Scandium-44: Diagnostic Feasibility in Tumor-Related Angiogenesis

Angiogenesis-related cell-surface molecules, including integrins, aminopeptidase N, vascular endothelial growth factor, and gastrin-releasing peptide receptor (GRPR), play a crucial role in tumour formation. Radiolabelled imaging probes targeting angiogenic biomarkers serve as valuable vectors in tumour identification. Nowadays, there is a growing interest in novel radionuclides other than gallium-68 (68Ga) or copper-64 (64Cu) to establish selective radiotracers for the imaging of tumour-associated neo-angiogenesis. Given its ideal decay characteristics (Eβ+average: 632 KeV) and a half-life (T1/2 = 3.97 h) that is well matched to the pharmacokinetic profile of small molecules targeting angiogenesis, scandium-44 (44Sc) has gained meaningful attention as a promising radiometal for positron emission tomography (PET) imaging. More recently, intensive research has been centered around the investigation of 44Sc-labelled angiogenesis-directed radiopharmaceuticals. Previous studies dealt with the evaluation of 44Sc-appended avb3 integrin–affine Arg-Gly-Asp (RGD) tripeptides, GRPR-selective aminobenzoyl–bombesin analogue (AMBA), and hypoxia-associated nitroimidazole derivatives in the identification of various cancers using experimental tumour models. Given the tumour-related hypoxia- and angiogenesis-targeting capability of these PET probes, 44Sc seems to be a strong competitor of the currently used positron emitters in radiotracer development. In this review, we summarize the preliminary preclinical achievements with 44Sc-labelled angiogenesis-specific molecular probes.


Tumour-Related Angiogenesis
The formation of new blood vessels from pre-existing ones-denoted as angiogenesis-is required for tumour maintenance and development as well as for metastasis formation [1,2]. Angiogenesis is regulated by a delicate balance between a host of pro-angiogenic and antiangiogenic factors [3]. Hence, intensive attention has been placed on the assessment of cancer-associated neovascularization [4,5]. Producing various angiogenic factors, proteases, heparanase, digestive enzymes, and chemotactic and stimulatory factors, tumour cells exert a direct effect on capillary endothelial cells, induce the assembly of activated immune cells, and trigger the activity of stromal cells; thus, they create a pro-angiogenic niche in the tumour microenvironment [2,6].
Non-invasive positron emission tomography (PET) or positron emission tomography/computed tomography (PET/CT) are regarded as the mainstay diagnostic tools in the detection of primary tumours and pertinent metastases. Furthermore, PET can be a useful means of angiogenesis-targeted drug development, authorization, and dose estimation [8,12]. Several PET radionuclides have been used in angiogenesis-directed diagnostical settings, including fluoride-18 ( 18 F), gallium-68 ( 68 Ga), copper-64 ( 64 Cu), and even the gamma emitter indium-111 ( 111 In). Although scandium (Sc) radioisotopes were already recognized as valuable radiometals for isotope diagnostic use in the late 1990s, they have been set aside for almost 20 years [13].
There are two major ways to produce 44 Sc. Via the proton irradiation of natural calcium or enriched 44 Ca targets (through a 44 Ca(p,n) 44 Sc nuclear reaction), the production of 44 Sc can be easily obtained by applying low-energy cyclotrons [17][18][19]. Cyclotron-based production provides outstanding radiochemical yield (RCY) and radiochemical purity (RCP) for clinical applications [18,20]. Of note, 44 Sc production via applying a cyclotron has been confirmed to be economically viable [17]. Although 44 Sc can also be eluated from titanum-44/scandium-44 ( 44 Ti/ 44 Sc) generator systems, the radioactive-waste handling of long-lived 44 Ti (62 ± 2 years) makes this approach challenging [21][22][23]. In addition, the demanding production of 44 Ti implies another obstacle regarding the routine usage of 44 Ti/ 44 Sc generators [17,24]. Difficulties around the production of 44 Sc may underpin why this radiometal came to the focus of exhaustive investigation as a potential labelling isotope so late. 44 Sc, with physical properties of T 1/2 : 3.97 h (lately reported as 4.04 h), E β + average : 632 KeV, E β + mean : 0.63 MeV, and I: 94.3%, has begun to emerge as a radiometal of particular interest in imaging, dosimetry, and treatment follow-up [20, 25,26]. Positron emitter 44 Sc also has a gamma co-emission of 1157 keV (99.9%), which may limit the radioactive dose that can be injected into patients [26]. However, this high-energy gamma radiation made the radiometal a precious candidate for the novel β + γ coincidence PET imaging [27]. Since the positron energy of the applied PET radionuclide is inversely proportional to the resolution of the reconstructed image, better spatial-image resolution and better quality could be obtained with 44 Sc relative to other radiometals-for example, 68 Ga ( 68 Ga: E β + max : 1.9 MeV and E β + mean : 0.89 MeV vs. 44 Sc: E β + max : 1474 KeV and E β + mean : 0.63 MeV) [28][29][30]. These advantageous physical characteristics together with its decay to nontoxic Ca make 44 Sc widely feasible in PET and PET/CT imaging [13,31]. Given its small ionic radius, the chemistry of 44 Sc is comparable to that of 68 Ga; thus, 44 Sc could be applied in several fields, including dosimetry investigations in theranostic settings and in trafficking ligands with longer pharmacokinetics and subsequent requirements for longer imaging times (even 24 h post administration) [32,33].
