PET Imaging of Triple-Negative Breast Cancer and Tetrathiomolybdate Treatment Using [64Cu]Copper(II) Chloride

PET Imaging of Triple-Negative Breast Cancer and Tetrathiomolybdate Treatment Using [64Cu]Copper(II) Chloride | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article PET Imaging of Triple-Negative Breast Cancer and Tetrathiomolybdate Treatment Using [64Cu]Copper(II) Chloride Michael Lewis, Claudia Chambers, Alexander Schaedler, Mojgan Golzy, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5633914/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose In the United States, breast cancer is the second leading cause of cancer-related death. Triple-negative breast cancer (TNBC) is of substantial concern, as it lacks the receptors usually targeted by conventional treatments. Triple-negative breast tumors have a high degree of copper metabolism for the synthesis of transporters, enzymes, and chaperones. Tetrathiomolybdate (TM) is a well-tolerated oral therapy that has been investigated for chelating copper from tumors in TNBC patients, resulting in extended remission. The overall goal of this research was to evaluate [ 64 Cu]CuCl 2 PET/CT imaging of copper utilization in this disease, in the presence and absence of TM. Procedures Uptake, internalization, and efflux studies were performed in TNBC cells versus normal cells. Biodistribution experiments were then conducted in TNBC xenograft-bearing mice that were administered TM versus controls. PET/CT imaging of mice carrying TNBC tumors was also performed in the presence and absence of TM. Finally, imaging was performed in a healthy cat and cats with mammary carcinoma. Results SUM149 TNBC cells selectively took up, internalized, and retained [ 64 Cu]CuCl 2 more avidly than normal fibroblasts. When SUM149-bearing mice were given TM, tumor uptake decreased and tracer accumulation shifted predominantly to the liver and kidneys, compared to control mice, in which large quantities of 64 Cu were excreted into the intestines. These results were supported by PET/CT imaging of the mice. PET/CT of companion cats gave results similar to those obtained in mice, with high accumulation of radioactivity observed in the liver and gallbladder and moderate intestinal and renal clearance. In a cat with mammary carcinoma, [ 64 Cu]CuCl 2 was highly conspicuous, even in close proximity to the liver. Conclusions Utilization of [ 64 Cu]CuCl 2 in triple-negative breast cancer can be detected efficiently in cell and animal models of this disease. The tracer was also used successfully to evaluate TM therapy in the SUM149 TNBC mouse model. Furthermore, PET/CT imaging of both mice and cats with breast cancer shows the potential to monitor treatment with TM in a facile, noninvasive manner. We are currently conducting a clinical trial of [ 64 Cu]CuCl 2 PET/CT in companion cats with mammary carcinoma, with the future goal of evaluating the efficacy of TM in feline patients. Triple-negative breast cancer 64Cu PET imaging tetrathiomolybdate chelation therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Breast cancer is the most common cancer and second leading cause of cancer-related death in American women. Furthermore, its incidence in the United States is rising at a rate of 0.5% per year. This year, 310,720 new cases of breast cancer will be diagnosed in American women, along with 42,250 deaths [ 1 ]. Stage I-II hormone receptor-positive breast cancer responds well to both hormone therapy and chemotherapy, with 5-year survival rates of 90%. However, survival of women with metastatic disease drops to 28% at 5 years. Of particular concern is triple-negative breast cancer (TNBC), which lacks estrogen receptor, progesterone receptor, and Her2/neu. TNBC accounts for 15% of all breast cancers. It is characterized by a high degree of heterogeneity, aggressive behavior, constitutive activation of oncogenes, and poor overall response to standard-of-care, including hormone therapy and trastuzumab [ 2 ]. Treatment of stage IV TNBC includes the use of chemotherapy drugs such as anthracyclines, taxanes, capecitabine, gemcitabine, and eribulin, all of which have considerable dose-limiting toxicity. Patients with metastatic disease might respond better to emerging alternative treatments, such as targeted drugs, immunomodulators, and antiangiogenic therapy. Triple-negative breast cancer is a copper-avid tumor type, relying on this element for cell survival and proliferation. Ammonium tetrathiomolybdate (TM, Decuprate®), which is approved for oral therapy of Wilson’s disease [ 3 ], can be used to deplete copper from tumors. It is possible that treatment with TM will be helpful in the subsets of patients with certain molecular and/or clinicopathologic characteristics that are considered high risk. Copper chelation therapy also has the potential to be monitored in a noninvasive manner, potentially part of a personalized medicine approach. Copper chelation therapy inhibits several copper-dependent enzymes, including ceruloplasmin, ascorbate oxidase, cytochrome oxidase, superoxide dismutase, tyrosinase, ATPase, and mitochondria-specific copper chaperones [ 4 – 6 ]. Furthermore, copper depletion suppresses collagen remodeling [ 7 ], inhibits angiogenesis [ 8 – 10 ], and induces apoptosis [ 11 ] in TNBC. In all, a combination of these effects prevents tumor invasion and metastasis [ 5 , 7 , 12 ]. In tumor-bearing mice, TM also renders tumors more susceptible to drugs such as cisplatin [ 13 ] and doxorubicin [ 14 ]. To date, most clinical trials of TM have focused on biomarker analysis to assess the efficacy of the drug. Chan et al. [ 15 ] monitored ceruloplasmin levels and LOXL2 expression to evaluate response to TM therapy in 75 patients, for two years or until relapse. Of those, 51 patients each received 1396 cycles of TM over the 2-year period, and levels of both biomarkers decreased and correlated with improved event-free survival. In another Phase II study, Liu and colleagues determined that collagen processing was altered by TM, as measured by LOXL2, Pro-C3, C1M, and C6M levels [ 16 ]. LOXL2 expression increased in patients experiencing progressive disease or death, compared to patients with no evidence of disease, in whom LOXL2 levels remained stable. Collagen formation, assessed by Pro-C3 expression, increased, while markers of collagen degradation, such as C1M and C6M, increased in patients with disease progression/death, whereas these markers remained stable in those in remission. Conversely, C1M expression increased in those with no evidence of disease but remained stable in progressive patients and cases resulting in death. Blockhuys et al. [ 6 ] examined the effect of the copper chaperone ATOX1 on TNBC and high-risk breast cancer patients. Their Phase II trial showed that ATOX1 levels increased in patients with better event-free survival after TM therapy. Those investigators found that as cytoplasmic ATOX1 levels increased, patients experienced better event-free survival. The opposite was observed in patients with nuclear ATOX1. Biomarker analysis requires considerable method development and time to complete the assays. An alternative assessment of response to treatment with TM might be performed by noninvasive imaging, providing a more facile way to evaluate copper metabolism and correlate it with patient outcome. [ 64 Cu]copper(II) chloride could be used as a true radiotracer to measure copper utilization by PET/CT. Studies in healthy humans [ 17 , 18 ] with [ 64 Cu]CuCl 2 have shown that the major organ of uptake was the liver, followed by the walls of the large intestine and the pancreas. Renal clearance was negligible. Lack of urinary excretion was particularly important in imaging prostate cancer [ 19 , 20 ], as no accumulation of radioactivity in the bladder occurred, and high contrast imaging of local disease was possible. In this report, we describe the use of [ 64 Cu]copper(II) chloride for PET/CT imaging in animal models of triple-negative breast cancer. To our knowledge, this tracer has not been evaluated in TNBC. In our in vitro studies, we found that TNBC cells preferentially take up copper. Moreover, PET/CT imaging of mice with human triple-negative xenografts and cats with naturally occurring mammary carcinoma showed specific and high contrast imaging in vivo, despite high accumulation of copper in the liver. Materials and Methods General Information All reagents were of the highest purity obtainable. Ultrapure water (18 MΩ-cm) was used for all experiments. Sterile phosphate-buffered saline (PBS) was purchased from Fisher Scientific (Pittsburgh, PA), and [ 64 Cu]Cu(II) chloride was obtained from Washington University in St. Louis and diluted to 0.1 µCi/µL with PBS. A PerkinElmer PerkinElmer Wizard 2 2480 (PerkinElmer Life Sciences, Gaithersburg, MD) automatic gamma counter was used to count radioactive samples. Tetrathiomolybdate (99.97% purity) was purchased from Millipore Sigma (St. Louis, MO). SUM149 triple-negative breast cancer cells were purchased from Applied Biological Materials (Richmond, BC) and NIH 3T3 cells were obtained from the American Type Culture Collection (ATCC, Rockland, MD). SUM149 cells were maintained in Ham’s F-12 media, containing 5% heat-inactivated fetal bovine serum (FBS), 10 mM HEPES, 1 µg/mL hydrocortisone, and 5 µg/mL insulin, cultured at 37°C and 5% CO 2 . NIH 3T3 cells were cultured in DMEM with 10% FBS. Four- to six-week-old female NCR nude mice were obtained from Taconic Biosciences (Rensselaer, NY). Cell Uptake, Internalization, and Efflux Assays Cell Culture Cell studies were conducted using modifications of our previously published procedures [ 21 ]. SUM149 cells were grown in Ham’s F-12 (Gibco™, Fisher Scientific) supplemented with 10% FBS (Gibco), insulin (5 µg/mL), hydrocortisone (1 µg/mL; both from Sigma-Aldrich, St. Louis, MO), and 1% penicillin-streptomycin. The cells were subsequently washed twice with serum free Minimum Essential Medium (MEM) before being drawn up to 8 × 10 6 cells per mL. NIH 3T3 cells were grown in Dulbecco's Modified Eagle Medium (DMEM), with the addition of 10% FBS. The cells were subsequently washed twice with serum-free MEM before being drawn up to 8 × 10 6 cells per mL. Uptake Cells were incubated (37°C, 5% CO 2 ) with 100,000 cpm of [ 64 Cu]CuCl 2 /200 µL. Uptake of copper was evaluated over a time course in triplicate (5, 15, 30, 60, 120, 180, 240 min). Uptake assays were accomplished by separating the cell pellet via centrifugation (15,000 rpm, 4°C, 5 min) and washing once with ice-cold PBS. Cell-associated radioactivity was determined as the ratio of radioactivity in the cell pellet divided by the total radioactivity. Internalization Cells were incubated (37°C, 5% CO 2 ) with 100,000 cpm of [ 64 Cu]CuCl 2 /200 µL. Internalization of copper was evaluated over a time course in triplicate (5, 15, 30, 60, 120, 180, 240 min). An acid wash was completed (0.9% NaCl 