Copper-67 radioimmunotheranostics for simultaneous immunotherapy and immuno-SPECT

Copper-67 (t1/2 = 2.58 days) decays by β− (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\text{E}_{{\upbeta^{-}\text{max}}}$$\end{document}Eβ-max: 562 keV) and γ-rays (93 keV and 185 keV) rendering it with potential for both radionuclide therapy and single-photon emission computed tomography (SPECT) imaging. Prompted by the recent breakthrough of 67Cu production with high specific activity, high radionuclidic purity, and sufficient quantities, the interest in the theranostic potential of 67Cu has been rekindled. This work addresses the practicability of developing 67Cu-labeled antibodies with substantially improved quality for cancer radioimmunotheranostics. Proof of concept is demonstrated with pertuzumab, a US-FDA-approved monoclonal antibody for combination therapies of HER2-positive breast cancer. With an average number of 1.9 chelators coupled to each antibody, we achieved a two-order of magnitude increase in radiolabeling efficiency compared to literature reports. In a preclinical therapeutic study, mice (n = 4–7/group) bearing HER2+ xenografts exhibited a 67Cu-dose dependent tumor-growth inhibition from 67Cu-labeled-Pertuzumab co-administered with trastuzumab. Furthermore, greater tumor size reduction was observed with 67Cu-labeled-pertuzumab formulations of higher specific activity. The potential of SPECT imaging with 67Cu radiopharmaceuticals was tested after 67Cu-labeled-Pertuzumab administration. Impressively, all tumors were clearly visualized by SPECT imaging with 67Cu-labeled-Pertuzumab even at day 5 post injection. This work demonstrates it is practical to use 67Cu radioimmunoconjugates for cancer radioimmunotheranostics.

www.nature.com/scientificreports/ of several layers of tumor cells and its shorter half-life (t 1/2 = 2.58 d) is more optimal for patient care. In addition to 67 Cu, copper has several other radioisotopes ( 60 Cu, 61 Cu, 62 Cu, and 64 Cu) that have been reported for the development of PET radiopharmaceuticals. Of them, 64 Cu has been widely used for preclinical and clinical PET studies due to its moderate half-life (t 1/2 = 12.7 h), low positron energy, and availability 13 . Given that copper radioisotopes are chemically identical, the same bifunctional chelators that have been developed for 64 Cu radiopharmaceuticals can be used directly for 67 Cu radiopharmaceuticals 13 . More importantly, PET imaging can be made readily available by simply swapping 67 Cu with 64 Cu in the same radiopharmaceutical when desired for pre-screen of patients for targeted 67 Cu-radionulide therapy or dosimetry estimation. Surprisingly to some extent, in the reported nuclear medicine applications of 67 Cu, its γ-emissions at 93 keV (16%) and 185 keV (49%) 14 are largely neglected for SPECT imaging. Of note, the branching ratios of 67 Cu's γ-emissions are much higher than those of 177 Lu's, which would provide the much-needed imaging sensitivity. Despite the potential for both imaging and therapy, the use of 67 Cu for targeted radionuclide therapy has been hampered for decades by its limited supply and low specific activity. The production of 67 Cu has been tested on a variety of nuclear reactions, such as 68 Zn(p, 2p) 67 Cu, 70 Zn(p, α) 67 Cu, 68 Zn(γ, p) 67 Cu, and 67 Zn(n,p) 67 Cu, in addition to heavy-ion fragmentation [15][16][17][18][19][20][21][22][23][24][25] . However, therapeutic quantities of 67 Cu at high specific activity were not achieved until recently 21 . A major breakthrough was reported in the 68 Zn(γ,p) 67 Cu reaction by irradiating a highly enriched 68 Zn (98.97%) target with high energy γ rays produced by bremsstrahlung conversion of electrons from electron accelerator 26,27 . The specific activity of 67 Cu so produced has reached over 5.55 GBq/μg (150 mCi/ μg) 26 , which is a big leap as compared to that (0.69 GBq/μg or 18.6 mCi/μg) obtainable by conventional 68 Zn(p, 2p) 67 Cu reaction 28 . Due to this breakthrough in 67 Cu production, no-carrier-added 67 Cu has been made available in large scales at the National Isotope Development Center 29 . Indeed, a clinical trial with 67 Cu-Octreotate was reported in 2019 30 , and a few preclinical theranostic studies with 67 Cu have been seen recently [31][32][33] . In this work, we evaluated the use of 67 Cu produced by the electron accelerator method with the aim to reassess the role of 67 Cu in radioimmunotherapy coupled with the up-to-date SPECT technology. A HER2 targeting antibody, pertuzumab, was used as a model antibody to construct the desired radioimmunotheranostic conjugate.