Prior studies have already dealt with the investigation of the feasibility of 44 Sc in pre-therapeutic dosimetry [34,35]. Khawar et al. published a paper indicating that the pharmacokinetics of [ 44 Sc]Sc-PSMA-617-based (PSMA (prostate-specific membrane antigen)) PET/CT imaging serves as a useful tool for the calculation of normal organ-absorbed doses and the maximum allowable activity in prostate-cancer patients prior to lutetium-177 ( 177 Lu)-labelled PSMA-617 ([ 177 Lu]Lu-PSMA-617) radiotherapy [36]. Pioneering clinical studies further strengthened the efficacy of 44 [35].
Similar to 68 Ga, 44 Sc is also able to form thermodynamically stable complexes with chelator DOTA (1,4,7,10-teraazacyclododecane-N,N ,N",N" -teraacetic acid) [40,41]. In addition to this, 44 Sc seems superior to 68 Ga in several facets. Given its longer half-life (T 1/2 44 Sc: 3.97 h vs. T 1/2 68 Ga: 68 min) and cyclotron-dependent extensive synthesis, 44 Sc can be easily shipped to remote nuclear medical facilities. This contributes not only to the wider distribution of the radioisotope but also to the accomplishment of proteinor antibody-based PET studies with lengthened examination times [42]. Furthermore, radiopharmaceuticals with longer pharmacokinetic characteristics can also be proposed due to the longer half-life of the radiometal [17]. Moreover, the pharmacokinetic properties of 44 Sc-appended imaging probes are largely comparable to those of the 68 Ga-labelled counterparts [43]. The nearly four-hour half-life of 44 Sc could be easily fitted to the pharmacokinetics of several targeting molecules, including peptides, antibodies, or their fragments, as well as oligonuclides, which makes facile radiopharmaceutical synthesis possible [13,17]. Additionally, long-lived 44 Sc favours delayed imaging and the achievement of appropriate tumour-to-background (T/M) ratios. The time of imaging is of critical importance from the point of view of patient management and the scheduling of examinations. Therefore, the use of 44 Sc-labelled radiopharmaceuticals would contribute to the establishment of a fluent workflow. 44 Sc-labelled PET radiopharmaceuticals could even be appropriate for intraoperative radio-guided surgery, including the detection of lymphatic metastases at later time points post injection [32]. Consequently, 44 Sc-based PET probes could stand out as valuable imaging agents. 47 Sc (T 1/2 = 3.35 days)-the therapeutic match of 44 Sc-emits a β − radiation of a maximum energy of 0.600 MeV (31.6%) and 0.439 MeV (68.4%), which could be exploited in targeted radiotherapy [33,44]. Therefore, 44 Sc/ 47 Sc has exquisite potential as a radiotheranostic pair in PET diagnostics and in therapeutic settings [33,45]. Besides 44 Sc and 47 Sc, scandium-43 ( 43 Sc) is another significant member of the group of Sc isotopes. Investigating the quantitative capabilities of 43 Sc/ 44 Sc and comparing it to 18 F and 68 Ga, Lima et al. published a paper indicating that the application of radiotracers labelled with the mixture of 43 Sc/ 44 Sc may be beneficial in clinical fields [46]. The findings of their phantom study proved that precise, quantitative PET/CT could be obtained by applying commercial PET systems [46]. Given that 43  The optimal short half-life (109.8 min) and the high positron abundance (β ≥ 97%) of 18 F made it the most commonly used radioisotope for the labelling of PET biomolecules [48]. Positron emitter 18 F possesses a relatively low positron energy (E max = 0.635 MeV and E mean = 0.250 MeV) and a short positron range within the tissue (maximum of 2.3 mm) [49,50]. These nuclear properties ensure ideal image quality and high spatial resolution obtained with 18 F-labelled tracers [51]. The 109.8 min-long half-life is beneficial for synthesis procedures as well as for the performance of examinations of a few hours [51]. Owing to the short longevity, 18 F-labelled radiopharmaceuticals can be safely administered without an increased risk of excess radiation. Moreover, another advantage is the facile cyclotron-based production, which provides immense quantities of the isotope at high specific activity [51]. Although a cyclotron is required to produce the radiometal, its decay characteristics make the distribution of 18 F-and 18 F-labelled radiotracers to distant nuclear medical laboratories without an on-site cyclotron possible. Even though 18 F is well suited for the labelling of a wide range of small and medium-sized molecules, the radiolabelling of peptides with 18 F is still cumbersome [51]. Hence, different isotopes have come into focus that address some of the difficulties associated with 18 F-based radiotracers.