500mL, 40 mM NaAc, 40 mM HAc, 1 g FBS, pH 4.5) to remove surface associated activity. The cell pellet was subsequently separated via centrifugation (15,000 rpm, 4°C, 5 min) and washed once with ice-cold PBS. Internalized cell-associated radioactivity was determined as the ratio of radioactivity in the cell pellet divided by the radioactivity in the combined supernatants. Efflux For efflux studies, cells were incubated (37°C, 5% CO 2 ) with 100,000 cpm of [ 64 Cu]CuCl 2 /200 µL. Radiopharmaceutical uptake was performed as described above for 2 h at 37°C and 5% CO 2 . Then cells were subsequently washed with ice cold PBS and isolated by centrifugation before resuspending in fresh medium. Efflux of copper from the cells was measured at subsequent time points ranging from 5 min to 4 h, after isolating aliquots of the cells in triplicate, washing the cells in ice cold PBS, and counting them separately from the supernatant, as described above. Biodistribution Studies All animal studies were conducted in accordance with the guidelines established by the University of Missouri Animal Care and Use Committee. NCR nude mice were implanted with 1 × 10 7 SUM149 cells in PBS:Matrigel (2:1), subcutaneously (SC) in the hind flank. Eight weeks after tumor inoculation, when tumors had grown to 50–100 mg, mice received an intravenous (IV) injection of 0.37 MBq (10 µCi) of [ 64 Cu]CuCl 2 in 100 µL of PBS. Prior to the study, half of the mice received regular drinking water, and half received 0.03 mg/mL of TM in drinking water [ 13 ] for one week. At 1, 4, and 24 h, biodistributions (n = 4) were obtained. Tissues taken included blood, liver, spleen, lung, heart, stomach, large and small intestines, muscle, bone, brain, pancreas, kidneys, and tumor. Urine and feces were collected as well. Tissues were weighed and counted in the gamma counter with a standard of the injected dose, such that % injected dose per gram of tissue (% ID/g) and % injected dose per organ (% ID) were determined as decay-corrected uptakes. Murine PET/CT Imaging An aliquot of 200 MBq (6 mCi) of [ 64 Cu]CuCl 2 in approximately 10 µL of 0.05 M HCl was diluted to 53.7 MBq/mL (1.45 mCi/mL) with PBS. Mice (n = 6) were injected IV via the tail vein with 100 µL of this solution. As in the biodistribution studies described above, half of the mice received TM in drinking water, and half drank regular water. Under isoflurane anesthesia, mice were placed in a prone position, and PET/CT imaging was performed on a Bruker Albira Si SPECT/PET/CT camera. Images were acquired over 15 min, with an axial field of view of 99 mm and a bed position offset at 105 mm. Next, CT imaging was performed for 13 min. PET/CT images were processed using PET ordered subset expectation maximization (OSEM) software. One iteration at 0.75 mm was used, with decay, random, and scatter corrections. Feline PET/CT Imaging Feline studies were also conducted in accordance with the guidelines established by the University of Missouri Animal Care and Use Committee. Companion cats were anesthetized, and an IV catheter was placed in the right forelimb. The cats were injected with 280 MBq (7.6 mCi) of [ 64 Cu]CuCl 2 in 0.5 mL of sterile PBS. At 20 h post-injection, cats were scanned using a Canon Celestion PET/CT camera. PET imaging parameters were 2–3 min per bed position, 4 mm slices, and 50% overlap between slices. The voxel size was 4 × 4 × 4 mm 3 , the field of view (FOV) was 240 mm, and the pixel size was 2.0. A coaxial CT scan was performed using settings of 120 kV/300 mA, 0.75 s rotation, detailed HP (11.0/PF 0.688), and the same FOV. PET data were reconstructed with time-of-flight 3-dimensional ordered subset expectation maximization (TOF 3D OSEM) with a Gaussian postfilter. CT images were reconstructed with a 3-dimensional adaptive iterative dose reduction (AIDR 3D) algorithm. Statistical Analysis Repeated measure analysis of variance (ANOVA), including the between-subject variable, was used to assess the effect of water treatment on outcomes of interest; i.e., %ID/g and %ID. The interaction between time (in hours) and water treatment was considered in each model. The R-square goodness of fit measure was used to assess the model. The partial sum of squares (Type III sum of squares) was used to specify the significant main effect of time, water treatment and their interactions on each outcome. The interaction term tested whether the effect of water treatment on the outcome is the same over the time interval, meaning that slopes of regression lines are significantly different for the two groups. Results Cell Studies Uptake, internalization, and efflux studies showed greater retention in SUM149 triple-negative breast cancer cells than in normal NIH 3T3 fibroblasts. As shown in Fig. 1 A, the amount of [ 64 Cu]CuCl 2 taken up by SUM149 cells was 19.2 ± 0.6% at 5 min, and this value increased to 40.3 ± 1.3% after 4 h of incubation. In contrast, uptake in fibroblasts was 9.33 ± 0.13%ID/g at 5 min and increasing to 16.6 ± 0.1% at 4 h, less than half that of the TNBC cells. SUM149 cells also internalized 64 Cu to a greater degree than 3T3 cells (Fig. 1 B), increasing from 9.0 ± 0.3% at 5 min to 43.9 ± 0.3% by 4 h of incubation. Internalization in the fibroblast cell line was 7.4 ± 0.1% at 5 min, but it only increased to 23.5 ± 0.2% at 4 h. Retention of 64 Cu in SUM149 cells was also greater than in 3T3 cells (Fig. 1 C), as efflux of copper from the TNBC cells was slower than that from the fibroblasts. At 5 min, retention of 64 Cu in SUM149 cells was 91.0 ± 6.7%, decreasing to 65.5 ± 0.9% after 4 h. By comparison, the amount of copper retained in 3T3 cells was 87.9 ± 2.5% at 5 min and 56.9 ± 0.7% after 4 h of incubation. Biodistribution Studies Biodistributions of [ 64 Cu]CuCl 2 were obtained in SUM149-bearing nude mice, with (Fig. 2 B) or without (Fig. 2 A) tetrathiomolybdate in drinking water for one week. In each case, the major organ of uptake was the liver. Control mice receiving water showed liver accumulations of 23.0 ± 2.6%ID/g, 24.1 ± 3.7%ID/g, and 13.9 ± 2.6%ID/g at 1, 4, and 24 h, respectively. Concomitantly, there was also a high degree of radioactivity in the gastrointestinal tract, which was most pronounced at 1 h and 4 h. Accretion of copper in the small intestine increased from 13.6 ± 1.2%ID/g at 1 h to 17.2 ± 2.9%ID/g at 4 h, after which it decreased to 4.5 ± 1.0%ID/g at 24 h. The uptakes of 64 Cu in the large bowel followed those in the small intestine over the time course of the study. Accumulations of 6.0 ± 0.4%ID/g at 1 h, 21.8 ± 2.3%ID/g at 4 h, and 5.4 ± 1.2%ID/g at 24 h were observed in the large intestine, demonstrating the transit of 64 Cu through the GI tract. The other major organ of uptake was the kidney. At 1 h, kidney accretion was 11.6 ± 1.2%ID/g, which remained relatively constant at 4 h (12.0 ± 2.2%ID/g) and then decreased at 24 h (8.4 ± 1.3%ID/g). Blood clearance of 64 Cu was also protracted, ranging from 2.5 ± 0.4%ID/g to 3.4 ± 0.9%ID/g at 4 h and 1.9 ± 0.1%ID/g at 24 h. All other organs showed relatively moderate levels of radioactivity. Mice that received TM had liver uptakes of 18.1 ± 5.8%ID/g at 1 h, 29.7 ± 4.4%ID/g at 4 h, and 23.5 ± 1.5%ID/g at 24 h, which were comparable to those in controls. Interestingly, high gut uptake was not observed in mice administered TM. Instead, clearance of 64 Cu shifted dramatically toward the renal pathway. While liver uptake remained relatively constant throughout the study, kidney uptake increased substantially to 15.8 ± 0.9%ID/g at 1 h, 20.9 ± 5.0%ID/g at 4 h, and 18.3 ± 1.8%ID/g at 24 h, considerably higher than in untreated mice. As in controls, prolonged retention of radioactivity from the blood was observed. Blood uptakes of 18.5 ± 1.2%ID/g, 11.6 ± 0.4%ID/g, and 2.2 ± 0.8%ID/g were observed at 1, 4, and 24 h, respectively. As in the case of untreated controls, accumulation of radioactivity in other organs was relatively low. Tumor uptake was initially similar in both groups of animals, then it decreased steadily in the TM group at later time points. At 1 h, tumor accretion was 4.5 ± 1.3%ID/g when animals received the drug, compared to 5.0 ± 0.8%ID/g in untreated mice. By 4 h post-injection, uptake in the tumor was 9.7 ± 2.6%ID/g for mice receiving plain water, versus 3.7 ± 0.3%ID/g in mice administered TM. While this value remained relatively constant at 24 h (3.0 ± 0.7%ID/g), tumor uptake in untreated animals was considerably higher (7.1 ± 2.5%ID/g). Statistical differences between mice receiving regular water versus mice receiving TM were the result of 1) administration of the drug (TM versus regular water), 2) time, and 3) an interaction of water and time. For example, in the liver there was no statistical difference between the two groups according to the type of water administered (P = 0.14) overall. However, a significant primary effect of time (P < 0.0001) and a significant time × water treatment interaction on outcome (P < 0.0032) were observed, meaning that a significantly different trend for the two water treatment groups existed. However, the regression lines of the two groups crossed each other, resulting in no significant overall mean outcomes difference (data not shown). The largest difference between the two groups in the biodistribution studies was uptake in the intestines. In the small bowel, the effects of TM in water (P < 0.0001) over the time course of the study (P < 0.0001) resulted in a significant interaction (P = 0.0004). In the large intestine, the type of water gave significantly different results (P = 0.011), but over the course of the study, time did not (P = 0.21). No significant interaction for large intestinal uptake (P = 0.15) was observed. The kidneys showed highly significant differences according to the presence or absence of TM (P = 0.0021) over time (P = 0.011), with a significantly different trend for the two treatments (P = 0.015). In the tumor, the effect of TM alone was quite noticeable. While time was not a significant factor in tumor uptake (P = 0.27), the administration of TM had a prominent effect (P = 0.023). However, the interaction of water and TM did not produce a significant effect (P = 0.95), meaning the drug had the primary effect on tumor uptake. Murine PET/CT Imaging PET/CT imaging of SUM149-bearing nude mice was conducted at 1, 4, and 24 h post-injection. These studies gave results that were consistent with those of the conventional biodistribution studies. At 1 h and 4 h, high uptake in the intestines obscured imaging of the xenograft, which was only seen after sufficient clearance at 24 h. In mice receiving regular water (Fig. 3 A), tumors were clearly detected, along with high liver uptake. Concomitant with high liver accumulation was pronounced uptake in the intestines. High liver uptake was also observed in animals receiving TM (Fig. 3 B); however, uptake in the intestines was minimal. Instead of GI clearance, excretion appeared to shift