Results
Pertuzumab conjugation, 67 Cu radiolabeling, and in vitro stability. The conjugation of p-SCN-Bn-NOTA (2-S-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid) to pertuzumab (20:1 molar ratio) was performed at pH 8.5 for 3 h at room temperature, followed by purification by fast protein liquid chromatography (FPLC). Under the condition, the average number of NOTA chelators per pertuzumab molecule was determined to be 1.9 by the isotope dilution method (Fig. 1). We conducted a series of radiolabeling tests varying the amount of the antibody conjugate for labeling with a fixed amount (27 MBq) of 67 Cu (Table 1) in order to obtain the highest achievable radiolabeling efficiency and specific activity of [ 67 Cu] Cu-NOTA-Pertuzumab. By maintaining the amount of NOTA-Pertuzumab at 5 µg and above, the radiolabeling yield was virtually quantitative. When the conjugate amount dropped to 2 µg, 1 µg, and 0.5 µg, the radiochemical yield decreased to 54%, 32%, and 13%, respectively. Accordingly, the highest achievable specific activity was attained at 8.6 GBq/mg (or 230 mCi/mg) at the end of synthesis when the amount of conjugate was 1 µg. The radiopharmaceutical purity was over 99% after purification as determined by both radio-TLC and radio-FPLC. The in vitro stability of [ 67 Cu]Cu-NOTA-Pertuzumab was found stable (> 97% intact) out to 7 days in rat serum, indicating no appreciable dissociation of 67 Cu from the conjugate in the presence of serum proteins.   Fig. 2A and Figure S1). The immunoreactive fraction of [ 67 Cu]Cu-NOTA-Pertuzumab was determined to be 80.6%, which is comparable to previously reported 67 Cu-labeled immunoconjugates 12,34 or pertuzumab conjugates 35 . It indicates that the immunoreactivity of pertuzumab with HER2 was maintained when conjugated with NOTA at the molar ratio of 1:1.9 (Pertuzumab:NOTA) (Fig. 2B) 34,37 . Shown in Fig. 3A and Figure    www.nature.com/scientificreports/ 25 days. As the radiation dose escalated to the medium and high doses, which correspond to 2 times and 3 times of the low dose, respectively, tumor size reduction was observed almost immediately after the dose administration. Interestingly, we observed a better treatment outcome in Group 3 than in Group 5, both of which received the same amount of 67 Cu-activity but the latter with 20 times lower specific activity.

Radiation toxicity resulted from treatment with [ 67 Cu]Cu-NOTA-Pertuzumab.
All the mice in the control group (Group 1) survived to day 22 after the start of treatment but they were sacrificed due to the drastically increased tumor burden (Fig. 3B). Impressively, the mice in the low dose group (Group 2) experienced a weight loss ( Figure S3) in the beginning but regained the weight back to normal as the control group within week 3, indicating that the single-dose injection of 3.7 MBq was tolerable. For the groups treated with escalated doses of 7.4 and 14.8 MBq, mice started to lose weight from day 4 post treatment in tandem with observed tumor burden reduction. The fact that the mice did not regain their weight back to normal indicates the unwanted systemic toxicity resulted from [ 67 Cu]Cu-NOTA-Pertuzumab administered at the medium and high doses. Consequently, the mice in Group 3 (medium dose) showed 50% fatality rate on day 18 and an average survival of 20.5 days during the period of study, while those in Group 4 (high dose) only survived 11.7 days on average. Not surprisingly, Group 5 (medium dose but with low specific activity) exhibited a similar toxicity or survival trend to Group 3, with 50% fatality rate on day 20 and an average survival of 21.8 days.  Group 3), the SPECT signal in the tumor was noticeably enhanced. In contrast, the signal intensity in the background tissues (e.g., liver) were much less than that in the tumor and decreased over time. Consequently, the tumor-to-background contrast became further enhanced over the time course, a highly desirable feature for clinical imaging data interpretation.

