy by significantly increasing the survival time of hamsters bearing solid tumors without acute toxicity (8).

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.

⁶⁴Cu was produced on a CS-15 biomedical cyclotron at Washington University School of Medicine, using published methods (2). ⁶⁴Cu-PTSM was synthesized as described previously (9, 16). Unless otherwise stated, all chemicals were purchased from Aldrich Chemical Company, Inc. (Milwaukee, WI). All solutions were prepared with distilled deionized water (Milli-Q; >18 megaohm resistivity). Radiochemical purity of ⁶⁴Cu-PTSM in all studies was >98% as determined by radio-TLC.

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 × 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.

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.

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).

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. χ² or Fisher’s exact tests were used to compare proportions and frequency data.

The data obtained from the biodistribution of ⁶⁴Cu-PTSM in nontumor-bearing hamsters are shown in Table 1. 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).

There were no premature deaths in any of the experimental or control groups, and growth of tumors occurred only at the port and midline incision sites. The results clearly demonstrated the ability of ⁶⁴Cu-PTSM to inhibit the implantation of tumors in wound sites in a dose-dependent manner (Table 2). 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).

Magnetic resonance images of hamster abdominal cavities clearly showed tumor development and growth. The presence of tumors in the GW39-treated animals was clearly seen in these images. The uptake of ⁶⁴Cu-PTSM and ⁶⁴Cu-TETA-1A3 in the abdomen of hamsters as monitored by microPET is shown in Fig. 1,A. 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. 1,B) 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. 1 C). 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.

⁶⁴Cu is a cyclotron-produced isotope that has applications both in PET and radiotherapy. The addition of ⁶⁴Cu to the radiotherapy arsenal is particularly innovative because it enables the accurate monitoring of biodistribution and biokinetics through concurrent PET imaging. To date, ⁶⁴Cu-radiotherapy studies have included the use of the ⁶⁴Cu-labeled MAb 1A3 (17) and ⁶⁴Cu-labeled somatostatin-based peptides (5, 6). Recently, we showed that the hypoxia-selective ⁶⁴Cu-ATSM inhibits the growth of GW39 tumors in hamsters with no acute toxicity (8).

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 β⁻ particle (40%), a 0.66-MeV β⁺ particle (19%), and a γ 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 β⁻ particles. Additionally, ⁶⁴Cu has a maximum recoil energy resulting from the nuclear transmutation of the copper ion (from β⁻ = 7.6 eV; from β⁺ = 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. 1,B). Furthermore, ¹⁸F-FDG imaging confirmed the growth of tumors at the sites of trocar placement and midline incision (Fig. 1 C). 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.

We thank Dr. Deborah W. McCarthy and Todd A. Perkins for production of ⁶⁴Cu, and Drs. Dan Ye, Richard Laforest, Douglas J. Rowland, Joon Young Kim, and Wenping Li for assistance. We also thank Suzanne Hickerson, Terry Sharp, and John Engelbach for excellent technical support.