to the kidneys, which was consistent with biodistribution results. Little to no tumor uptake was seen in mice administered TM. These results suggest that copper chelation can not only prevent accumulation of the element in the xenograft, but also increase renal clearance. Feline PET/CT Imaging In the first study, a healthy volunteer companion cat was scanned at 20 h post-injection of [ 64 Cu]CuCl 2 . The imaging results, shown in Fig. 4 , were also consistent with mouse PET/CT. The major off-target organs were the liver and gallbladder, while the intestines and kidneys showed moderate activity. A moderate level of radioactivity was also observed in the bladder, as urinary excretion was a significant route of elimination. Based on the imaging results in the normal cat, we have opened a clinical trial of [ 64 Cu]CuCl 2 PET/CT imaging in companion cats with mammary carcinoma. An example is shown in Fig. 5 . Again, the liver was the major organ of uptake (Fig. 5 A). In this patient, a chain of tumors in four mammary glands was observed. When images of the patient were analyzed in transaxial mode (Fig. 5 B), all of the lesions were very conspicuous, even in close proximity to the highly radioactive liver. Discussion In the present studies, we evaluated the tumor uptake and retention of [ 64 Cu]copper(II) chloride in cell and animal models of TNBC, and we determined the effect of tetrathiomolybdate treatment in tumor-bearing mice. At present, the only common treatments for metastatic TNBC consist of dose-limiting chemotherapy regimens. Recently, pembrolizumab (Keytruda®) has received FDA approval to treat TNBC, but this antibody only targets PD-L1-positive tumors that account for 20–37% of triple-negative breast cancers. Furthermore, pembrolizumab also has highly toxic side effects. These side effects include loss of immune tolerance, resulting in organ failure or even death. This antibody also has the potential to cause adverse events in the form of infusion reactions, which can be severe or life-threatening. In contrast, TM therapy is an oral drug that is well-tolerated and can be administered several times a day. In one study, Liu et al. [ 5 ] found that TM therapy resulted in 71.7% event-free survival and 74.2% overall survival in TNBC patients receiving TM over a median period of 9.4 years, with a median of 3423 treatment cycles. Another clinical trial performed by Sahota and coworkers demonstrated that TM therapy gave 90% event-free survival at 6.9 years for Stage II/III TNBC and 66.7% progression-free survival for Stage IV TNBC [ 22 ]. Chan et al. achieved similar results in their study, with 69% event-free survival of Stage IV triple-negative patients after 6.3 years [ 15 ]. In triple-negative breast cancer, measurement of copper biomarkers has been used by two groups to evaluate patients and to monitor TM therapy. Chan et al. showed that TM therapy was safe and well-tolerated, and it resulted in lower levels of ceruloplasmin and LOXL2, as determined by ELISA [ 15 ]. Furthermore, Blockhuys and coworkers used immunohistochemistry to demonstrate that TM treatment also reduces expression of ATOX1, which correlates with improved event-free survival [ 6 ]. Herein we report the use of [ 64 Cu]CuCl 2 PET/CT as a modality for noninvasive imaging of mouse and feline models of TNBC. Our cell studies showed that [ 64 Cu]CuCl 2 is preferentially taken up by TNBC cells, as opposed to normal fibroblasts. Uptake in SUM149 triple-negative breast cancer cells was approximately 2.5 times higher than in NIH 3T3 fibroblasts, while internalization of 64 Cu was about 2 times higher in the triple-negative cell line. Copper-64 was also retained to a greater degree in SUM149 cells, with an approximate difference of 9% in efflux compared to the normal cell line. Evaluation of biodistribution data demonstrated preferential uptake of [ 64 Cu]CuCl 2 in the liver, where hepatic uptake reached a maximum of 32.7% of the total dose at 1 h and cleared to 17.7%ID by 24 h, while approximately 1% of the dose targeted the tumor. Most of the excretion occurred through the hepatobiliary pathway. In mice treated with TM, liver accumulation was initially lower at 1 h, reached a peak at 4 h, and then decreased by 24 h. Kidney uptake of [ 64 Cu]CuCl 2 in untreated mice decreased throughout the course of the study. However, renal accumulation of the tracer was higher in animals receiving TM, where kidney accretion actually increased over time. The reason for this shift to renal clearance upon receiving TM is unknown and would be worth further investigation. In the tumor, uptake in mice administered TM was considerably lower. At 1 h post-injection, tumor accumulation in treated animals was approximately the same as in control mice, but by 4 h the uptake of [ 64 Cu]CuCl 2 was about 6%ID/g higher in the untreated group and remained higher in these animals at 24 h. The data indicate that TM therapy results in lower uptake and retention of 64 Cu in tumor-bearing mice. PET/CT imaging of SUM149-bearing mice was consistent with this trend as well. The optimal imaging time was 24 h post-injection, due to the high excretion of 64 Cu in the intestines. While tumors were clearly detected in mice receiving plain water at 24 h, little to no signal from the xenograft was observed in mice given TM. Imaging of other organs was also consistent with the conventional biodistribution data. While the liver and intestines in untreated mice were clearly visible, the shift to renal clearance in animals given TM was readily apparent, with high contrast imaging of the kidneys. PET/CT imaging of companion cats afforded results that mirrored those obtained in the SUM149 murine model. In the normal cat, the liver was the major organ of uptake, and excretion into the gallbladder was especially prominent. Accumulation in the gastrointestinal tract, kidneys, and bladder were modest in comparison. In the cat with mammary carcinoma, high liver and gallbladder accumulation was also observed, along with considerable excretion into the intestine. The chain of mammary tumors in the abdomen was also clearly displayed, but liver uptake obscured imaging of more cranial lesions. In order to detect tumors in close proximity to the liver, evaluation of imaging in transaxial mode allowed high resolution in this region. Conclusion In these studies, we demonstrated that PET/CT imaging using [ 64 Cu]CuCl 2 in animal models of triple-negative breast cancer allowed detection of tumors with high sensitivity and resolution. Consistent with higher selective uptake in TNBC cells, we found that [ 64 Cu]CuCl 2 could detect triple-negative tumors in murine and feline models of breast cancer. We have also shown that [ 64 Cu]CuCl 2 has potential to serve as a true radiotracer to monitor copper chelation therapy with TM. At present, we are conducting a clinical trial of [ 64 Cu]CuCl 2 PET/CT imaging in cats with mammary carcinoma. Our goal is to correlate standardized uptake values (SUVs) to copper biomarkers and receptor status in feline patients. In the future, we plan to conduct similar imaging studies in feline mammary carcinoma patients receiving TM. Declarations Acknowledgments The authors are grateful for excellent technical support from Katie Tucker. Funding This work was supported by a grant from the University of Missouri Molecular Imaging and Theranostics Center (to MRL). Competing Interests The authors declare that they have no competing financial interests or personal relationships. Author Contributions All authors, experimental design and execution; MRL, original draft, supervision, project administration, methodology, investigation, formal analysis, data curation; CGC, methodology, investigation, formal analysis; AWS, methodology, investigation, formal analysis; MG, methodology, formal analysis; LDW and TLC, methodology, investigation, data curation; KS, methodology, formal analysis; CP, supervision and project administration; VAY, JML, CG, JLT, CAM, and JNB, methodology, investigation, formal analysis, data curation, project administration; CJS, manuscript review and editing, project administration. Ethics Approval All animal studies were conducted in accordance with the guidelines established by the University of Missouri Animal Care and Use Committee. Data Availability Analysis of data supporting our findings is included in the article. The raw data generated by these studies is available from the corresponding author on request. References Siegel RL, Giaquinto AN, Jemal A (2023) Cancer statistics, 2024. CA Cancer J Clin 74:12-49 Jin J, Tao Z, Cao J, Li T, Hu X (2021) DNA damage response inhibitors: An avenue for TNBC treatment. Biochim Biophys Acta 1875:188521 De Fabregues O, Viñas, Palasí A, Quintana M, Cardona I et al (2020) Ammonium tetrathiomolydate in the decoppering phase treatment of Wilson's disease with neurological symptoms: A case series. Brain Behav 10:e01596 Alvarez HM, Xue Y, Robinson CD, Canalizo-Hernández MA, Marvin RG et al (2010) Tetrathiomolybdate inhibits copper trafficking proteins through metal cluster formation. Science 327:331-334 Ramchandani D, Berisa M, Tavarez DA, Li Z, Miele M et al (2021) Copper depletion modulates mitochondrial oxidative phosphorylation to impair triple negative breast cancer metastasis. Nature Commun 12:7311 Blockhuys S, Hildesjö C, Olsson H, Vahdat L, Wittung-Strafshede P (2021) Evaluation of ATOX1 as a potential predictive biomarker for tetrathiomolybdate treatment of breast cancer patients with high risk of recurrence. Biomedicines 9:1887 Liu YL, Bager CV, Willumsen N, Ramchandani D, Kornhauser N et al (2021) Tetrathiomolybdate (TM)-associated copper depletion influences collagen remodeling and immune response in the pre-metastatic niche of breast cancer. NPJ Breast Cancer 108 Joimel U, Gest C, Soria J, Pritchard L-L, Alexandre J et al (2010) Stimulation of angiogenesis resulting from cooperation between macrophages and MDA-MB-231 breast cancer cells: Proposed molecular mechanism and effect of tetrathiomolybdate. BMC Cancer 10:375 McCarty MF, Block KI (2005) Multifocal angiostatic therapy: An update. Integr Cancer Ther 4:301-314 Pan Q, Kleer CG, van Golen KL, Irani J, Bottema KM et al (2002) Copper defiency induced by tetrthiomolybdate suppresses tumor growth and angiogenesis. Cancer Res 62:4854-4859 Karginova O, Weekley CM, Raoul A, Alsayed A, Wu T et al (2019) Inhibition of copper transport induces apoptosis in triple-negative breast cancer cells and supresses tumor angiogenesis. Mol Cancer Ther 18:873-885 Pan Q, Bao LW, Merajver SD (2003) Tetrathiomolybdate inhibits angiogenesis and metastasis through supression of the nf k b signaling cascade. Mol Cancer Res 1:701-706 Chisholm CL, Wang H, Wong AH-H, Vazquez-Ortiz G, Chen W et al (2016) Ammonium tetrathiomolybdate treatment targets the copper transporter ATP7A and enhances sensitivity of breast cancer to cisplatin. Oncotarget 7:84439-84452 Pan Q, Bao LW, Kleer