Immuno-SPECT/CT imaging with [ 67 Cu]Cu
Quantitative tumor uptake from the SPECT imaging data are presented as absolute radioactivity concentration (µCi/mL) (Fig. 4B). Clearly, the dose escalation from Group 2 to Group 4 resulted in significantly increased radioactivity concentration in tumors (Group 2: 0.47 ± 0.13 MBq/mL at day 2 and 0.22 ± 0.05 MBq/mL at day 5; Group 3: 1.31 ± 0.25 MBq/mL at day 2 and 0.70 ± 0.22 MBq/mL at day 5; and Group 4: 2.20 ± 0.50 MBq/mL at day 2 and 1.28 ± 0.45 MBq/mL at day 5) (p < 0.05 between each data set at day 2 or day 5). A comparative quantitative analysis between Group 3 and Group 5 showed that higher specific activity of [ 67 Cu]Cu-NOTA-Pertuzumab led to noticeably increased radioactivity concentration in tumors on day 2 (Group 3: 1.31 ± 0.25 MBq/mL vs. Group 5: 1.09 ± 0.11 MBq/mL). Interestingly, the tumor radioactivity concentration difference between Group 3 and Group 5 diminished at day 5 (Group 3: 0.70 ± 0.22 MBq/mL vs. Group 5: 0.73 ± 0.10 MBq/mL; p > 0.05). Reflected in the SPECT images, the higher specific activity Group 3 showed higher visual tumor uptake intensity than the lower specific activity Group 5 on day 2, but the difference disappeared on day 5.

Discussion
Copper-67 has been long considered as an "ideal" radionuclide for radioimmunotherapy due to its highly desirable decay features 12,39 . However, a rather limited number of studies were reported in either preclinical investigation or clinical trial settings in radioimmunotherapy 40 , because of the low production yield, high cost, and limited availability of 67 Cu 21 . The recent advancement in 67 Cu production, represented by the 68 Zn(γ,p) 67 Cu reaction, has elicited rapid developments of 67 Cu radiopharmaceuticals for cancer theranostics with focuses on 67 Cu labeled small molecules and peptides for radionuclide therapy [30][31][32][33] . Particularly important to antibody-based radiopharmaceuticals, 67 Cu with high specific activity would revitalize the conventional radioimmunotherapy hampered by the low specific activity of 67 Cu. Coupled with its SPECT imaging capability, 67 Cu would allow the development of radioimmunotheranostics featuring both clinical potentials of radioimmunotherapy and immuno-SPECT.
Prior to the current source of 67 Cu from the nuclear reaction of 68 Zn(γ,p) 67 Cu, the obtainable specific activity of 67 Cu from conventional production methods was in the range of 2-10 Ci/mg or 74-370 GBq/mg 37 . Accordingly, the specific activities of most 67 Cu-labeled antibodies previously reported were in the range of 37-56 MBq (1-1.5 mCi)/mg of antibody 34,37 , which were too low to have practical applications in radioimmunotherapy, in particular when the antigen expression levels are low and thus can be readily saturated. Reported with the specific activity up to 150 Ci/mg or 5,550 GBq/mg 26 , the currently available source of 67 Cu should be able to enhance the practicality of using 67 Cu for radioimmunotherapy.
We performed the γ-spectroscopic analysis of the high specific activity 67 Cu on a high purity germanium (HPGe) system. It confirmed that the 67 Cu solution as-received was pure without any measurable radionuclidic contaminants two days after production ( Figure S4). Because of the high quality of 67 Cu in terms of high specific activity and high radionuclidic purity, we observed a remarkably high radiolabeling efficiency for radiolabeling of NOTA-Pertuzumab with the 67 Cu, which is at least two order of magnitude greater than that reported in the literature with conventional 67 Cu. Consequently, we were able to attain the specific activity of [ 67 Cu]Cu-NOTA-Pertuzumab up to 8.6 GBq/mg (230 mCi/mg), two orders of magnitude higher than what reported for conventional 67 Cu-labeled antibodies. Of note, with the molar ratio of NOTA-to-Pertuzumab at 1.9:1, the immunoconjugate of NOTA-Pertuzumab maintained over 80% immunoreactivity of its parent antibody, pertuzumab.
