⁶⁴Cu-DOTHA₂-PSMA was synthesized, radiolabeled and characterized as previously described with a 116 ± 30 MBq/nmol molar activity (31).

¹⁷⁷Lu-PSMA-617 preparation. The synthesis of the PSMA scaffold was achieved as previously described by Benešová et al. (32). Purity of the peptide was verified by HPLC and greater than 95% ( Supplementary Figures 1 and 2 ). Both nuclear magnetic resonance spectroscopy and mass spectroscopy data were consistent with those reported in the literature (32). No-carrier added ¹⁷⁷LuCl₃ in 0.04 M HCl was obtained from Isotopia Molecular Imaging Ltd (Israël) and ITG Isotope Technologies Garching GmbH (Germany). Labeling of DOTA-PSMA was performed in 0.1 M ammonium acetate buffer pH 5.5 at 90°C for 15 min. Quality control of the drug was performed by Radio-TLC with sodium citrate buffer, pH 5.5, as eluent and always revealed labeling yields greater than 99% with an effective molar activity of 50 MBq/nmol. The radioligand solution was used without further purification steps.

All experiments utilized NOD-Rag1^null  IL2rg^null (NRG) mice (The Jackson Laboratory, 4-8 weeks old, and 18-26 g at time of tumor implantation) (33). For survival assays, human prostate adenocarcinoma LNCaP cells (ATCC) xenografts were implanted 4 ± 1 weeks before the experiment by injecting 200 µL of a 1:1 matrigel (Fisher Scientific)/phoshate-buffered saline (Wisent) mixture containing 8 × 10⁶ LNCaP cells subcutaneously on mice shoulders. LNCaP PSMA expression was previously verified (31). Protocols were approved by the Animal Ethics Committee of the Université de Sherbrooke according to the Canadian Council on Animal Care guidelines.

To estimate maximal tolerated injected activity (IA) for survival assays, 6 NRG non-tumor-bearing mice were divided into 3 groups to test 70 MBq, 120 MBq and 150 MBq of ⁶⁴Cu-DOTHA₂-PSMA in 0.3 mL of saline (bolus injection by tail vein, constant molar activity). Starting IA was based on reported activities with ⁶⁴Cu therapy in relatively similar conditions and scaled to mouse weight if needed (23–28). Mouse weight and general well-being were monitored daily 19 to 21 days. Maximal tolerated IA was chosen based on absence of general toxicity.

When tumor reached a maximal diameter of 5 to 8 mm, LNCaP tumor-bearing mice (n = 30) were divided into 3 treatment groups with comparable tumor size distribution. NRG Mice were injected in the tail vein with 0.3 mL of saline containing previously determined maximal tolerated IA of ⁶⁴Cu-DOTHA₂-PSMA, equimolar amount of nonradioactive ^natCu-DOTHA₂-PSMA as a control or the maximum tolerated IA of ¹⁷⁷Lu-PSMA-617 based on literature [120 MBq (34, 35)]. Survival end points were the ethical limit points: sustained weight loss > 20%, tumor diameter > 1 cm, behavior alterations suggestive of pain, tumor ulceration to the skin, non-manageable toxicity signs such as diarrhea or vomiting. Mouse weight, tumor size measured by caliper, and well-being were followed every 1 to 4 days for a maximum of 62 days. Tumor volume was calculated as an ellipsoid volume = (length × width × height)/2, estimating height = width (36). To determine the effect of radiotherapy on control of tumor growth, we suggest two calculated values: time-to-regrowth (TTR) for the interval to regrow after reaching the nadir and time-to-initial volume (TTIV) for the interval to regrow over the initial volume (calculations’ details in Supplementary material ).

General toxicity was assessed by the follow-up of the mouse weight. Mice food was mixed with water for treated mice. To obtain a general evaluation of blood toxicity, red blood cells (RBCs) were counted in survival experiment mice every 3 days from 2 to 18 days p.i. based on literature results (half the group per time point to spread out individual collections to every 6 days, leading in practice to 3 to 7 samples per time point) (sampling details in Supplementary material ) (25, 26, 28). Results were compared to values measured in non-treated mice (n = 13).

