Hypoxia imaging and theranostic potential of [64Cu][Cu(ATSM)] and ionic Cu(II) salts: a review of current evidence and discussion of the retention mechanisms

Background Tumor hypoxia (low tissue oxygenation) is an adverse condition of the solid tumor environment, associated with malignant progression, radiotherapy resistance, and poor prognosis. One method to detect tumor hypoxia is by positron emission tomography (PET) with the tracer [64Cu][Cu-diacetyl-bis(N(4)-methylthiosemicarbazone)] ([64Cu][Cu(ATSM)]), as demonstrated in both preclinical and clinical studies. In addition, emerging studies suggest using [64Cu][Cu(ATSM)] for molecular radiotherapy, mainly due to the release of therapeutic Auger electrons from copper-64, making [64Cu][Cu(ATSM)] a “theranostic” agent. However, the radiocopper retention based on a metal-ligand dissociation mechanism under hypoxia has long been controversial. Recent studies using ionic Cu(II) salts as tracers have raised further questions on the original mechanism and proposed a potential role of copper itself in the tracer uptake. We have reviewed the evidence of using the copper radiopharmaceuticals [60/61/62/64Cu][Cu(ATSM)]/ionic copper salts for PET imaging of tumor hypoxia, their possible therapeutic applications, issues related to the metal-ligand dissociation mechanism, and possible explanations of copper trapping based on studies of the copper metabolism under hypoxia. Results We found that hypoxia selectivity of [64Cu][Cu(ATSM)] has been clearly demonstrated in both preclinical and clinical studies. Preclinical therapeutic studies in mice have also demonstrated promising results, recently reporting significant tumor volume reductions and improved survival in a dose-dependent manner. Cu(II)-[Cu(ATSM)] appears to be accumulated in regions with substantially higher CD133+ expression, a marker for cancer stem cells. This, combined with the reported requirement of copper for activation of the hypoxia inducible factor 1 (HIF-1), provides a possible explanation for the therapeutic effects of [64Cu][Cu(ATSM)]. Comparisons between [64Cu][Cu(ATSM)] and ionic Cu(II) salts have showed similar results in both imaging and therapeutic studies, supporting the argument for the central role of copper itself in the retention mechanism. Conclusions We found promising evidence of using copper-64 radiopharmaceuticals for both PET imaging and treatment of hypoxic tumors. The Cu(II)-[Cu(ATSM)] retention mechanism remains controversial and future mechanistic studies should be focused on understanding the role of copper itself in the hypoxic tumor metabolism.


Background
Tumor hypoxia is an adverse condition of the tumor microenvironment defined by low tissue oxygenation as a result of an imbalance between cellular oxygen supply and consumption [1]. The cause of tumor hypoxia includes poor perfusion due to lack of blood flow, inadequate oxygen diffusion due to increased distance with tumor expansion, or reduced transport capacity of oxygen in blood (anemia) [2]. Tumor hypoxia is typically associated with malignant progression, metastasis, resistance to chemo-and/or radiotherapy, recurrence, and overall poor prognosis [3][4][5]. Tumor hypoxia can also induce aggravating biological changes in the tumor microenvironment that result in change of gene expression patterns, alter the malignant potential of the tumor, and lead to increased tumor aggressiveness [6]. Detection of tumor hypoxia is therefore of great importance in order to optimize the treatment strategy and improve overall prognosis [2,7].