Given the fact that only a very limited number of radioimmunotherapy studies were previously reported with 67 Cu 34,37 , there is a lack of references to the proper radiotherapeutic dose range of 67 Cu radiopharmaceuticals. To provide a proof-of-concept, we set out to start with 3.7 MBq per mouse for a single intravenous administration, followed by dose escalation to 7.4 and 14.8 MBq. Indeed, the 3.7 MBq low dose treatment group showed a marked therapeutic effect measured by tumor-growth inhibition while all mice survived the treatment. The dose escalation to 7.4 and 14.8 MBq resulted in greater tumor burden reduction but a considerable toxicity emerged in the medium and high dose groups. As such, to overcome this toxicity issue, a dose fractionation strategy needs to be considered for future studies. To evaluate the added-value of the high specific activity of 67 Cu, we conducted a comparative treatment study, in which Group 3 (1.10 GBq/mg) and Group 5 (53 MBq/ mg) represent the currently achievable specific activity of 67 Cu-labeled immunoconjugates and the conventional specific activity range (37-56 MBq/mg), respectively. We found that mice in Group 3 showed higher tumor accumulation at day 2, indicating high specific activity is beneficial to targeted delivery of radiation dose. This observation can be attributed to the fact that the antigen's binding sites for [ 67 Cu]Cu-NOTA-Pertuzumab are limited. Consequently, low specific activity [ 67 Cu]Cu-NOTA-Pertuzumab might lead to low targeted delivery of 67 Cu thus eliciting unwanted systemic toxicity because of the binding site saturation 41 . Indeed, we observed more effective tumor-growth inhibition for the high specific activity group (Group 3) than for its low specific activity counterpart. Of note, the specific activity of [ 67 Cu]Cu-NOTA-Pertuzumab in Group 3 in this work was set conservatively at 1.10 GBq/mg, which was only about one seventh of the maximum achievable, in order to evaluate the practicality of radioimmunotherapy with [ 67 Cu]Cu-NOTA-Pertuzumab. This is because a too high specific activity is not always desirable due to the fact that low expression of the targeted antigen may exist in non-target organs and passive non-specific binding may occur as well 42 .
Another major goal of this work was to impart the diagnostic value of 67 Cu to radioimmunotherapy. Traditionally, the quantification capability of SPECT is limited as compared to PET largely due to the inherent physical detection sensitivity of γ-rays 43 . To date, the advancement of technology has rendered SPECT with the capability of absolute quantification of signals in regions of interest in terms of activity concentration (e.g., kBq/cc or equally μCi/mL) and normalized uptake, such as the standardized uptake value (SUV) [43][44][45] . Indeed, quantitative SPECT has been reported for disease diagnosis, patient stratification, radionuclide therapy dosimetry, and longitudinal follow-up imaging 43,46,47 . These advancements would potentially propel 67 Cu into clinical applications, such as early diagnosis, dosimetry, prognosis, and dosage planning. To make it a reality, we managed to calibrate our small animal SPECT/CT scanner with 67 Cu and generate the corresponding acquisition and analysis protocols so as to acquire and quantify 67 Cu-SPECT images while performing the radioimmunotherapy studies. Shown in Fig. 4, we were able to perform immuno-SPECT imaging with [ 67 Cu]Cu-NOTA-Pertuzumab along with quantitative data analysis, unequivocally indicating the feasibility of incorporating the diagnostic value of 67 Cu into radiotherapies with 67 Cu radiopharmaceuticals.

Conclusion
The recent breakthrough of 67 Cu production offers great opportunities to revitalize 67 Cu radiopharmaceuticals by making available a reliable source of 67 Cu with high specific activity, high radionuclidic purity, and sufficient quantity. Our work presented herein with [ 67 Cu]Cu-NOTA-Pertuzumab demonstrates the potential as well as the practicality of using 67 Cu for the design and development of cancer radioimmunotheranostics. CuCl 2 solution was purchased from Idaho Accelerator Center, Idaho State University. The Na 125 I solution was purchased from Perkin-Elmer. The fast protein liquid chromatography (FPLC) was performed on a GE ÄKTAF-PLC system equipped with a Superdex 200 Increase 10/300 GL column and a Raytest in-line radio-detector. The mobile phase was 0.05 M phosphate buffer (pH 7.2) with 0.15 M NaCl and 2 mM ethylenediaminetetraacetic acid (EDTA). The flow rate was 1 mL/min. Radio-TLC analysis was performed on a Rita Star Radioisotope TLC Analyzer (Straubenhardt, Germany) to monitor the radiolabeling reaction using instant thin-layer chromatography (iTLC) paper and 1 × PBS as the developing solution.