To evaluate sub-acute toxicity, kidneys, liver and salivary glands of survival experiment mice were collected and stained with hematoxylin and eosin (H&E), Masson’s trichrome and light periodic acid-Schiff for the kidneys (preparation details in Supplementary material ). Digitized slides were blindly analyzed by two persons (i.e. a certified pathologist and a medical student), to identify potential signs of radiation injury as suggested by Fajardo (37) and detailed in Supplementary Table 1 . Changes could be parenchymal (e.g. necrosis, apoptosis, atrophy), stromal (e.g. fibrosis), vascular (e.g. edema, ischemia, hemorrhage, endothelial wall atypia and foam cells plaques) or inflammatory. Fibrosis was graded and averaged between results of both analysts if discordant. Proportion of live (at time of fixation) tumor cells over total tumor tissue was assessed using a grid. Results were compared between groups and to the results from two non-treated tumor-bearing mice ineligible to survival assays (oversize tumor).

Kinetic values were obtained based on previously published healthy organ biodistribution and tumor PET data (31). Dosimetry was calculated for 25 g mouse model, human model, and sphere model for tumor in OLINDA/EXM 2.2.3 (Hermes Medical Solution). Detailed methodology is provided as supplementary material.

Results were reported as mean ± standard deviation unless mentioned otherwise. Tests used were two-tailed Student T test (Holm-Sidak correction for multiple comparisons, Welch test when applicable for non-consistent standard deviations) and two-tail correlation test with calculation of Pearson r coefficients. Survival results were analyzed by Kaplan-Meier curves stratified by treatment groups and comparison was made using Mantel-Cox log-rank test. For all tests, adjusted p < 0.05 was the threshold for significance. Excel and GraphPad Prism 8 were used for calculations.

In practice, IA tested were: 68.3 MBq (67.8 – 68.8 MBq, n = 2), 117 MBq (115 – 119 MBq, n = 2), and 156 MBq (154 – 159 MBq, n = 2) ( Figure 2 and Supplementary Table 2 ). Mice in the first two groups mainly gained weight from time of injection and showed normal behavior. One mouse in the last group lost up to 7% of its initial weight at first then stagnated around 100% of initial weight without external intervention. Mice from the last group showed signs of fear or stress for a total of 5 and 8 days out of 21 days follow-up but were active and vigorous. In consideration of these data and for practical reasons, we did not escalate to a further IA and determined 150 MBq as maximal tolerated IA to be injected in survival experiments.

Survival curves are displayed in Figure 3A and survival experiments details including mice and tumors individual characteristics are available in Supplementary Tables 3 – 6 . Survival assays mice injected with 146 ± 14 MBq of ⁶⁴Cu-DOTHA₂-PSMA group (n = 12) survived significantly longer than controls injected with the same amount of ^natCu-DOTHA₂-PSMA (1.23 nmol, n = 10) (p < 0.001, hazard ratio: 0.208 (95%CI: 0.0646 – 0.670)). Their survival was not significantly different from mice injected with 123 ± 13 MBq of ¹⁷⁷Lu-PSMA-617 (n = 7) (p = 0.09, hazard ratio 0.490 (95%CI: 0.165 – 1.45)). Median survival was 35.5 days with ⁶⁴Cu- DOTHA₂-PSMA (interquartile range (IQR): 26.5 days), 5 days with ^natCu-DOTHA₂-PSMA (IQR: 1 day) and 30 days with ¹⁷⁷Lu-PSMA-617 (IQR: 4 days). Average tumor volumes at injection were 108 ± 55 mm³, 93.3 ± 56.1 mm³, and 107 ± 35 mm³ for ⁶⁴Cu-DOTHA₂-PSMA, ^natCu-DOTHA₂-PSMA and ¹⁷⁷Lu-PSMA-617 groups, respectively (no significant difference between groups (p > 0.91)). One ⁶⁴Cu-DOTHA₂-PSMA treated mouse and one ¹⁷⁷Lu-PSMA-617 treated mouse were retrospectively rejected from survival experiments after injection, as they should not have been included in the protocol because of an oversized tumor at the beginning of treatment. They were, however, included in toxicity results because this rejection criteria did not affect healthy organs or general well-being.