Conventionally, assessment of tumor hypoxia has been achieved by using invasive techniques such as measuring the oxygen partial pressure (pO 2 ) with intratumoral polarographic sensors (an oxygen probe), developed in the 1990s [3,8]. Although invasive methods such as polarographic electrodes have the advantage of high sensitivity and are often regarded as gold standards, these methods also suffer from limited tumor accessibility, disruption of the tumor microenvironment, and difficulties to distinguish necrotic regions of the tumor, in addition to the troublesome and painful procedures [9]. As a result, using invasive techniques has questionable oxygen level representation of the tumor as a whole as well as difficulties to visualize variations of oxygen concentration within the tumor [5]. A number of noninvasive techniques can be used to image tumor hypoxia, including positron emission tomography (PET), functional magnetic resonance imaging (fMRI), electron paramagnetic resonance (EPR), and optical spectroscopy. Though based on different principles, these techniques share similar advantages that allow serial tracking of hypoxic regions and accessibility that are otherwise challenging for invasive techniques [5]. In blood oxygen level dependent (BOLD) fMRI, the detection of hypoxia is based on an indirect mechanism by measuring the magnetic susceptibility difference between deoxyhemoglobin (paramagnetic) and oxy-hemoglobin (diamagnetic). The measurement is influenced by for instance the change of hemoglobin concentration and interconversions, thus the result is considered as qualitative assessments of oxygenation dynamics but not direct oxygen levels [10]. In contrast, detection of tumor hypoxia in PET is a direct measurement, presenting higher sensitivity and is directly quantitative. Compared to the more commonly used fluorine-based PET tracers, tracers based on copper allow a rapid visualization of hypoxia (in contrast to [ 18 F]F-MISO), or better penetration of the blood-brain barrier and no urinary bladder uptake (in contrast to [ 18 F]FAZA) [11,12], in addition to longer half-lives that contributes to better image quality and more convenient clinical practice. Here, we focus on reviewing recent advances and challenges in imaging of tumor hypoxia using PET with one of the most promising copper tracers, [ 64

Copper-labelled ATSM
There has been a number of preclinical studies on radi-  [13]. Selectivity of copper-labelled ATSM in hypoxic tumor regions has since been explored. In a comparative in vivo and in vitro study between [ 64  which are characterized as cancer stem cells (CSCs) and enriched under hypoxia due to survival advantages, as well as promoted self-renewal ability through the activation of the hypoxia inducible factor-1α (HIF-1α) [20][21][22]. In addition, it has also been suggested that [ 64 Cu][Cu(ATSM)] is a direct indicator of an overreduced intracellular state caused by hypoxia, and thus, an indirect indicator of hypoxia [23]. Collectively, these preclinical studies have provided evidence to suggest the use of radiolabeled copper [Cu(ATSM)] not only as a tracer for imaging of tumor hypoxia, but potentially as a tracer of clonogenic cancer stem cells or stem-like cells which accumulate in regions characterized by a highly reductive biochemical environment.

Ionic copper-64
Using simple [ 64 Cu]Cu-dichloride ([ 64 Cu]CuCl 2 ), Peng et al. were able to visualize mouse hepatoma xenografts with small animal PET, where the copper uptake is believed to be mediated by the copper transporter 1 (CTR-1) [24]. Later, the same group observed increased tumor uptake of copper-64 in a human prostate cancer xenograft mouse model and concluded that [ 64 Cu]CuCl 2 PET may be useful for the detection of local recurrence in the prostate bed [25]. Similarly, Zhang et al. later visualized human hepatocellular carcinoma in a xenograft model using [ 64 Cu]CuCl 2 as the PET tracer [26]. Concerning the stability of ligand-bound copper-64 tracers from other studies, Jørgensen et al. investigated the uptake of copper-64 from [ 64 Cu]CuCl 2 in five different xenograft models (from colorectal cancer, glioblastoma, head and neck cancer, neuroendocrine lung carcinoma, and ovarian cancer) and found high tumor uptake of copper-64 in four out of the five models, with the exception of ovarian cancer having moderate uptake [27].