CG, Brewer GJ, Merajver SD (2003) Antiangiogenic tetrathiomolybdate enhances the efficacy of doxorubicin against breast carcinoma. Mol Cancer Ther 2:617-622 Chan N, Willis A, Kornhauser N, Ward MM, Lee SB et al (2017) Influencing the tumor microenvironment: A phase II study of copper depletion using tetrathiomolybdate in patients with breast cancer at high risk for recurrence and in preclinical models of lung metastases. Clin Cancer Res 23:666-676 Liu YL, Liv Bager C, Willumsen N, Ramchandani D, Kornhauser N et al (2021) Tetrathiomolybdate (TM)-associated copper depletion influences collagen remodeling and immune response in the pre-metastatic niche of breast cancer. NPJ Breast Cancer 7:108 Avila-Rodriguez MA, Rios C, Carrasco-Hernandez J, Manrique-Arias JC, Martinez-Hernandez R et al (2017) Biodistribution and radiation dosimetry of [ 64 Cu]copper dichloride: First-in-human study in healthy volunteers. EJNMMI Res 7:98 Kjægaard K, Sandahl TB, Frisch K, Vase KH, Keiding S et al (2020) Intravenous and oral copper kinetics, biodistribution and dosimetry in healthy humans studied by [ 64 Cu]copper PET/CT. EJNMMI Radiopharm Chem 5:15 Chakravarty R, Shetty P, Nair KVV, Rajeswari A, Jagadeesan KC et al (2020) Reactor produced [ 64 Cu]CuCl 2 as a pet radiopharmaceutical for cancer imaging: From radiochemistry laboratory to nuclear medicine clinic. Ann Nucl Med 34:899-910 Mascia M, Villano C, De Francesco V, Schips L, Marchioni M et al (2021) Efficacy and safety of the 64 Cu(II)Cl 2 PET/CT for urological malignancies. Clin Nucl Med 46:443-448 Jia F, Daibes Figueroa S, Gallazzi F, Balaji BS, Hannink M et al (2008) Molecular imaging of bcl-2 expression in small lymphocytic lymphoma using 111 In-labeled PNA-peptide conjugates. J Nucl Med 49:430-438 Sahota S, Kornhauser N, Willis A, Ward MM, Cigler T et al (2017) A phase II study of copper-depletion using tetrathiomolybdate (TM) in patients (pts) with breast cancer (bc) at high risk for recurrence: Updated results. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5633914","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":420300910,"identity":"ee997eba-ca09-4e0f-96a7-e2509ba79805","order_by":0,"name":"Michael Lewis","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYBACAyA+wNgmwcDPwNgAFmEDYgnCWs5JMEg2kKKFgfEfkHEASRS/FvYew4M/t1nIG1873PzxR8XhfD4G5oO3efBp4TljcJh3m4ThttuJbdI8Zw5btjGwJVvj1SKRu+Ew4zaJBDOgFmbGtsMGbAw8ZtJ4tci/3XDwZ5tEgvHsxOaPP8Fa+L/h1yLBu+EAL1CLgXRigwQvxBY2/Fp48j8cBmoxnAHxS7oBGzObseUcPFrs248lA91TJ88/O/0xMMSsDeTbmx/eeINHCxbATJryUTAKRsEoGAVYAACLvEk/DSv41QAAAABJRU5ErkJggg==","orcid":"","institution":"Curators of the University of Missouri: University of Missouri","correspondingAuthor":true,"prefix":"","firstName":"Michael","middleName":"","lastName":"Lewis","suffix":""},{"id":420300911,"identity":"72ff7025-b7d0-4322-8269-594fe4860eae","order_by":1,"name":"Claudia Chambers","email":"","orcid":"","institution":"Curators of the University of Missouri: University of Missouri","correspondingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"","lastName":"Chambers","suffix":""},{"id":420300912,"identity":"58f62d06-9334-4beb-a15b-dfea2e529579","order_by":2,"name":"Alexander Schaedler","email":"","orcid":"","institution":"Curators of the University of Missouri: University of Missouri","correspondingAuthor":false,"prefix":"","firstName":"Alexander","middleName":"","lastName":"Schaedler","suffix":""},{"id":420300913,"identity":"eb8cfd46-0e6c-43fb-a1a5-9c161a5497a7","order_by":3,"name":"Mojgan Golzy","email":"","orcid":"","institution":"Curators of the University of Missouri: University of Missouri","correspondingAuthor":false,"prefix":"","firstName":"Mojgan","middleName":"","lastName":"Golzy","suffix":""},{"id":420300914,"identity":"6954f5b9-a5f9-4c80-89df-c33048245406","order_by":4,"name":"Lisa Watkinson","email":"","orcid":"","institution":"Curators of the University of Missouri: University of Missouri","correspondingAuthor":false,"prefix":"","firstName":"Lisa","middleName":"","lastName":"Watkinson","suffix":""},{"id":420300915,"identity":"35cbdcc1-dca1-4a9e-85a2-66d27c44b13b","order_by":5,"name":"Terry Carmack","email":"","orcid":"","institution":"Curators of the University of Missouri: University of Missouri","correspondingAuthor":false,"prefix":"","firstName":"Terry","middleName":"","lastName":"Carmack","suffix":""},{"id":420300916,"identity":"e44a3768-5dc9-4540-8d38-e04012a44951","order_by":6,"name":"Vivian Yang","email":"","orcid":"","institution":"Curators of the University of Missouri: University of Missouri","correspondingAuthor":false,"prefix":"","firstName":"Vivian","middleName":"","lastName":"Yang","suffix":""},{"id":420300917,"identity":"f425abfb-7864-430e-bf6e-85afbbf8a01c","order_by":7,"name":"Kanishka Sikligar","email":"","orcid":"","institution":"Curators of the University of Missouri: University of Missouri","correspondingAuthor":false,"prefix":"","firstName":"Kanishka","middleName":"","lastName":"Sikligar","suffix":""},{"id":420300918,"identity":"798f3801-fcbc-46e9-b471-e380b96b762d","order_by":8,"name":"Joni Lunceford","email":"","orcid":"","institution":"Curators of the University of Missouri: University of Missouri","correspondingAuthor":false,"prefix":"","firstName":"Joni","middleName":"","lastName":"Lunceford","suffix":""},{"id":420300919,"identity":"fd8caf46-8ae5-4d31-9ffd-e08f359b2bfb","order_by":9,"name":"Colleen Garrett","email":"","orcid":"","institution":"Curators of the University of Missouri: University of Missouri","correspondingAuthor":false,"prefix":"","firstName":"Colleen","middleName":"","lastName":"Garrett","suffix":""},{"id":420300920,"identity":"298c4ea3-b168-437a-b9e0-411867857c56","order_by":10,"name":"Christos Papageorgiou","email":"","orcid":"","institution":"Curators of the University of Missouri: University of Missouri","correspondingAuthor":false,"prefix":"","firstName":"Christos","middleName":"","lastName":"Papageorgiou","suffix":""},{"id":420300921,"identity":"4d59538c-c22d-433f-9d97-efbf3a12b962","order_by":11,"name":"Jessica Talbott","email":"","orcid":"","institution":"Curators of the University of Missouri: University of Missouri","correspondingAuthor":false,"prefix":"","firstName":"Jessica","middleName":"","lastName":"Talbott","suffix":""},{"id":420300922,"identity":"700b0b1c-ea2b-4bc8-b465-9a1ff86459f3","order_by":12,"name":"Charles Maitz","email":"","orcid":"","institution":"Curators of the University of Missouri: University of Missouri","correspondingAuthor":false,"prefix":"","firstName":"Charles","middleName":"","lastName":"Maitz","suffix":""},{"id":420300923,"identity":"eb8a6c70-0ad8-48cd-b689-7fab261ef24e","order_by":13,"name":"Jeffrey Bryan","email":"","orcid":"","institution":"Curators of the University of Missouri: University of Missouri","correspondingAuthor":false,"prefix":"","firstName":"Jeffrey","middleName":"","lastName":"Bryan","suffix":""},{"id":420300924,"identity":"eee1ef22-3c00-4ee8-ac7b-82eeb36ea5f7","order_by":14,"name":"Charles Smith","email":"","orcid":"","institution":"Curators of the University of Missouri: University of Missouri","correspondingAuthor":false,"prefix":"","firstName":"Charles","middleName":"","lastName":"Smith","suffix":""}],"badges":[],"createdAt":"2024-12-12 19:44:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5633914/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5633914/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":77616755,"identity":"4092e39a-409f-4577-a777-829817a47c9a","added_by":"auto","created_at":"2025-03-03 15:06:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":45341,"visible":true,"origin":"","legend":"\u003cp\u003eUptake (A), internalization (B), and efflux (C) of [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e in SUM149 TNBC cells versus NIH 3T3 fibroblasts\u003c/p\u003e","description":"","filename":"Lewisetal.MIB2024Figure11.png","url":"https://assets-eu.researchsquare.com/files/rs-5633914/v1/1039c740833cf15dc0ee895e.png"},{"id":77616756,"identity":"9202a8f4-7997-4f2e-a8d3-e35b7fb4fb5d","added_by":"auto","created_at":"2025-03-03 15:06:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":40609,"visible":true,"origin":"","legend":"\u003cp\u003eBiodistribution of [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e in SUM149-bearing nude mice, given regular drinking water (A) or water containing TM (B)\u003c/p\u003e","description":"","filename":"Lewisetal.MIB2024Figure21.png","url":"https://assets-eu.researchsquare.com/files/rs-5633914/v1/63f5c092fc3bd0d9f1686191.png"},{"id":77616760,"identity":"32efeb77-c27f-48b8-954c-f93b98f530f4","added_by":"auto","created_at":"2025-03-03 15:06:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":579176,"visible":true,"origin":"","legend":"\u003cp\u003ePET/CT imaging of SUM149-bearing mice at 24 h post-injection, in the absence of TM (A) and in the presence of TM (B); L = liver, I = intestines, K = kidney, T = tumor\u003c/p\u003e","description":"","filename":"Lewisetal.MIB2024Figure31.png","url":"https://assets-eu.researchsquare.com/files/rs-5633914/v1/fcd00932bbbc39a54b7f5ef8.png"},{"id":77618328,"identity":"362c0259-1b89-4003-ab5b-10db032c54bf","added_by":"auto","created_at":"2025-03-03 15:14:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":186870,"visible":true,"origin":"","legend":"\u003cp\u003ePET/CT imaging of [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e in a healthy volunteer cat at 20 h post-injection. (A) sagittal slice; (B) coronal slice; L = liver, GB = gallbladder, I = intestines, K = kidney, B = bladder\u003c/p\u003e","description":"","filename":"Lewisetal.MIB2024Figure41.png","url":"https://assets-eu.researchsquare.com/files/rs-5633914/v1/68bb57ff4585f68fff37c1cd.png"},{"id":77616758,"identity":"e47410b7-3383-4313-80b0-a5fcb3356380","added_by":"auto","created_at":"2025-03-03 15:06:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":151748,"visible":true,"origin":"","legend":"\u003cp\u003ePET/CT imaging of [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e in a feline mammary carcinoma patient at 20 h post-injection. (A) sagittal slice; (B) transaxial slice; L = liver, I = intestines, T = tumor\u003c/p\u003e","description":"","filename":"Lewisetal.MIB2024Figure51.png","url":"https://assets-eu.researchsquare.com/files/rs-5633914/v1/2446616e00221072786489af.png"},{"id":79615155,"identity":"f3168982-b21c-4de2-bc53-325133854b70","added_by":"auto","created_at":"2025-03-31 19:04:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1623270,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5633914/v1/ff5ed7af-92ef-48b6-8862-b181f73e327d.pdf"}],"financialInterests":"","formattedTitle":"PET Imaging of Triple-Negative Breast Cancer and Tetrathiomolybdate Treatment Using [64Cu]Copper(II) Chloride","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBreast cancer is the most common cancer and second leading cause of cancer-related death in American women. Furthermore, its incidence in the United States is rising at a rate of 0.5% per year. This year, 310,720 new cases of breast cancer will be diagnosed in American women, along with 42,250 deaths [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Stage I-II hormone receptor-positive breast cancer responds well to both hormone therapy and chemotherapy, with 5-year survival rates of 90%. However, survival of women with metastatic disease drops to 28% at 5 years. Of particular concern is triple-negative breast cancer (TNBC), which lacks estrogen receptor, progesterone receptor, and Her2/neu. TNBC accounts for 15% of all breast cancers. It is characterized by a high degree of heterogeneity, aggressive behavior, constitutive activation of oncogenes, and poor overall response to standard-of-care, including hormone therapy and trastuzumab [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Treatment of stage IV TNBC includes the use of chemotherapy drugs such as anthracyclines, taxanes, capecitabine, gemcitabine, and eribulin, all of which have considerable dose-limiting toxicity. Patients with metastatic disease might respond better to emerging alternative treatments, such as targeted drugs, immunomodulators, and antiangiogenic therapy.