The cause of euthanasia was reaching tumor size limit for all but one mouse in ¹⁷⁷Lu-PSMA-617 group (maximal weight loss, day 14) and two mice in the ⁶⁴Cu-DOTHA₂-PSMA group (pain behavior, day 60 and reaching follow-up limit, day 62). Two ⁶⁴Cu-DOTHA₂-PSMA and two ^natCu-DOTHA₂-PSMA mice had a second tumor of similar size at time of therapy that did not reach 10 mm tumor diameter limit, but mainly showed similar evolution patterns.

Individual and group average tumor volume progression is displayed in Figures 3C–F . In comparison to the control group, TTR and TTIV ( Figure 3G ) were significantly higher in groups treated with ⁶⁴Cu-DOTHA₂-PSMA (24.1 ± 14.6 days (p < 0.001) and 33.7 ± 16.5 days (p < 0.001), respectively) or with ¹⁷⁷Lu-PSMA-617 (17.1 ± 8.6 days (p = 0.003) and 22.9 ± 10.4 days (p = 0.003), respectively). TTR and TTIV values for the control group were both of 0.857 ± 1.406 days. No statistically significant difference was observed between ⁶⁴Cu-DOTHA₂-PSMA and ¹⁷⁷Lu-PSMA-617 (TTT: p = 0.26; TTIV: p = 0.24).

Within individual treatment groups ¹⁷⁷Lu-PSMA-617 and ⁶⁴Cu-DOTHA₂-PSMA, there is no correlation between IA (absolute and per gram) and survival (R² < 0.3). ⁶⁴Cu-DOTHA₂-PSMA treated tumors with bigger initial volume had shorter TTR, TTIV and survival, but correlation was low (R² < 0.5). For ¹⁷⁷Lu-PSMA-617 treated tumors, there were no correlation between initial tumor volume and survival or TTR, but there was a moderate positive correlation between initial tumor volume and TTIV (R² = 0.611, p = 0.04). This correlation was mainly dependent on the mouse with the smallest initial tumor (49.0 mm³) being euthanized early for maximal weight loss and not for reaching the maximal tumor size. When testing without this mouse, there was no correlation (R² = 0.179).

General toxicity was assessed through weight follow-up. Only one mouse from the ¹⁷⁷Lu-PSMA-617 group maintained a weight loss until reaching the ethical limit (20% weight loss). As depicted in Figure 3B , mice from all groups tended to lose weight at the beginning of study, but later maintained their weight or gained back at least their initial weight.

Normal range obtained from non-treated mice was 1.19 ± 0.21 × 10¹⁰ RBCs/mL (95% confidence interval (CI): 1.07 × 10¹⁰ to 1.30 × 10¹⁰ RBCs/mL, n = 13), Supplementary Figure 3 and Table 7 . Samples from ⁶⁴Cu-DOTHA₂-PSMA treated mice showed no significant difference from measured normal range, with values at days 8 and 17 lower than normal (all p > 0.35). Samples from ¹⁷⁷Lu-PSMA-617 treated mice showed a significantly lower count on day 8 with 9.13 ± 0.49 × 10⁹ RBCs/mL (p < 0.001) and values staying low until day 14. Control ^natCu-DOTHA₂-PSMA treated mice RBCs counts could only be obtained on day 2 (n = 4) given the low median survival and did not significantly differ from normal range (p = 0.98).

Histopathological analysis of healthy organs showed little to no signs of radiotoxicity, as displayed in Figure 4 for comparison between control, non-treated mice and ⁶⁴Cu-DOTHA₂-PSMA treated mice (detailed numerical values for all groups in Supplementary Tables 8 , 9 ). In kidneys ( Figures 4A–D ), liver ( Figures 4E–G ), salivary glands ( Figures 4H–J ), fibrosis increase in thickness and length was mainly found in the perivascular area, including the portal area for the liver and in addition to surrounding excretory ducts for the salivary glands (fibrosis score: Figure 4N ). There were at most rare signs of interstitial fibrosis in the kidneys. In the liver, rare septal fibrosis was formed without important extensions between portal spaces and central veins to form nodules. Fibrosis was increased with ⁶⁴Cu-DOTHA₂-PSMA in comparison to the non-treated control in the kidneys, liver and salivary gland (p < 0.001, p = 0.006 and p < 0.001, respectively). For ¹⁷⁷Lu-PSMA-617 group, fibrosis was significantly increased in comparison to non-treated controls in the liver (p = 0.009). For ^natCu-DOTHA₂-PSMA, fibrosis was increased in comparison to non-treated mice in the kidneys, liver and salivary glands (p < 0.001, p = 0.009 and p < 0.001, respectively). There were no significant differences between ⁶⁴Cu-DOTHA₂-PSMA, ¹⁷⁷Lu-PSMA-617 and ^natCu-DOTHA₂-PSMA groups in all organs. In all healthy organs, there was neither necrosis, hemorrhage, cells or structure atrophy, edema (present surrounding the gland in one sample of ^natCu-DOTHA₂-PSMA), endothelial wall atypia or foam cells plaques, nor glomerular changes for the kidney. Limited, rare and locally circumscribed inflammation was noted in liver only, similar in all groups.