To investigate the role of copper itself in the retention mechanism, Hueting et al. intriguingly discovered in an in vivo study that when comparing the behavior of [ 64 Cu]Cu-acetate and [ 64 Cu][Cu(ATSM)] in EMT6 and HT1080 mouse xenograft models, the uptake and distribution of copper-64 showed mirroring performance of the two tracers [28]. Furthermore, when increasing the inhalation of O 2 at two hours retention, the uptake of both tracers was also correspondingly reduced [28].  [28]. Using an octanol-extraction method to analyze the amount of intact [ 64 Cu][Cu(ATSM)] in mouse whole blood, the same group reported that only 3% of the copper complex remained intact 30 min after injection, while the rest existed as serum-bound copper-64, having radioactivity comparable to the extraction from mice injected with [ 64 Cu]Cu-acetate [28]. In summary, the results from these authors seem to question the validity of the previously proposed copper retention mechanism based on the instability of [ 64 Cu][Cu(ATSM)] complex in vivo [28]. Such instability will lead to the dismantling of [ 64 Cu]Cu(II) from the complex before reaching the tumor cells, despite the mechanism has been demonstrated in vitro [28]. Ferrari et al. further investigated the theranostic possibility of [ 64 Cu]CuCl 2 in a glioblastoma xenograft model and found good visualization of tumors, and noted that the ability of copper ions to enter cancer cells may not simply depend on the complexing ligand, but rather be tightly linked to copper itself [29].

Clinical studies with [ * Cu][Cu(ATSM)] and ionic copper-64
The possibility of using radiolabeled copper [Cu(ATSM)] and ionic Cu(II) salts for PET imaging of tumor has also been demonstrated in clinical studies with different cancer types, including lung, cervical, rectal, head and neck, brain, and prostate cancer. One of the reasons these cancer types were chosen in the studies with [ * Cu][Cu(ATSM)] PET imaging is because of the difficulties to detect hypoxia due to the physical inaccessibility to use invasive methods [30][31][32][33]. Although the exact mechanism and relations to tissue hypoxia explaining the retention of either [ 64 2 have the advantages of much faster pharmacokinetics, better signal-to-noise ratio, and no interference due to absence of urinary bladder uptake [36,37,42]. Furthermore, ionic [ 64 Cu]Cu 2+ have also demonstrated improved diagnostic sensitivity in a recent study involving 50 patients with prostate cancer, with 82% detection rate compared to 74% using multiparametric MRI, which is the standard radiologic procedure, and 56% using [ 18 F]-choline-PET, although the authors did not report comparisons with the more recently developed prostate-specific membrane antigen (PSMA)-based PET tracer [37]. Despite early experiments were mainly based on radiolabeled copper [ * Cu][Cu(ATSM)], later studies have also involved ionic radiocopper and reached similar results. Notably, if the copper retention indeed followed solely the ligand-based trapping mechanism, using ionic radiocopper should not cause any selective accumulation in hypoxic tumors. Combining the clinical and in vivo preclinical results, it is clear that copper itself also plays an important role in the hypoxic selectivity and therefore should be emphasized.

Role of Cu-64 in therapeutic applications
Recently, several preclinical studies have reported the possibility of using [ 64 Cu][Cu(ATSM)] or [ 64 Cu]CuCl 2 , as therapeutic agents, in addition to imaging tumor hypoxia with PET. The ability of copper-64 for molecular radiotherapy is based on its complex decay scheme, which includes β + (0.656 MeV, 19%), β − (0.573 MeV, 40%), electron capture (41%), and gamma emission (1.346 MeV, 0.5%), with a half-life of 12.7 h [42]. In particular, electron capture will generate a cascade of Auger electrons with high linear energy transfer (LET) in tissue, which has been demonstrated to induce tumor cell death with high efficiency due to the release of the electrons in close proximity to the DNA [43,44]. Auger electrons from copper-64 are considered high LET radiation with~2 keV of average energy and with~126 nm average range in tissue, which is also a desirable property as these electrons travel much shorter than the diameter of tumor cells compared to other common β − emission radionuclide therapies where the electrons from β − emissions have much longer range [45][46][47].