Cell cultures and animal models. All animal studies were performed in accordance with relevant guidelines and regulations through a protcol approved by the Institutional Animal Care and Use Committee (IACUC) at UT Southwestern, which adheres to the ARRIVE guidelines. Two cell lines (HCC1954 − HER2+ and MDA-MB-231 − HER2-) were used in this study. MDA-MB-231 cells was cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, 100 μg/mL streptomycin, and 1% L-glutamine in a humidified incubator with 5% CO 2 at 37 °C. All cell culture reagents were purchased from Invitrogen. The HCC1954 cell line were kindly provided by Dr. E. Sally Ward at UT Southwestern. The HCC1954 cell line was cultured in T-media (Invitrogen Corporation, CA) supplemented with 5% FBS and 1 × penicillin/streptomycin (PS). Female SCID mice (6-8 weeks of age) were purchased from the Wakeland Colony at UT Southwestern. To establish the tumor model, HCC1954 cell suspension was injected subcutaneously (0.5 × 10 6 cells per injection with 50% BD matrigel, injection volume 100 µL) at the shoulder position. After injection, animals were monitored twice a week by general observations. The tumor volume was determined by the formula of Volume = (Width 2 × Length)/2 by caliper measurements.
Antibody conjugation. Conjugation of p-SCN-Bn-NOTA to pertuzumab was performed following previously reported methods 22 . Briefly, pertuzumab was firstly dialyzed to 0.05 M phosphate buffer (pH 7.2) with 0.15 M NaCl and then added a phosphate buffer (pH 8.5) containing 20-fold molar excess of p-SCN-Bn-NOTA. The resultant solution was stirred at room temperature for 3 h to form NOTA-Pertuzumab conjugate, which was subsequently purified by FPLC. The pooled fractions of NOTA-Pertuzumab conjugate were concentrated using Amicon Ultra 4 mL Centrifugal Filters (10 KDa cut-off) and in the same time the storage medium changed to 0.2 M ammonium acetate buffer (pH 6.5). The obtained immunoconjugate was stored at 4 °C for following uses. The protein concentration after modification was determined by the Coomassie (Bradford) Protein Assay.
Analysis of the radiochemical purity of 67 Cu. An aliquot (1 mL) of 67 Cu was analyzed using a high purity germanium detector (HPGe) (Canberra, USA) for radiochemical impurities. The HPGe was calibrated for energy and efficiency using a 1 mL mixed gamma source (Eckert and Ziegler, USA). Identification of gamma rays was determined using the LBL gamma search database 48 . Radiolabeling. The radiolabeling of NOTA-Pertuzumab with 67 Cu was typically carried out by adding 89 MBq (2.4 mCi) of 67 CuCl 2 in 0.1 M HCl to 3.2 µL of NOTA-Pertuzumab (25 µg/µL) pre-diluted with 0.05 mL 1 M NH 4 OAc buffer. The pH of the reaction medium was maintained at 5.0 -5.5 and the reaction was allowed to proceed for 30 min at 30 °C. The reaction mixture was sampled and then quenched with 5 µL of 5 mM diethylene triamine pentetic acid (DTPA) for 5 min at room temperature to determine the radiochemical yield via radio-iTLC, followed by radiochemical purity measurement by FPLC. The product, [ 67 Cu]Cu-NOTA-Pertuzumab, obtained with > 99% radiochemical yield & purity was used for in vitro and in vivo studies. To determine the highest achievable radiochemical yield and specific activity, the 67 Cu activity (27 MBq) and total reaction mixture volume were maintained the same while varying the amount of NOTA-Pertuzumab (0.5, 1, 2, 5, 25, 125 µg).
In vitro stability. The in vitro stability test was performed in the rat serum. Briefly, [ 67 Cu]Cu-NOTA-Pertuzumab (1.5 MBq, 5 μL) was added into 100 μL of rat serum (n = 3) and incubated at 37 °C. A 10 μL of sample was taken out and mixed with 2 μL of 5 mM DTPA from day 2 to day 7, respectively. The resulting solution was then analyzed by radio-TLC and radio-FPLC.