LNCaP tumors ( Figures 4K–M ) showed necrosis, apoptosis, fibrosis and bleeding, in higher proportion in tumors treated with radioactivity. Hence, the ratios of alive tumor cells to total tumor tissue in ⁶⁴Cu-DOTHA₂-PSMA and ¹⁷⁷Lu-PSMA-617 groups were lower than with ^natCu-DOTHA₂-PSMA group (p < 0.001 and p = 0.04, respectively) and with non-treated control group (p = 0.002 and p = 0.04, respectively) ( Figure 4O ). Tumoral fibrosis was significantly increased in ⁶⁴Cu-DOTHA₂-PSMA and ¹⁷⁷Lu-PSMA-617 groups in comparison to ^natCu-DOTHA₂-PSMA (p < 0.001 for both) and to non-treated mice for ⁶⁴Cu-DOTHA₂-PSMA (p = 0.005) ( Figure 4N ).

There was no correlation between IA or IA/g with fibrosis score per groups for all organs (R²: 0 – 0.38). In healthy organs, there was no correlation between survival (and therefore age) and fibrosis score (R²: 0.001 – 0.16). Concordance between the fibrosis score results of both analysts were of 85%, 82%, 67% and 67% for the kidneys, liver, salivary glands and tumors, respectively. Differences, when present, were of one point or less.

Mouse dosimetry results are presented in Table 1 with kinetics values used for calculations presented in Supplementary Table 10 . Tumor dose was 2.67 × 10² mGy/MBq (95% CI: 1.94 × 10² – 3.4 × 10² mGy/MBq), using average tumor weight of 0.1 g. In mouse, highest organ dose was estimated to the liver, leading to a tumor to liver ratio of 0.730 (0.464 – 1.09). Second highest ratio was to the stomach wall (estimated from stomach kinetics), at 1.84 (1.18 – 2.70). Tumor-to-kidney ratio was 2.23 (1.42 – 3.30).

Human extrapolation of dosimetry presented in Table 2 (kinetic values in Supplementary Table 11 ) yielded effective dose of 3.14 × 10^-2 mSv/MBq (95% CI: 2.67 × 10^-2 – 3.61 × 10^-2 mSv/MBq).

⁶⁴Cu-DOTHA₂-PSMA maximal tolerated IA was estimated to be 150 MBq in NRG mice. This value is slightly higher than that reported for ¹⁷⁷Lu-PSMA-617 in immunodeficient mice (34, 35) suggesting that the ⁶⁴Cu-radioligand is well tolerated in immunodeficient mice.

⁶⁴Cu-DOTHA₂-PSMA showed therapeutic efficacy. It showed survival and growth delay (TTR and TTIV values) superior to control and similar to ¹⁷⁷Lu-PSMA-617, the lead radioligand for prostate cancer radioligand therapy. Other non-PSMA ⁶⁴Cu radioligands in literature showed improvement in survival and tumor size control, but comparison is limited given their application with another target (23–30). For ¹⁷⁷Lu-PSMA-617 with a slightly higher volume endpoint (1000 mm³), Fendler et al. found similar survival results to ours using 120 MBq (34) and Kuo et al. found higher median survival (58 days) even with the use of low IA (18.5 MBq) (38). Banerjee et al. found a 130 days median survival in a different prostate cancer model (PSMA+ PC3 PIP cells) with a higher tumoral volume endpoint (1800 mm³) (35).