The effectiveness of copper-64 as a therapeutic agent has been experimentally demonstrated in a number of in vitro and in vivo studies with various solid tumors. As early as in 2001, Lewis [48]. The study further confirmed with the alkali comet assay that significant DNA damage was observed in cells exposed to [ 64 Cu][Cu(ATSM)]. Combined with the lack of increase of apoptotic and necrotic cells, the damage was considered to be direct intracellular radiation damage [48]. These authors also hypothesized that the electrons from β − emission contributed to tumor cell killing, since the majority of the intracellular [ 64 Cu][Cu(ATSM)] was found to remain in the post-mitochondrial supernatant [48]. However, other studies have demonstrated that Auger electrons are cytotoxic even without internalization, due to nuclides remaining on the cell membrane [49,50]. Correlative studies using [ 64  Cu-ATSM rapidly accumulated in tumors of patients with lung cancer but not in the lung of healthy volunteers. The level of radiocopper reached abnormally intense plateau levels within a few minutes after intravenous administration in tumors with different distribution from [ 18 F]-FDG. [39] Lung cancer (NSCLC) 19 PET imaging with 60  [40] Cervical cancer 15 Patients with hypoxic cancer as determined by 60 Cu-ATSM correlates with overexpressed VEGF, EGFR, COX-2, CA-9, as well as high apoptotic and worse prognosis. [110] Cervical cancer 38 Patients were monitored through a period from 3 to 79 months. With a threshold of T/M = 3.5 the 3-year progression free and cause-specific survival rate of patients with normoxic tumors (T/M < 3.5, 71%) is significantly higher than that of hypoxic tumor (T/M > 3.5, 28%). Advantages of 60 Cu-ATSM over 18 F-FMISO include much faster pharmacokinetics which results in better target-to-background ratio, and no interference due to urinary bladder uptake. [32] Rectal 19 60 Cu-ATSM PET may be predictive of survival and possible tumor response to neoadjuvant chemoradiotherapy in rectal cancer. [34] Cervical cancer 10 PET is effective in detecting local recurrence and lymph node metastasis, although the evidence is still limited and further clinical trials are necessary to obtain more conclusive results [37,58]. Glioblastoma multiforme (GBM), the most common and aggressive primary brain tumor in adults which typically is associated with poor prognosis, is known to have hypoxic regions that are resistant to chemo-and radiotherapy, as well as frequent recurrences after surgery [59][60][61]. to bevacizumab treatment has shown to reduce tumor size and lead to prolonged survival, without major adverse effects [64].
A list of preclinical investigations on the therapeutic effects of copper-64 is summarized in Table 2 The maximum tumor-to-background ratio was reached 1 hour after injection. Mean SUV max in lymph nodes and bone metastases were significantly higher than local relapse. Discounting the Auger electrons, the mean absorbed dose for PCa lesions per administration was~0.06 mGy/ MBq. The therapeutic effect of [ 64 Cu]CuCl 2 is mainly dependent on the Auger electron emission instead of beta radiation. [58] * Atomic mass number and perhaps the CSCs [19,57], thereby significantly inhibit tumor growth with few adverse effects. The optimal administration method is likely to use multiple smaller doses to reduce possible adverse effects while keeping sustained internal radiation DNA damages of hypoxic tumor cells [29,62]. Though the copper retention mechanism remains not fully understood, the trapping of copper-64 in hypoxic regions combined with the decay scheme of copper-64 with high LET, short-range Auger electrons clearly contributes to such therapeutic effects. In addition to be a stand-alone therapeutic agent, copper-64 radiopharmaceuticals have also been shown to address tumor hypoxia in combinatory cancer treatments (e.g., bevacizumab [64]), which opens new avenues of possibilities.

Controversial copper uptake mechanism
Although the effectiveness of using [ 64 )] is more than a hypoxia tracer; its accumulation in tumor cells is also linked to the redox potential and reactive oxygen species which reflects the overreduced cellular state [66]. Although the exact reduction mechanism of copper in the tumor is debated, the foundation of the ligand-based dissociation mechanism is that the Cu(II)-[Cu(ATSM)] complex remains intact after the administration in the blood plasma, until encountering a hypoxic region of the tumor where the Cu(II) can be irreversibly reduced to Cu(I), and subsequently be trapped. However, studies using ionic copper-64 have raised questions on the in vivo retention mechanism. As mentioned, a direct in vitro and in vivo comparison between  showed a greater tumor/brain ratio [68].