\u003c/p\u003e \u003cp\u003eTriple-negative breast cancer is a copper-avid tumor type, relying on this element for cell survival and proliferation. Ammonium tetrathiomolybdate (TM, Decuprate\u0026reg;), which is approved for oral therapy of Wilson\u0026rsquo;s disease [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], can be used to deplete copper from tumors. It is possible that treatment with TM will be helpful in the subsets of patients with certain molecular and/or clinicopathologic characteristics that are considered high risk. Copper chelation therapy also has the potential to be monitored in a noninvasive manner, potentially part of a personalized medicine approach.\u003c/p\u003e \u003cp\u003eCopper chelation therapy inhibits several copper-dependent enzymes, including ceruloplasmin, ascorbate oxidase, cytochrome oxidase, superoxide dismutase, tyrosinase, ATPase, and mitochondria-specific copper chaperones [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Furthermore, copper depletion suppresses collagen remodeling [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], inhibits angiogenesis [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and induces apoptosis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] in TNBC. In all, a combination of these effects prevents tumor invasion and metastasis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In tumor-bearing mice, TM also renders tumors more susceptible to drugs such as cisplatin [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and doxorubicin [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo date, most clinical trials of TM have focused on biomarker analysis to assess the efficacy of the drug. Chan et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] monitored ceruloplasmin levels and LOXL2 expression to evaluate response to TM therapy in 75 patients, for two years or until relapse. Of those, 51 patients each received 1396 cycles of TM over the 2-year period, and levels of both biomarkers decreased and correlated with improved event-free survival. In another Phase II study, Liu and colleagues determined that collagen processing was altered by TM, as measured by LOXL2, Pro-C3, C1M, and C6M levels [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. LOXL2 expression increased in patients experiencing progressive disease or death, compared to patients with no evidence of disease, in whom LOXL2 levels remained stable. Collagen formation, assessed by Pro-C3 expression, increased, while markers of collagen degradation, such as C1M and C6M, increased in patients with disease progression/death, whereas these markers remained stable in those in remission. Conversely, C1M expression increased in those with no evidence of disease but remained stable in progressive patients and cases resulting in death. Blockhuys et al. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] examined the effect of the copper chaperone ATOX1 on TNBC and high-risk breast cancer patients. Their Phase II trial showed that ATOX1 levels increased in patients with better event-free survival after TM therapy. Those investigators found that as cytoplasmic ATOX1 levels increased, patients experienced better event-free survival. The opposite was observed in patients with nuclear ATOX1.\u003c/p\u003e \u003cp\u003eBiomarker analysis requires considerable method development and time to complete the assays. An alternative assessment of response to treatment with TM might be performed by noninvasive imaging, providing a more facile way to evaluate copper metabolism and correlate it with patient outcome. [\u003csup\u003e64\u003c/sup\u003eCu]copper(II) chloride could be used as a true radiotracer to measure copper utilization by PET/CT. Studies in healthy humans [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] with [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e have shown that the major organ of uptake was the liver, followed by the walls of the large intestine and the pancreas. Renal clearance was negligible. Lack of urinary excretion was particularly important in imaging prostate cancer [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], as no accumulation of radioactivity in the bladder occurred, and high contrast imaging of local disease was possible.\u003c/p\u003e \u003cp\u003eIn this report, we describe the use of [\u003csup\u003e64\u003c/sup\u003eCu]copper(II) chloride for PET/CT imaging in animal models of triple-negative breast cancer. To our knowledge, this tracer has not been evaluated in TNBC. In our in vitro studies, we found that TNBC cells preferentially take up copper. Moreover, PET/CT imaging of mice with human triple-negative xenografts and cats with naturally occurring mammary carcinoma showed specific and high contrast imaging in vivo, despite high accumulation of copper in the liver.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGeneral Information\u003c/h2\u003e \u003cp\u003eAll reagents were of the highest purity obtainable. Ultrapure water (18 MΩ-cm) was used for all experiments. Sterile phosphate-buffered saline (PBS) was purchased from Fisher Scientific (Pittsburgh, PA), and [\u003csup\u003e64\u003c/sup\u003eCu]Cu(II) chloride was obtained from Washington University in St. Louis and diluted to 0.1 \u0026micro;Ci/\u0026micro;L with PBS. A PerkinElmer PerkinElmer Wizard\u003csup\u003e2\u003c/sup\u003e 2480 (PerkinElmer Life Sciences, Gaithersburg, MD) automatic gamma counter was used to count radioactive samples. Tetrathiomolybdate (99.97% purity) was purchased from Millipore Sigma (St. Louis, MO).\u003c/p\u003e \u003cp\u003eSUM149 triple-negative breast cancer cells were purchased from Applied Biological Materials (Richmond, BC) and NIH 3T3 cells were obtained from the American Type Culture Collection (ATCC, Rockland, MD). SUM149 cells were maintained in Ham\u0026rsquo;s F-12 media, containing 5% heat-inactivated fetal bovine serum (FBS), 10 mM HEPES, 1 \u0026micro;g/mL hydrocortisone, and 5 \u0026micro;g/mL insulin, cultured at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. NIH 3T3 cells were cultured in DMEM with 10% FBS. Four- to six-week-old female NCR nude mice were obtained from Taconic Biosciences (Rensselaer, NY).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell Uptake, Internalization, and Efflux Assays\u003c/h3\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture\u003c/h2\u003e \u003cp\u003eCell studies were conducted using modifications of our previously published procedures [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. SUM149 cells were grown in Ham\u0026rsquo;s F-12 (Gibco\u0026trade;, Fisher Scientific) supplemented with 10% FBS (Gibco), insulin (5 \u0026micro;g/mL), hydrocortisone (1 \u0026micro;g/mL; both from Sigma-Aldrich, St. Louis, MO), and 1% penicillin-streptomycin. The cells were subsequently washed twice with serum free Minimum Essential Medium (MEM) before being drawn up to 8 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per mL. NIH 3T3 cells were grown in Dulbecco's Modified Eagle Medium (DMEM), with the addition of 10% FBS. The cells were subsequently washed twice with serum-free MEM before being drawn up to 8 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per mL.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eUptake\u003c/h3\u003e\n\u003cp\u003eCells were incubated (37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e) with 100,000 cpm of [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e/200 \u0026micro;L. Uptake of copper was evaluated over a time course in triplicate (5, 15, 30, 60, 120, 180, 240 min). Uptake assays were accomplished by separating the cell pellet via centrifugation (15,000 rpm, 4\u0026deg;C, 5 min) and washing once with ice-cold PBS. Cell-associated radioactivity was determined as the ratio of radioactivity in the cell pellet divided by the total radioactivity.\u003c/p\u003e\n\u003ch3\u003eInternalization\u003c/h3\u003e\n\u003cp\u003eCells were incubated (37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e) with 100,000 cpm of [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e/200 \u0026micro;L. Internalization of copper was evaluated over a time course in triplicate (5, 15, 30, 60, 120, 180, 240 min). An acid wash was completed (0.9% NaCl 500mL, 40 mM NaAc, 40 mM HAc, 1 g FBS, pH 4.5) to remove surface associated activity. The cell pellet was subsequently separated via centrifugation (15,000 rpm, 4\u0026deg;C, 5 min) and washed once with ice-cold PBS. Internalized cell-associated radioactivity was determined as the ratio of radioactivity in the cell pellet divided by the radioactivity in the combined supernatants.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEfflux\u003c/h2\u003e \u003cp\u003eFor efflux studies, cells were incubated (37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e) with 100,000 cpm of [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e/200 \u0026micro;L. Radiopharmaceutical uptake was performed as described above for 2 h at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. Then cells were subsequently washed with ice cold PBS and isolated by centrifugation before resuspending in fresh medium. Efflux of copper from the cells was measured at subsequent time points ranging from 5 min to 4 h, after isolating aliquots of the cells in triplicate, washing the cells in ice cold PBS, and counting them separately from the supernatant, as described above.