On the whole, toxicity results pointed to limited toxicity for ⁶⁴Cu-DOTHA₂-PSMA, similar to ¹⁷⁷Lu-PSMA-617. Mice weight was maintained above limit endpoints for all but one ¹⁷⁷Lu-PSMA-617 treated mouse, suggesting a low toxicity for both ⁶⁴Cu-DOTHA₂-PSMA and ¹⁷⁷Lu-PSMA-617. Thought the only significant drop in red blood cells was observed in ¹⁷⁷Lu-PSMA-617 mice samples at 8 days p.i., period of days 8 to 17 could be an interval of potential myelotoxicity for both radioactive compounds. Regarding kidneys, liver and salivary glands, only non-significant fibrosis in normal location was found with no other sign of radiation injuries nor pathological processes (e.g. nodule formation in liver, significant interstitial fibrosis in kidneys) (37, 39, 40). The difference between the control group injected with ^natCu-DOTHA₂-PSMA and the non-treated controls suggests an interaction of the ligand with the PSMA enzyme influencing its function (41). However, this is not likely to have important toxic implications at injected activities similar to the present study given that all fibrosis noted was non-pathological fibrosis. The fibrosis scale was obtained by blind comparison of samples, but on the whole the difference between scores were low and fibrosis was present in low quantity.

For LNCaP tumors, pathological analysis at varying time after implantation could have a confounder effect on the results. Non-treated LNCaP tumors can demonstrate necrosis and fibrosis without treatment (42). This phenomenon was witnessed in both control groups, but in a significantly smaller proportion than the ones noticed in the groups injected with radioactivity. In clinical pathology practice, important proportion of dead tumor cells is considered more likely to be induced at least in part by treatment. Furthermore, in mice treated with radioligands, necrosis was found over all tumor bulk and not only in the middle of a highly alive section. For healthy organs, few weeks difference in age between controls and treated mice is less likely to explain the difference between survival groups and non-treated controls since there is no correlation between time of death and fibrosis score.

For comparison regarding toxicity, preclinical studies of other ⁶⁴Cu radioligand therapy compounds noted no to only transient weight loss and no to only mild signs in pathological examination (23–27, 29). Transient drop in RBCs was noted in a study 7 days p.i. at its highest IA (28) while no significant drop in RBCs was noted with others (23–26). For preclinical studies of ¹⁷⁷Lu-PSMA-617, no weight loss was reported at a lower IA (38) and no change in RBCs was noted at 4 and 8 weeks p.i (34, 35).. With ¹⁷⁷Lu-PSMA-617, Banerjee et al. noted mild changes in salivary glands, minimal changes in kidneys and significant changes in lacrimal glands and testes 8 weeks p.i (35).

Dosimetry calculations yielded an effective dose within normal limits and showed that the main organs at risk for toxicity are the liver and gastrointestinal tract. High dose in liver for human and low tumor-to-liver ratio in mouse were expected from previous distribution results (31). Interestingly, kidneys and salivary glands dose estimations in humans were low for ⁶⁴Cu-DOTHA₂-PSMA, with favorable tumor-to-kidney ratio in mouse. In a theranostic perspective, human dosimetry estimation of ⁶⁴Cu-DOTHA₂-PSMA’s effective dose is within safe levels for imaging with diagnostic IA. For instance, if one was to inject 200 MBq as for ⁶⁸Ga-PSMA-617 human scan, effective dose to the patient would be 6.28 mSv. This is similar to ¹⁸F-fluorodesoxyglucose PET for tumor imaging (~6.7 mSv), ⁶⁸Ga-PSMA-11 and ⁶⁸Ga-PSMA-617 (43–45). For comparison regarding therapy, human studies with ¹⁷⁷Lu-PSMA-617 showed that organs receiving among the highest calculated doses are the kidneys (0.39 to 0.88 mGy/MBq) and salivary glands (0.44 to 1.17 mGy/MBq), respectively 7 to 16 times and 23 to 62 times higher values than the ⁶⁴Cu-DOTHA₂-PSMA human model absorbed doses estimations (46–48). Lacrymal gland dose estimation is unknown for ⁶⁴Cu-DOTHA₂-PSMA, since it is unavailable in the OLINDA/EXM human model used. It was, however, the highest dose in VISION phase III trial dosimetry sub-study (2.1 ± 0.5 mGy/MBq) (48). Liver, spleen and effective doses are similar for both radioligands, even with the important liver uptake for ⁶⁴Cu-DOTHA₂-PSMA (46, 47). Red marrow doses were higher for ¹⁷⁷Lu-PSMA-617 than estimated for ⁶⁴Cu-DOTHA₂-PSMA, but this could be in part due to bone metastasis in patients for ¹⁷⁷Lu-PSMA-617 (46–48).