In vitro, elevated accumulation of [ 64 Cu][Cu(ATSM)] was detected in severe hypoxia (pO 2 < 0.5%), which further increased with decreasing oxygen concentration. Accumulation of [ 64 Cu]CuCl 2 was elevated at pO 2 < 5%, but with no further significant increase at lower oxygen concentrations [68]. The authors concluded that while both [ 64 Cu][Cu(ATSM)] and [ 64 Cu]CuCl 2 could be considered as hypoxia-selective tracers, non-hypoxic accumulation in copper transporters should also be accounted for [68]. Interestingly, in an investigation of the possibility to use with enhanced efflux, while knockdown of MDR1 showed inverse effects, which further implies a more complex copper retention mechanism than what was previously proposed [69].
These preclinical results are critical for the understanding of clinical studies using ionic radiocopper salts for PET imaging of tumors, in which the correlation between tumor oxygenation and the radiocopper uptake is much more difficult to address directly. The direct comparisons in preclinical studies have challenged the conventional understanding of the copper uptake mechanism in tumors, in which the role of copper-64 itself in the copper metabolism pathway should be addressed and the function of the ligand is in question due to its potential lack of stability in blood. In fact, an in-  (Table 1). To better understand the copper uptake mechanism, it is necessary to revisit the role of copper and its transportation in mammals.

Copper metabolism
Copper is an essential element in mammals and is required as a cofactor for a number of cuproenzymes, mainly distributed in liver, muscle, bond, and blood [70]. Copper uptake and distribution is regulated by proteins including CTR-1, copper transporter 2 (CTR-2), copper chaperones such as antioxidant protein 1 (Atox1), copper chaperone for superoxide dismutase (CCS), cytochrome c oxidase copper chaperone 17 (Cox17), and the copper transporting adenosine triphosphatases (ATPases) ATP7A and ATP7B, as shown in Fig. 1 [71][72][73]. Free copper ions are typically absent in blood or cytoplasm, since any presence of ionic copper in either Cu(I) or Cu(II) will rapidly associate with three known high-affinity protein carriers including albumin, transcuprein, and ceruloplasmin [70,74,75]. Uptake of copper from blood is mainly through the high-affinity CTR-1, which transports copper in the form of Cu(I) into the cells as bioavailable copper in the cytosol [76][77][78]. Explicit evidence has also shown that copper in blood plasma is carried by proteins but not low molecular weight amino acids or complexes [70,79]. It has been demonstrated that transport of copper from blood uptake contains two phases. In the first phase after injection, copper will be absorbed rapidly by albumin (which contains high-affinity copper(II) binding sites [74,75]) and transcuprein, and again exclusively bind to proteins but not low molecular weight complexes or as free form, reaching a minimum level in plasma within approximately 2 h [80,81]. In this phase, the main destinations of copper are the liver (~40%), muscles (~18%), and the kidneys (~6%) [80]. Reemergence of copper in plasma starts from 6 h to approximately 1 day after initial injection at which point the blood copper concentration reaches another maximum, this time incorporated with ceruloplasmin and being transported to other tissues [80].
To transport copper into the cells, albumin or transcuprein delivers copper to the extracellular domain of the plasma membrane transporter CTR-1, which then imports copper across the cell membrane and further deliver to copper chaperone proteins [82,83]. Copper(II) is believed to be reduced to Cu(I) during this stage by metalloreductases, but the exact reduction process and maintenance of Cu(I) with the presence of oxygen before reaching CTR-1 remains unclear [76]. Copper chaperone protein Atox1 delivers Cu(I) to ATP7A (neuron/astrocyte cells) or ATP7B (liver), before it is delivered to the target cuproenzymes, such as cytochrome c oxidase (CCO) in mitochondrial via Cox17 or copper/zincsuperoxide dismutase via CCS [83].