The short half-life and related transport challenges, however, may limit the widespread implementation of 68 Ga into clinical and preclinical settings. Owing to the short longevity of the radiometal, radiolabelling procedures are only possible in laboratories equipped with in-house 68 Ge/ 68 Ga-generator systems. In addition, due to its short half-life, 68 Galabelling can be applied solely in case of small molecules and peptides featured with a rapid pharmacokinetic profile [17,56]. Furthermore, 68 Ga-labelling is only suitable for PET examinations of relatively short duration. Because of the breakthrough of 68 Ge and sorbent material, the purification and concentration of 68 Ga is warranted following elution, which could also restrict its applicability in routine diagnostic usage [57]. Image noise associated with the elevated positron energy of the radionuclide constitutes another shortcoming of 68 Ga imaging [32]. These facts render 68 Ga-labelled radiotracers of limited attractiveness for centralized distribution.
The application of 64 Cu may bridge the limitations derived from 68 Ga-associated imaging. Given its longer half-life (T 1/2 : 12.7 h) and generator-independent production, the use of 64 Cu (β + emission, E average = 278 keV, abundance: 19%) seems to be economically more viable for PET imaging [58,59]. Due to its longer-lived nature, the easy transport to distant laboratories without an on-site cyclotron facilitates the integration of 64 Cu-appended radiopharmaceuticals into diagnostics. Moreover, the 12.7 h half-life of the radiometal can be easily tailored to biomarker-targeting small and large molecules, peptides, antibodies, and nanomolecules with prolonged elimination kinetics [60]. Additionally, its longer halflife makes 64 Cu-based radiochemical procedures facile [60]. The coordination chemistry of the radiometal enables complexation with various chelating agents, which further supports the frictionless performance of radiolabelling [60]. Therefore, 64 Cu is extremely useful in the establishment of a broad set of radiotracers for diagnostic purposes. However, the use of 64 Cu is not without shortcomings. Its concomitant β − emission (β − = 39.0%, E = 190.2 keV) and relatively low positron-branching ratio (17.6%) mean a meaningful extra radiation danger for patients [58,61,62]. Furthermore, in case of complexation, chelators must be customized to the complex redox chemistry of 64 Cu [54].
Beyond 44 Sc, the use of zirconium-89 ( 89 Zr) has also been exhaustively investigated in PET imaging [63]. Given its favourable decay half-life (T 1/2 = 3.3 days; 78.4 h), appropriate radiochemistry, and the accessibility of different chelating agents for complexation with the radioisotope, 89 Zr is welcomed for the radiolabelling of PET-based imaging molecules [63]. The relatively long half-life of the radiometal could be easily adjusted to the pharmacokinetic profile of monoclonal antibodies (mABs); therefore, 89 Zr seems to be well suited for the radiolabelling of mABs and for PET immunoimaging applications [63][64][65]. Several mAbs, including anti-human epidermal growth factor receptor 2 (HER2) trastuzumab, anti-epidermal growth factor receptor (EGFR) mAb cetuximab, anti-PSMA mAb J591, and anti-vascular endothelial growth factor (VEGF) bevacizumab, were successfully labelled with 89 Zr for the PET imaging of breast cancer, squamous-cell carcinoma, prostate tumours, and ovarian tumours, respectively, at both preclinical and clinical levels [66][67][68][69].