Irradiation tolerance is considered to be higher for radioligand therapy than for external beam radiotherapy (47, 49). However, external beam radiotherapy dose limits are sometimes still used to guide radioligand therapy safety. Considering limits or standards of 23 Gy for the kidneys, 30 Gy for the liver, 20 − 25 Gy to the salivary glands, 2 Gy to the red marrow, 40 Gy to the intestines and 12 Gy for whole-lung irradiation (46, 47, 50–53), the injection of 44 GBq of ⁶⁴Cu-DOTHA₂-PSMA over several cycles such as for ¹⁷⁷Lu-PSMA-617 (13) would not reach organ doses limits. Up to a total of 187 GBq could be injected before reaching the medullary and liver limit doses (calculations based on organ absorbed doses). We do not suggest using this high injected activity and it is simply underlined here to put ⁶⁴Cu-DOTHA₂-PSMA dosimetry profile in perspective. Clinical studies should be conducted to determine ⁶⁴Cu-DOTHA₂-PSMA biodistribution and dosimetry in humans to choose an appropriate injected activity.

Mouse dosimetry was obtained mainly to calculate ratios between tumor and healthy organs irradiation. In 2018, Kuo et al. reported mouse dosimetry for ¹⁷⁷Lu-PSMA-617 with a methodology highly similar to ours using OLINDA/EXM 2.0 while we used the latest version 2.2.3 (38). Their results per organs for ¹⁷⁷Lu-PSMA-617 were in average ~500 times lower than ours for ⁶⁴Cu-DOTHA₂-PSMA. We recalculated ¹⁷⁷Lu-PSMA-617 dosimetry with OLINDA/EXM 2.2.3 based on their kinetics values and obtained results closer to ⁶⁴Cu-DOTHA₂-PSMA, notably 5.43 × 10² mGy/MBq, 3.08 × 10² mGy/MBq, and 5.85 mGy/MBq for tumor, kidneys and liver, respectively. Other organs doses ranged from 2.54 mGy/MBq to 14.2 mGy/MBq, except for the bladder dose calculated with a different voiding model than our methodology. The difference between ¹⁷⁷Lu-PSMA-617’s results reported with OLINDA/EXM 2.0 and those obtained with the version 2.2.3 is notable. With 2.2.3, the tumor-to-kidney ratio is 1.76, slightly lower than for ⁶⁴Cu-DOTHA₂-PSMA, and the tumor-to-liver ratio is 92.8, importantly higher than for ⁶⁴Cu-DOTHA₂-PSMA.

Taking into consideration mouse and human models dosimetry results and analysis, the liver rather than the kidneys or the salivary glands would be at greater risk with ⁶⁴Cu-DOTHA₂-PSMA when compared to ¹⁷⁷Lu-PSMA-617, though the liver dose is similar for both compounds. This dosimetry pattern could be a challenge for clinical use. On the other hand, it could also be of interest to complete the clinical arsenal with a radioligand with different toxicity profile to offer an endotherapeutic option to more patients (e.g. with renal insufficiency or important prior bladder irradiation) or for patients experiencing side effects (e.g. xerostomia). Their irradiation should also be considered as radioligand therapy is being increasingly studied at earlier stages of the disease (e.g. adjuvant to prostatectomy) (13). Patient living longer could experience different side effects over the long term or eventual use adjuvant to external beam radiotherapy could modify the risk profile, with increasing risk to pelvic organs in the irradiation field.

It is important to note that dosimetry estimates to organs and tumor do not take account of dose rates and repetitions of irradiation, which can both influence the therapeutic outcome and toxicity (23, 24, 28, 29, 54).