In a copper transport perspective, regulation of CTR-1 is crucial, and in a hypoxic environment, the HIFs mediate the signaling cascade and trigger a series of cellular responses in both normal and cancer tissues [84]. HIFs respond to hypoxia by altering the gene expression involving at least 150 proteins that control critical cellular functions such as metabolism and survival [84]. Under normoxia, HIF-1α is regularly produced and degraded, achieving an equilibrium; under hypoxia, HIF-1α is stabilized, activating transcriptions in combination with other coactivators, including factors that promote the self-renewal capability, multipotency, adaptivity, as well as inhibition of differentiation of CSCs, in effect giving CSCs survival advantages and enhanced self-renewed proliferation [20,22,84]. Notably, copper is required for the activation of HIF-1 via HIF-1α binding to the hypoxia-responsive element and the formation of the HIF-1 transcriptional complex [85,86]. As a result, these studies predict a favorable environment for CSCs and its self-renewing proliferation under hypoxia with the activation of HIF-1, leading to an overall worsened cancer prognosis as well as potential resistance to chemoradiotherapy. Indeed, such correlation has been widely observed and reported in various cancers, including ovarian cancer [87], cervical cancer [88,89], breast cancer [90], lung cancer [91], bladder cancer [92], and glioma [93].
Similarly, hypoxia has been shown to stimulate the expression of CTR-1 [94], and upregulation of CTR-1 has also been repeatedly reported in hypoxic tumors [25, 29, Fig. 1 Simplified illustration of copper transport mechanism. Cu(II) is carried by albumin or transcuprein in blood, and is believed to be reduced to Cu(I) by reductases near the surface of cells. Copper transporter 1 (CTR-1) is the major high-affinity copper transporter, which transports Cu(I) into the cells and deliver to the copper chaperone Cox17, the antioxidant protein 1 (Atox1), or the copper chaperone for superoxide dismutase (CCS). Atox1 further deliver copper to either the copper transporting adenosine triphosphatase (ATPase) ATP7A in neuron/astrocyte cells, or the ATPase ATP7B in the liver, which either pass the copper to target cuproenzymes, or to excretion [95][96][97][98][99]. Peng et al. reported a significantly higher [ 64 Cu]CuCl 2 uptake in tumor tissue compared to normal tissue, meanwhile observing a substantial increase of CTR-1 expression in tumor but not in normal tissue [25]. Cai et al. provided [97]. Remarkably, CTR-1 transports copper in the form of Cu(I) instead of copper(II) [76][77][78], thus, if the upregulation of CTR-1 indeed is responsible for the transport of copper to the hypoxic tumor sites, copper(II) must have been disassociated and reduced to Cu(I) in blood, before it is delivered to the tumor through the copper secretory pathway.

Dosimetry
One important aspect to consider when using copper-64-based radiopharmaceuticals for PET diagnostics, and especially for therapeutic applications, is radiation dosimetry. However, such evaluation is difficult due to the complexity of the copper-64 decay scheme, especially when considering absorbed dose from high-LET Auger electron emissions. An early study on the therapeutic effect of [ 64 Cu][Cu-(ATSM)] by Lewis et al. suggested that the absorbed dose from Auger electrons is dependent on the distance between copper-64 ions and the cell nucleus, since the Auger electrons have low energy and very short range in tissue but can induce significant DNA damage in close proximity to the cell nucleus [46]. In an analysis of [ 60 [101]. For a more comprehensive list of dosimetry estimations in different organs, we refer readers to the original article in reference [101].
A similar dosimetry estimation was also reported by Panichelli et al. in a clinical study of brain tumors involving 19 patients with [ 64 Cu]CuCl 2 PET. The highest dose-absorbing organ was the liver, with an absorbed dose of 0.321 mGy/MBq, while the full body dose was estimated to be 0.0312 mGy/MBq [38]. In a recent clinical study using [ 64 Cu]CuCl 2 to detect prostate cancer relapse involving 50 patients, Piccardo et al. evaluated the absorbed dose to each organ. The authors reiterated that the critical organ is the liver, as reported by prior study [36], and found that the effective dose for [ 64 [37]. However, it is worth noting that the absorbed dose for the liver is considerably higher in [ 64 Cu]CuCl 2 PET with 0.271 mGy/MBq, compared to 18 F-choline (0.0610 mGy/MBq) and 68 Ga-PSMA (0.0309 mGy/MBq) [37]. The authors argued that the increased radiation exposure is negligible, although the effects to liver was not specifically discussed [37].