Determination of average chelator number conjugated per antibody. The average number of NOTA chelators per pertuzumab molecule was determined by the isotope dilution method 49 . Briefly, a series of standardized nat CuCl 2 dilutions were prepared to react with the NOTA-Pertuzumab conjugate. For each nat CuCl 2 concentration, 10 μg (69 pmol) of the NOTA-Pertuzumab conjugate was added to 56 μL of 0.1 M NH 4 OAc, pH 5.0, followed by adding 40 μL of the standardized nat CuCl 2 solution and 1 μL of 64 CuCl 2 (4.4 MBq). The reaction mixture was incubated at 37 °C for 1 h, after which 10 μL of 5 mM DTPA was added to remove non-specifically bound 64 Cu(II)/Cu(II). After 5 min, 1 μL of the reaction mixture was spotted for radio-TLC analysis. The plate was analyzed by a radio-TLC scanner. The number of chelators per antibody molecule was calculated based on a graph of the yield vs. the corresponding nat CuCl 2 concentration processed by GraphPad Prism 7.0 software 49 . Cell uptake experiments were performed after the verification of positive HER2 expression in HCC1954 cells and negative HER2 expression in MDA-MB-231 cells. Cells were seeded in 24-well plates with 2.5 × 10 5 cells per well and incubated overnight in a humidified incubator at 37 °C with 5% CO 2 . Cells were then incubated with 250 μL fresh complete media containing 25 ng/mL of [ 67 Cu]Cu-NOTA-Pertuzumab. The plates were incubated at 25 °C for 1.5 h with gentle rocking. After incubation, the cells were washed with PBS in triplicate and then trypsinized. Trypsinized cells were placed in culture tubes and the radioactivity associated with the cells was counted. Bound radioactivity was calculated as the ratio of bound to the total radioactivity added per well.
Radioimmunotherapy, toxicity assay. Radioimmunotherapy studies were performed on female SCID mice bearing HCC1954 tumor subcutaneously. When tumors grew to approximately 200 mm 3 by average, the mice were randomized into 5 different groups and intravenously received 0.1 mg of trastuzumab 30 min prior to the tail vein injection of either the Pertuzumab only (Group 1) or [ 67 Cu]Cu-NOTA-Pertuzumab (Group 2, 3, 4, and 5) ( Table 2). Tumor growth was monitored with a digital caliper 2-3 times per week until the end of the study. Tumor volume (mm 3 ) was calculated using the formula of ½ × (length × width 2 ). The average difference in volume of the tumors was graphed vs the number of days after treatment in order to monitor the tumor response to therapy. The percent change in tumor volume over time was determined by the tumor size normalized to the beginning of treatment.

SPECT/CT imaging. SPECT/CT imaging was performed at day 2 and day 5 post injection of [ 67 Cu]
Cu-NOTA-Pertuzumab on a NanoSPECT/CT Plus System (Bioscan, Washington, DC, USA). The field of view (FOV) of the SPECT/CT was adjusted to have the mouse in the center. CT imaging was performed using 360 projections per rotation with 45 kVp, 1000 ms exposure, and the binning factor of 1:4. The SPECT data were collected with 4 detector arrays collimated with multi-pinhole apertures giving a post-reconstruction resolution of 0.73 mm. SPECT images were acquired for 80 s in a standard anterior projection in list mode, at a rate of 1 frame per second. A calibration file was created using the known amount of 67 Cu and then used to validate the following 67 Cu imaging protocol. The SPECT imaging reconstruction was carried out using HiSPECT NG (Scivis wissenschaftliche Bildverarbeitung GmbH, Germany) with 35% smoothing, 100% resolution, and 3 × 3 iterations (Standard mode). The quantification of the 67 Cu activity accumulated in tumor was performed using the Invicro's VivoQuant 2.0 software package (Mediso, Boston, USA). After co-registration of the CT and SPECT images, a cylindrical region of interest (ROI) was drawn, encompassing the tumor and liver in all tomographic planes containing the organs. The total activity in the tumor was quantified as percentage injected dose per gram (%ID/g), which is defined as: %ID/g = Activity (mCi/mL) Total Injected Dose (mCi) ×Density ×100 assuming the density of tissue = 1 g/mL www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.