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBiodistribution Studies\u003c/h3\u003e\n\u003cp\u003e All animal studies were conducted in accordance with the guidelines established by the University of Missouri Animal Care and Use Committee. NCR nude mice were implanted with 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e SUM149 cells in PBS:Matrigel (2:1), subcutaneously (SC) in the hind flank. Eight weeks after tumor inoculation, when tumors had grown to 50\u0026ndash;100 mg, mice received an intravenous (IV) injection of 0.37 MBq (10 \u0026micro;Ci) of [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e in 100 \u0026micro;L of PBS. Prior to the study, half of the mice received regular drinking water, and half received 0.03 mg/mL of TM in drinking water [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] for one week. At 1, 4, and 24 h, biodistributions (n\u0026thinsp;=\u0026thinsp;4) were obtained. Tissues taken included blood, liver, spleen, lung, heart, stomach, large and small intestines, muscle, bone, brain, pancreas, kidneys, and tumor. Urine and feces were collected as well. Tissues were weighed and counted in the gamma counter with a standard of the injected dose, such that % injected dose per gram of tissue (% ID/g) and % injected dose per organ (% ID) were determined as decay-corrected uptakes.\u003c/p\u003e\n\u003ch3\u003eMurine PET/CT Imaging\u003c/h3\u003e\n\u003cp\u003eAn aliquot of 200 MBq (6 mCi) of [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e in approximately 10 \u0026micro;L of 0.05 M HCl was diluted to 53.7 MBq/mL (1.45 mCi/mL) with PBS. Mice (n\u0026thinsp;=\u0026thinsp;6) were injected IV via the tail vein with 100 \u0026micro;L of this solution. As in the biodistribution studies described above, half of the mice received TM in drinking water, and half drank regular water. Under isoflurane anesthesia, mice were placed in a prone position, and PET/CT imaging was performed on a Bruker Albira Si SPECT/PET/CT camera. Images were acquired over 15 min, with an axial field of view of 99 mm and a bed position offset at 105 mm. Next, CT imaging was performed for 13 min. PET/CT images were processed using PET ordered subset expectation maximization (OSEM) software. One iteration at 0.75 mm was used, with decay, random, and scatter corrections.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFeline PET/CT Imaging\u003c/h2\u003e \u003cp\u003e Feline studies were also conducted in accordance with the guidelines established by the University of Missouri Animal Care and Use Committee. Companion cats were anesthetized, and an IV catheter was placed in the right forelimb. The cats were injected with 280 MBq (7.6 mCi) of [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e in 0.5 mL of sterile PBS. At 20 h post-injection, cats were scanned using a Canon Celestion PET/CT camera. PET imaging parameters were 2\u0026ndash;3 min per bed position, 4 mm slices, and 50% overlap between slices. The voxel size was 4 \u0026times; 4 \u0026times; 4 mm\u003csup\u003e3\u003c/sup\u003e, the field of view (FOV) was 240 mm, and the pixel size was 2.0. A coaxial CT scan was performed using settings of 120 kV/300 mA, 0.75 s rotation, detailed HP (11.0/PF 0.688), and the same FOV. PET data were reconstructed with time-of-flight 3-dimensional ordered subset expectation maximization (TOF 3D OSEM) with a Gaussian postfilter. CT images were reconstructed with a 3-dimensional adaptive iterative dose reduction (AIDR 3D) algorithm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eRepeated measure analysis of variance (ANOVA), including the between-subject variable, was used to assess the effect of water treatment on outcomes of interest; i.e., %ID/g and %ID. The interaction between time (in hours) and water treatment was considered in each model. The R-square goodness of fit measure was used to assess the model. The partial sum of squares (Type III sum of squares) was used to specify the significant main effect of time, water treatment and their interactions on each outcome. The interaction term tested whether the effect of water treatment on the outcome is the same over the time interval, meaning that slopes of regression lines are significantly different for the two groups.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCell Studies\u003c/h2\u003e \u003cp\u003eUptake, internalization, and efflux studies showed greater retention in SUM149 triple-negative breast cancer cells than in normal NIH 3T3 fibroblasts. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, the amount of [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e taken up by SUM149 cells was 19.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6% at 5 min, and this value increased to 40.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3% after 4 h of incubation. In contrast, uptake in fibroblasts was 9.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13%ID/g at 5 min and increasing to 16.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1% at 4 h, less than half that of the TNBC cells. SUM149 cells also internalized \u003csup\u003e64\u003c/sup\u003eCu to a greater degree than 3T3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), increasing from 9.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3% at 5 min to 43.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3% by 4 h of incubation. Internalization in the fibroblast cell line was 7.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1% at 5 min, but it only increased to 23.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2% at 4 h. Retention of \u003csup\u003e64\u003c/sup\u003eCu in SUM149 cells was also greater than in 3T3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), as efflux of copper from the TNBC cells was slower than that from the fibroblasts. At 5 min, retention of \u003csup\u003e64\u003c/sup\u003eCu in SUM149 cells was 91.0\u0026thinsp;\u0026plusmn;\u0026thinsp;6.7%, decreasing to 65.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9% after 4 h. By comparison, the amount of copper retained in 3T3 cells was 87.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5% at 5 min and 56.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7% after 4 h of incubation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eBiodistribution Studies\u003c/h2\u003e \u003cp\u003eBiodistributions of [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e were obtained in SUM149-bearing nude mice, with (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) or without (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) tetrathiomolybdate in drinking water for one week. In each case, the major organ of uptake was the liver. Control mice receiving water showed liver accumulations of 23.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6%ID/g, 24.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7%ID/g, and 13.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6%ID/g at 1, 4, and 24 h, respectively. Concomitantly, there was also a high degree of radioactivity in the gastrointestinal tract, which was most pronounced at 1 h and 4 h. Accretion of copper in the small intestine increased from 13.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2%ID/g at 1 h to 17.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9%ID/g at 4 h, after which it decreased to 4.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0%ID/g at 24 h. The uptakes of \u003csup\u003e64\u003c/sup\u003eCu in the large bowel followed those in the small intestine over the time course of the study. Accumulations of 6.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4%ID/g at 1 h, 21.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3%ID/g at 4 h, and 5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2%ID/g at 24 h were observed in the large intestine, demonstrating the transit of \u003csup\u003e64\u003c/sup\u003eCu through the GI tract. The other major organ of uptake was the kidney. At 1 h, kidney accretion was 11.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2%ID/g, which remained relatively constant at 4 h (12.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2%ID/g) and then decreased at 24 h (8.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3%ID/g). Blood clearance of \u003csup\u003e64\u003c/sup\u003eCu was also protracted, ranging from 2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4%ID/g to 3.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9%ID/g at 4 h and 1.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1%ID/g at 24 h. All other organs showed relatively moderate levels of radioactivity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMice that received TM had liver uptakes of 18.1\u0026thinsp;\u0026plusmn;\u0026thinsp;5.8%ID/g at 1 h, 29.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.4%ID/g at 4 h, and 23.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5%ID/g at 24 h, which were comparable to those in controls. Interestingly, high gut uptake was not observed in mice administered TM. Instead, clearance of \u003csup\u003e64\u003c/sup\u003eCu shifted dramatically toward the renal pathway. While liver uptake remained relatively constant throughout the study, kidney uptake increased substantially to 15.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9%ID/g at 1 h, 20.9\u0026thinsp;\u0026plusmn;\u0026thinsp;5.0%ID/g at 4 h, and 18.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8%ID/g at 24 h, considerably higher than in untreated mice. As in controls, prolonged retention of radioactivity from the blood was observed. Blood uptakes of 18.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2%ID/g, 11.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4%ID/g, and 2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8%ID/g were observed at 1, 4, and 24 h, respectively. As in the case of untreated controls, accumulation of radioactivity in other organs was relatively low.\u003c/p\u003e \u003cp\u003eTumor uptake was initially similar in both groups of animals, then it decreased steadily in the TM group at later time points. At 1 h, tumor accretion was 4.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3%ID/g when animals received the drug, compared to 5.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8%ID/g in untreated mice. By 4 h post-injection, uptake in the tumor was 9.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6%ID/g for mice receiving plain water, versus 3.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3%ID/g in mice administered TM. While this value remained relatively constant at 24 h (3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7%ID/g), tumor uptake in untreated animals was considerably higher (7.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5%ID/g).\u003c/p\u003e \u003cp\u003eStatistical differences between mice receiving regular water versus mice receiving TM were the result of 1) administration of the drug (TM versus regular water), 2) time, and 3) an interaction of water and time. For example, in the liver there was no statistical difference between the two groups according to the type of water administered (P\u0026thinsp;=\u0026thinsp;0.14) overall. However, a significant primary effect of time (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and a significant time \u0026times; water treatment interaction on outcome (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0032) were observed, meaning that a significantly different trend for the two water treatment groups existed. However, the regression lines of the two groups crossed each other, resulting in no significant overall mean outcomes difference (data not shown).