Despite evaluation in several preclinical and clinical studies, the accurate dosimetry of copper-64 to tumor and organs remains yet to be established. As previously mentioned, a detailed and accurate dosimetry description is challenging because of the short-range high-LET Auger electron emission, which is believed to be more radiotoxic than β − /β + emission and being the main contributor of the therapeutic effect. However, the distancedependent dose deposit nature complicated the evaluation, since the degree of internalization of copper-64 ions must be taken into account.

Reconsidering the copper pathways under tumor hypoxia
Based on our review of copper-64 radiopharmaceuticals in PET imaging and therapeutic purposes, we propose an emphasized role of copper itself in the copper uptake in hypoxic tumors, as shown in Fig. 2. Starting with ionic copper(II) in blood, either from direct injection of ionic copper compounds or through dismantling of copper(II) complexes, copper(II) is quickly reduced to Cu(I) by reductases [102][103][104][105], immediately binding to copper-binding proteins. On the other hand, tumor hypoxia stimulates CTR-1 expression [29,94], which then binds to Cu(I) and is transported into the tumor cells. With Cu(I) available, hypoxic tumor cells activates HIF-1 [22,23,84,87], triggering a series of effects including promotion of CD133 + CSCs via survival advantages [20,22,23,57,84,87,93], which also enhances the selfrenewal ability and inhibits differentiation of CSCs [22,84,106]. In effect, this increases the tumor proliferation and generates oxidative stress, resulting in worsened tumor hypoxia [19,36,84,[107][108][109]. In the meantime, this picture can also help us to understand the efficiency of copper-64 radiopharmaceuticals in preclinical molecular radiotherapy studies. Clonogenic hypoxic tumors activate HIF-1 and have elevated CTR-1 expression, thus significantly increasing the uptake of copper-64. Localized high concentration of copper-64 emits high-LET Auger electrons at close proximity of these proliferating cancer cells, causing substantial DNA damages and eventually lead to significant tumor reduction.

Conclusions
Detecting and quantifying tumor hypoxia is one of the most critical yet challenging tasks for cancer management. In this review, we have summarized preclinical and clinical evidence that support the possibility of using both [ 64 Cu][Cu(ATSM)] and ionic 64 Cu(II) salts for imaging of tumor hypoxia using PET. These evidences support the possibility of using copper-64-PET to detect and quantify hypoxia in solid tumors in clinical practice and thereby enable the potential to provide more individualized and optimized cancer treatment. In addition, the possibility to use copper-64 as a therapeutic agent provides a promising approach to target clonogenic cancer cells, cancer stem cells, and stem-like cells that are challenging in conventional treatment, and which may contribute to improved patient outcomes. Together, copper-64 radiopharmaceuticals [ * Cu][Cu(ATSM)]/ [ * Cu]CuCl 2 have the potential to serve as theranostic agents that can simultaneously provide both diagnostics and treatment for cancer patients.
On the other hand, the current understanding of the copper retention mechanism remains to be fully resolved before using copper for therapeutic purposes in humans. Emerging evidence has challenged the understanding of the metal-ligand dissociation mechanism of Cu(II)-[Cu(ATSM)], specifically questioning the validity of the assumption that the complex can stay intact in blood. Several studies have also addressed the importance to  (HIF-1). c Activation of HIF-1 promotes cancer stem cells (CSCs) via survival advantages, which d enhances the self-renewal ability and inhibitsdifferentiation of CSCs. e In effect this is in favor of tumor proliferation, which f generates oxidative stress, and g further worsen tumor hypoxia. On the other hand, h tumor hypoxia stimulates copper transporter 1 (CTR-1) expression, increases the production of CTR-1 to i transport incoming Cu(I), j upregulating the HIF-1 expression, where Cu(I) is required understand the role of copper itself in the retention, although the exact pathways remain unclear and requires further investigations. In summary, the emerging preclinical and clinical studies as well as potential clinical applications of copper-64 radiopharmaceuticals in PET imaging and theranostics would benefit from further mechanistic investigations in order to identify the exact copper retention mechanism.