Some limitations and sources of variability should be underlined for the present study. Variation in group size can be explained by the few days variability in LNCaP xenograft growth parallel to radioisotope production and importation logistics. Variation in IA or tumor size at time of the experiment, which is expected for preclinical studies, did not significantly impact survival assays and toxicity results as verified by correlation tests. Fibrosis scores concordance rates between both analysts’ results were high to intermediate, with only one-point difference when present. Concordance in future studies results could be increased by making the fibrosis scale more precise thanks to the experience acquired in this project.

In hindsight, the implantation of one tumor per shoulder in the first treated mice was a suboptimal method. Although this is a common practice in LNCaP implantation to increase the probability of experiment success [estimated 50% success rate in xenografting (42, 55)], it also yielded more complicated survival analyses. For instance, if two tumors were present at the time of therapy, both did not necessarily reach the size limit on the very same day, even if response to treatment were mainly similar for both tumors. Consequently, four mice had to be euthanized when a first tumor reached the ethical limit, and therefore, growth follow-up for the second tumor was stopped and the time to size endpoint was not obtained. In an attempt to mitigate this issue, we selected TTIV and TTR methods for growth calculation, which also had the advantage to include in the analysis mice that reached limits other than tumor size. One tumor was implanted per mouse for subsequent radioligand therapy rounds.

The animal model has other limitations. The important immunosuppression required for tumor implantation in mice may lead to an underestimation of signs of toxicity through decreased inflammation (33). Furthermore, ⁶⁴Cu-DOTHA₂-PSMA binding can vary between mouse and human PSMA, which limits the interpretation of toxicity results in healthy PSMA-rich tissues. Although conduction of toxicity evaluation in survival assays mice is not ideal, it does allow for the assessment of a broad profile.

Future studies could evaluate the immunoreactivity of ⁶⁴Cu-DOTHA₂-PSMA as well as apoptosis and DNA double-strand breaks on tumor tissue after treatment. The PSMA expression and Ki67 marker expression could also be measured to confirm the level of PSMA positivity of surviving tumor cells and to have information on the proliferative capacity of the tumor cells respectively. Different injected activities and multidoses for radioligand therapy could be evaluated. Multidose of ⁶⁴Cu-DOTHA₂-PSMA could have an impact on tumor size control and toxicity, based on dose fractionation principles and similarly to previously published preclinical results with ⁶⁴Cu and ¹⁷⁷Lu and to ¹⁷⁷Lu-PSMA-617 clinical protocols (14, 23–25, 27–30). Copper-67 (⁶⁷Cu) has a similar β^- emission profile to ¹⁷⁷Lu with a 2.6 days half-life and can be used in theranostic pair with copper-64 (⁶⁴Cu) for PET imaging (20). ⁶⁴Cu/⁶⁷Cu-SAR-bisPSMA is currently under clinical trial for prostate cancer radioligand therapy (SECuRE trial, NCT04868604). Further research is also needed for a more complete toxicity profile, using healthy and immunocompetent mice followed over months with a wider panel of blood cells and specific time points of euthanasia for pathologic evaluation as groups. Other experiences could be added such as blood and urine markers of kidney and liver functions e.g. AST, ALT, blood urea nitrogen, creatinine and glomerular filtration rate. Finally, another interesting future research could evaluate the link between pathological findings, radioligand distribution in tissue, emission range and microdosimetry. For instance, the presence of radioligand in blood circulation could irradiate vasculature and be linked to perivascular fibrosis.

In conclusion, ⁶⁴Cu-DOTHA₂-PSMA showed efficiency for radionuclide therapy in comparison to control and similarly to the most clinically studied PSMA radioligand, ¹⁷⁷Lu-PSMA-617. Insights on toxicity suggest a safety profile similar to the one of ¹⁷⁷Lu-PSMA-617, but further confirmation studies are required. Dosimetry estimation yielded a low dose to the kidneys and salivary glands. Higher doses estimations were obtained for the liver and gastrointestinal tract, a perceived challenge to clinical use. Nevertheless, ⁶⁴Cu-DOTHA₂-PSMA offers the possibility to complement the current clinical arsenal with a theranostic radioligand that has different mechanism of action (both β^- particle and high LET Auger electron), clearance and dosimetry profile than previous radioligands. It could act as another alternative to ¹⁷⁷Lu-PSMA-617, similarly to ²²⁵Ac-PSMA-617, to allow personalization of radioligand therapy based on patients’ comorbidities, prior treatments or experienced side effects.