\u003c/p\u003e \u003cp\u003eThe largest difference between the two groups in the biodistribution studies was uptake in the intestines. In the small bowel, the effects of TM in water (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) over the time course of the study (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) resulted in a significant interaction (P\u0026thinsp;=\u0026thinsp;0.0004). In the large intestine, the type of water gave significantly different results (P\u0026thinsp;=\u0026thinsp;0.011), but over the course of the study, time did not (P\u0026thinsp;=\u0026thinsp;0.21). No significant interaction for large intestinal uptake (P\u0026thinsp;=\u0026thinsp;0.15) was observed. The kidneys showed highly significant differences according to the presence or absence of TM (P\u0026thinsp;=\u0026thinsp;0.0021) over time (P\u0026thinsp;=\u0026thinsp;0.011), with a significantly different trend for the two treatments (P\u0026thinsp;=\u0026thinsp;0.015). In the tumor, the effect of TM alone was quite noticeable. While time was not a significant factor in tumor uptake (P\u0026thinsp;=\u0026thinsp;0.27), the administration of TM had a prominent effect (P\u0026thinsp;=\u0026thinsp;0.023). However, the interaction of water and TM did not produce a significant effect (P\u0026thinsp;=\u0026thinsp;0.95), meaning the drug had the primary effect on tumor uptake.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMurine PET/CT Imaging\u003c/h2\u003e \u003cp\u003ePET/CT imaging of SUM149-bearing nude mice was conducted at 1, 4, and 24 h post-injection. These studies gave results that were consistent with those of the conventional biodistribution studies. At 1 h and 4 h, high uptake in the intestines obscured imaging of the xenograft, which was only seen after sufficient clearance at 24 h. In mice receiving regular water (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), tumors were clearly detected, along with high liver uptake. Concomitant with high liver accumulation was pronounced uptake in the intestines. High liver uptake was also observed in animals receiving TM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB); however, uptake in the intestines was minimal. Instead of GI clearance, excretion appeared to shift to the kidneys, which was consistent with biodistribution results. Little to no tumor uptake was seen in mice administered TM. These results suggest that copper chelation can not only prevent accumulation of the element in the xenograft, but also increase renal clearance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eFeline PET/CT Imaging\u003c/h2\u003e \u003cp\u003eIn the first study, a healthy volunteer companion cat was scanned at 20 h post-injection of [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e. The imaging results, shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, were also consistent with mouse PET/CT. The major off-target organs were the liver and gallbladder, while the intestines and kidneys showed moderate activity. A moderate level of radioactivity was also observed in the bladder, as urinary excretion was a significant route of elimination.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on the imaging results in the normal cat, we have opened a clinical trial of [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e PET/CT imaging in companion cats with mammary carcinoma. An example is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Again, the liver was the major organ of uptake (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In this patient, a chain of tumors in four mammary glands was observed. When images of the patient were analyzed in transaxial mode (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), all of the lesions were very conspicuous, even in close proximity to the highly radioactive liver.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present studies, we evaluated the tumor uptake and retention of [\u003csup\u003e64\u003c/sup\u003eCu]copper(II) chloride in cell and animal models of TNBC, and we determined the effect of tetrathiomolybdate treatment in tumor-bearing mice. At present, the only common treatments for metastatic TNBC consist of dose-limiting chemotherapy regimens. Recently, pembrolizumab (Keytruda\u0026reg;) has received FDA approval to treat TNBC, but this antibody only targets PD-L1-positive tumors that account for 20\u0026ndash;37% of triple-negative breast cancers. Furthermore, pembrolizumab also has highly toxic side effects. These side effects include loss of immune tolerance, resulting in organ failure or even death. This antibody also has the potential to cause adverse events in the form of infusion reactions, which can be severe or life-threatening.\u003c/p\u003e \u003cp\u003eIn contrast, TM therapy is an oral drug that is well-tolerated and can be administered several times a day. In one study, Liu et al. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] found that TM therapy resulted in 71.7% event-free survival and 74.2% overall survival in TNBC patients receiving TM over a median period of 9.4 years, with a median of 3423 treatment cycles. Another clinical trial performed by Sahota and coworkers demonstrated that TM therapy gave 90% event-free survival at 6.9 years for Stage II/III TNBC and 66.7% progression-free survival for Stage IV TNBC [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Chan et al. achieved similar results in their study, with 69% event-free survival of Stage IV triple-negative patients after 6.3 years [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In triple-negative breast cancer, measurement of copper biomarkers has been used by two groups to evaluate patients and to monitor TM therapy. Chan et al. showed that TM therapy was safe and well-tolerated, and it resulted in lower levels of ceruloplasmin and LOXL2, as determined by ELISA [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Furthermore, Blockhuys and coworkers used immunohistochemistry to demonstrate that TM treatment also reduces expression of ATOX1, which correlates with improved event-free survival [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHerein we report the use of [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e PET/CT as a modality for noninvasive imaging of mouse and feline models of TNBC. Our cell studies showed that [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e is preferentially taken up by TNBC cells, as opposed to normal fibroblasts. Uptake in SUM149 triple-negative breast cancer cells was approximately 2.5 times higher than in NIH 3T3 fibroblasts, while internalization of \u003csup\u003e64\u003c/sup\u003eCu was about 2 times higher in the triple-negative cell line. Copper-64 was also retained to a greater degree in SUM149 cells, with an approximate difference of 9% in efflux compared to the normal cell line.\u003c/p\u003e \u003cp\u003eEvaluation of biodistribution data demonstrated preferential uptake of [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e in the liver, where hepatic uptake reached a maximum of 32.7% of the total dose at 1 h and cleared to 17.7%ID by 24 h, while approximately 1% of the dose targeted the tumor. Most of the excretion occurred through the hepatobiliary pathway. In mice treated with TM, liver accumulation was initially lower at 1 h, reached a peak at 4 h, and then decreased by 24 h. Kidney uptake of [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e in untreated mice decreased throughout the course of the study. However, renal accumulation of the tracer was higher in animals receiving TM, where kidney accretion actually increased over time. The reason for this shift to renal clearance upon receiving TM is unknown and would be worth further investigation.\u003c/p\u003e \u003cp\u003eIn the tumor, uptake in mice administered TM was considerably lower. At 1 h post-injection, tumor accumulation in treated animals was approximately the same as in control mice, but by 4 h the uptake of [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e was about 6%ID/g higher in the untreated group and remained higher in these animals at 24 h. The data indicate that TM therapy results in lower uptake and retention of \u003csup\u003e64\u003c/sup\u003eCu in tumor-bearing mice. PET/CT imaging of SUM149-bearing mice was consistent with this trend as well. The optimal imaging time was 24 h post-injection, due to the high excretion of \u003csup\u003e64\u003c/sup\u003eCu in the intestines. While tumors were clearly detected in mice receiving plain water at 24 h, little to no signal from the xenograft was observed in mice given TM. Imaging of other organs was also consistent with the conventional biodistribution data. While the liver and intestines in untreated mice were clearly visible, the shift to renal clearance in animals given TM was readily apparent, with high contrast imaging of the kidneys.\u003c/p\u003e \u003cp\u003ePET/CT imaging of companion cats afforded results that mirrored those obtained in the SUM149 murine model. In the normal cat, the liver was the major organ of uptake, and excretion into the gallbladder was especially prominent. Accumulation in the gastrointestinal tract, kidneys, and bladder were modest in comparison.\u003c/p\u003e \u003cp\u003eIn the cat with mammary carcinoma, high liver and gallbladder accumulation was also observed, along with considerable excretion into the intestine. The chain of mammary tumors in the abdomen was also clearly displayed, but liver uptake obscured imaging of more cranial lesions. In order to detect tumors in close proximity to the liver, evaluation of imaging in transaxial mode allowed high resolution in this region.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn these studies, we demonstrated that PET/CT imaging using [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e in animal models of triple-negative breast cancer allowed detection of tumors with high sensitivity and resolution. Consistent with higher selective uptake in TNBC cells, we found that [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e could detect triple-negative tumors in murine and feline models of breast cancer. We have also shown that [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e has potential to serve as a true radiotracer to monitor copper chelation therapy with TM. At present, we are conducting a clinical trial of [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e PET/CT imaging in cats with mammary carcinoma. Our goal is to correlate standardized uptake values (SUVs) to copper biomarkers and receptor status in feline patients. In the future, we plan to conduct similar imaging studies in feline mammary carcinoma patients receiving TM.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003eThe authors are grateful for excellent technical support from Katie Tucker.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis work was supported by a grant from the University of Missouri Molecular Imaging and Theranostics Center (to MRL).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing financial interests or personal relationships.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003eAll authors, experimental design and execution; MRL, original draft, supervision, project administration, methodology, investigation, formal analysis, data curation; CGC, methodology, investigation, formal analysis; AWS, methodology, investigation, formal analysis; MG, methodology, formal analysis; LDW and TLC, methodology, investigation, data curation; KS, methodology, formal analysis; CP, supervision and project administration; VAY, JML, CG, JLT, CAM, and JNB, methodology, investigation, formal analysis, data curation, project administration; CJS, manuscript review and editing, project administration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u0026nbsp;\u003c/strong\u003eAll animal studies were conducted in accordance with the guidelines established by the University of Missouri Animal Care and Use Committee.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003eAnalysis of data supporting our findings is included in the article. The raw data generated by these studies is available from the corresponding author on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSiegel RL, Giaquinto AN, Jemal A (2023) Cancer statistics, 2024. CA Cancer J Clin 74:12-49\u003c/li\u003e\n\u003cli\u003eJin J, Tao Z, Cao J, Li T, Hu X (2021) DNA damage response inhibitors: An avenue for TNBC treatment. Biochim Biophys Acta 1875:188521\u003c/li\u003e\n\u003cli\u003eDe Fabregues O, Vi\u0026ntilde;as, Palas\u0026iacute; A, Quintana M, Cardona I et al (2020) Ammonium tetrathiomolydate in the decoppering phase treatment of Wilson\u0026apos;s disease with neurological symptoms: A case series. Brain Behav 10:e01596\u003c/li\u003e\n\u003cli\u003eAlvarez HM, Xue Y, Robinson CD, Canalizo-Hern\u0026aacute;ndez MA, Marvin RG et al (2010) Tetrathiomolybdate inhibits copper trafficking proteins through metal cluster formation. Science 327:331-334\u003c/li\u003e\n\u003cli\u003eRamchandani D, Berisa M, Tavarez DA, Li Z, Miele M et al (2021) Copper depletion modulates mitochondrial oxidative phosphorylation to impair triple negative breast cancer metastasis. Nature Commun 12:7311\u003c/li\u003e\n\u003cli\u003eBlockhuys S, Hildesj\u0026ouml; C, Olsson H, Vahdat L, Wittung-Strafshede P (2021) Evaluation of ATOX1 as a potential predictive biomarker for tetrathiomolybdate treatment of breast cancer patients with high risk of recurrence. Biomedicines 9:1887\u003c/li\u003e\n\u003cli\u003eLiu YL, Bager CV, Willumsen N, Ramchandani D, Kornhauser N et al (2021) Tetrathiomolybdate (TM)-associated copper depletion influences collagen remodeling and immune response in the pre-metastatic niche of breast cancer. NPJ Breast Cancer 108\u003c/li\u003e\n\u003cli\u003eJoimel U, Gest C, Soria J, Pritchard L-L, Alexandre J et al (2010) Stimulation of angiogenesis resulting from cooperation between macrophages and MDA-MB-231 breast cancer cells: Proposed molecular mechanism and effect of tetrathiomolybdate. BMC Cancer 10:375\u003c/li\u003e\n\u003cli\u003eMcCarty MF, Block KI (2005) Multifocal angiostatic therapy: An update. Integr Cancer Ther 4:301-314\u003c/li\u003e\n\u003cli\u003ePan Q, Kleer CG, van Golen KL, Irani J, Bottema KM et al (2002) Copper defiency induced by tetrthiomolybdate suppresses tumor growth and angiogenesis. Cancer Res 62:4854-4859\u003c/li\u003e\n\u003cli\u003eKarginova O, Weekley CM, Raoul A, Alsayed A, Wu T et al (2019) Inhibition of copper transport induces apoptosis in triple-negative breast cancer cells and supresses tumor angiogenesis. Mol Cancer Ther 18:873-885\u003c/li\u003e\n\u003cli\u003ePan Q, Bao LW, Merajver SD (2003) Tetrathiomolybdate inhibits angiogenesis and metastasis through supression of the nf\u003csub\u003ek\u003c/sub\u003eb signaling cascade. Mol Cancer Res 1:701-706\u003c/li\u003e\n\u003cli\u003eChisholm CL, Wang H, Wong AH-H, Vazquez-Ortiz G, Chen W et al (2016) Ammonium tetrathiomolybdate treatment targets the copper transporter ATP7A and enhances sensitivity of breast cancer to cisplatin. Oncotarget 7:84439-84452\u003c/li\u003e\n\u003cli\u003ePan Q, Bao LW, Kleer CG, Brewer GJ, Merajver SD (2003) Antiangiogenic tetrathiomolybdate enhances the efficacy of doxorubicin against breast carcinoma. Mol Cancer Ther 2:617-622\u003c/li\u003e\n\u003cli\u003eChan N, Willis A, Kornhauser N, Ward MM, Lee SB et al (2017) Influencing the tumor microenvironment: A phase II study of copper depletion using tetrathiomolybdate in patients with breast cancer at high risk for recurrence and in preclinical models of lung metastases. Clin Cancer Res 23:666-676\u003c/li\u003e\n\u003cli\u003eLiu YL, Liv Bager C, Willumsen N, Ramchandani D, Kornhauser N et al (2021) Tetrathiomolybdate (TM)-associated copper depletion influences collagen remodeling and immune response in the pre-metastatic niche of breast cancer. NPJ Breast Cancer 7:108\u003c/li\u003e\n\u003cli\u003eAvila-Rodriguez MA, Rios C, Carrasco-Hernandez J, Manrique-Arias JC, Martinez-Hernandez R et al (2017) Biodistribution and radiation dosimetry of [\u003csup\u003e64\u003c/sup\u003eCu]copper dichloride: First-in-human study in healthy volunteers. EJNMMI Res 7:98\u003c/li\u003e\n\u003cli\u003eKj\u0026aelig;gaard K, Sandahl TB, Frisch K, Vase KH, Keiding S et al (2020) Intravenous and oral copper kinetics, biodistribution and dosimetry in healthy humans studied by [\u003csup\u003e64\u003c/sup\u003eCu]copper PET/CT. EJNMMI Radiopharm Chem 5:15\u003c/li\u003e\n\u003cli\u003eChakravarty R, Shetty P, Nair KVV, Rajeswari A, Jagadeesan KC et al (2020) Reactor produced [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e as a pet radiopharmaceutical for cancer imaging: From radiochemistry laboratory to nuclear medicine clinic. Ann Nucl Med 34:899-910\u003c/li\u003e\n\u003cli\u003eMascia M, Villano C, De Francesco V, Schips L, Marchioni M et al (2021) Efficacy and safety of the \u003csup\u003e64\u003c/sup\u003eCu(II)Cl\u003csub\u003e2\u003c/sub\u003e PET/CT for urological malignancies. Clin Nucl Med 46:443-448\u003c/li\u003e\n\u003cli\u003eJia F, Daibes Figueroa S, Gallazzi F, Balaji BS, Hannink M et al (2008) Molecular imaging of \u003cem\u003ebcl-2\u003c/em\u003e expression in small lymphocytic lymphoma using \u003csup\u003e111\u003c/sup\u003eIn-labeled PNA-peptide conjugates. J Nucl Med 49:430-438\u003c/li\u003e\n\u003cli\u003eSahota S, Kornhauser N, Willis A, Ward MM, Cigler T et al (2017) A phase II study of copper-depletion using tetrathiomolybdate (TM) in patients (pts) with breast cancer (bc) at high risk for recurrence: Updated results. J Clin Oncol 35:suppl.2557\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Triple-negative breast cancer, 64Cu, PET imaging, tetrathiomolybdate, chelation therapy","lastPublishedDoi":"10.21203/rs.3.rs-5633914/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5633914/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eIn the United States, breast cancer is the second leading cause of cancer-related death. Triple-negative breast cancer (TNBC) is of substantial concern, as it lacks the receptors usually targeted by conventional treatments. Triple-negative breast tumors have a high degree of copper metabolism for the synthesis of transporters, enzymes, and chaperones. Tetrathiomolybdate (TM) is a well-tolerated oral therapy that has been investigated for chelating copper from tumors in TNBC patients, resulting in extended remission. The overall goal of this research was to evaluate [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e PET/CT imaging of copper utilization in this disease, in the presence and absence of TM.\u003c/p\u003e\u003ch2\u003eProcedures\u003c/h2\u003e \u003cp\u003eUptake, internalization, and efflux studies were performed in TNBC cells versus normal cells. Biodistribution experiments were then conducted in TNBC xenograft-bearing mice that were administered TM versus controls. PET/CT imaging of mice carrying TNBC tumors was also performed in the presence and absence of TM. Finally, imaging was performed in a healthy cat and cats with mammary carcinoma.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSUM149 TNBC cells selectively took up, internalized, and retained [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e more avidly than normal fibroblasts. When SUM149-bearing mice were given TM, tumor uptake decreased and tracer accumulation shifted predominantly to the liver and kidneys, compared to control mice, in which large quantities of \u003csup\u003e64\u003c/sup\u003eCu were excreted into the intestines. These results were supported by PET/CT imaging of the mice. PET/CT of companion cats gave results similar to those obtained in mice, with high accumulation of radioactivity observed in the liver and gallbladder and moderate intestinal and renal clearance. In a cat with mammary carcinoma, [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e was highly conspicuous, even in close proximity to the liver.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eUtilization of [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e in triple-negative breast cancer can be detected efficiently in cell and animal models of this disease. The tracer was also used successfully to evaluate TM therapy in the SUM149 TNBC mouse model. Furthermore, PET/CT imaging of both mice and cats with breast cancer shows the potential to monitor treatment with TM in a facile, noninvasive manner. We are currently conducting a clinical trial of [\u003csup\u003e64\u003c/sup\u003eCu]CuCl\u003csub\u003e2\u003c/sub\u003e PET/CT in companion cats with mammary carcinoma, with the future goal of evaluating the efficacy of TM in feline patients.\u003c/p\u003e","manuscriptTitle":"PET Imaging of Triple-Negative Breast Cancer and Tetrathiomolybdate Treatment Using [64Cu]Copper(II) Chloride","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-03 15:06:09","doi":"10.21203/rs.3.rs-5633914/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"91f4e62a-9a14-45b9-af6d-6fa1578a713d","owner":[],"postedDate":"March 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-31T18:55:59+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-03 15:06:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5633914","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5633914","identity":"rs-5633914","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}