Due to their decay characteristics (positron emission: E max and E average , 897 keV and 396.9 keV, respectively), PET images obtained with 89 Zr-labelled compounds are of adequate spatial resolution [70][71][72]. However, similar to 44 Sc, 89 Zr has spontaneous high-energy gamma radiation of 908.97 keV, which accounts for a major drawback of its usage [70]. Although the concomitant gamma radiation does not influence either the quality or the quantification of the PET scans, it needs to be taken into account when patient doses are determined [73,74]. Another potential disadvantage could be the limited availability of the radiometal [63].
Taking the abovementioned facts into account, 44 Sc may therefore be a valuable substitute for the currently applied 68 Ga and 64 Cu in the establishment of peptide-based targeted PET radiopharmaceuticals. In this review, we provide a comprehensive overview of the role of 44 Sc-labelled peptide-based radiopharmaceuticals in the molecular imaging of cancer-related angiogenesis ( Figure 1). Table 1 summarises the preclinical studies with 44 Sc-labelled PET radiotracers that selectively target angiogenic biomarkers. In Table 2   Pro-angiogenic VEGF-A 165 /NRP-1 complex formation Investigation of physicochemical properties and affinity for NRP-1 NRP-1 44 Scradiocompounds ( 44 Sc-1, 44 Sc-1bis, 44 Sc-2, 44

Integrin a v b 3
A v β 3 integrin has a central role in angiogenesis, neovascularisation, tumour growth, and related metastatic spread [80]. Built up by noncovalently assembled α and β transmembrane subunits, the a v b 3 integrin heterodimer is able to adhere to a wide set of target molecules, including extracellular matrix (ECM) or soluble ligands and different cell-surface molecules [81,82]. The interaction of a v b 3 integrin with ECM proteins, fibroblast growth factor-2 (FGF2), metalloproteinase MMP-2, activated platelet-derived growth factor (PDGF), insulin, and vascular endothelial growth factor (VEGF) receptors favours cellular adhesion, cell proliferation, migration, and invasiveness, as well as the inhibition of apoptotic processes [83,84].
Since a v b 3 integrin is upregulated in a vast array of cancer cells, tumour endothelial cells, and new-born blood vessels, it is recorded as an impressive biomarker of angiogenesis in oncological tumour diagnostics [82]. In addition, the expression level of a v b 3 integrin correlates well with angiogenic activity, which makes the receptor suitable for the trafficking of antiangiogenic therapy.
Arg-Gly-Asp (RGD) tripeptides specifically bind to integrin receptors [85]. The affinity of an RGD sequence containing peptides to a v b 3 integrin has increased its attractiveness for the development of RGD-based PET and single-photon emission computed tomography (SPECT) radioindicators in the detection of tumour-associated angiogenesis. Intensive focus has been placed upon the evaluation of 18  More recently, 44 Sc-based RGD radiopharmaceuticals have gained increasing interest for tumour-imaging scenarios.
Hernandez et al. used 44 Sc that was obtained from a cyclotron by the proton irradiation of natural Ca metal targets for the labelling of α v β 3 integrin-affine dimeric cyclic arginineglycine-aspartic acid (cRGD) 2 to assess the peptide selectivity of [ 44 Sc]Sc-DOTA-c(RGD) 2 at both the in vivo and in vitro levels [17]. The tumour-targeting capability, the specificity, and the binding potential of this radioisotope were assessed in vitro, in vivo, and ex vivo by performing competitive-cell binding assay, small-animal PET examinations, receptorblocking studies, and organ-distribution experiments. For the in vivo evaluation of the α v β 3 -targeting capacity of [ 44 Sc]Sc-DOTA-c(RGD) 2 , U87MG human glioblastoma-bearing female athymic nude mice were intravenously (iv.) administrated with 5.5-7.4 MBq of the radiotracer and then underwent microPET/microCT acquisition at different time points (0.5, 2, and 4 h post injection). The α v β 3 integrin overexpression of the tumour cells and the vasculature of the U87MG experimental models is well established. In a bid to assess the α v β 3 selectivity of cRGD, (cRGD) 2 , and DOTA-(cRGD) 2 in vitro, a competitive cellbinding assay was performed using integrin-targeting radioligand 125 I-echistatin. Moreover, in vivo and ex vivo receptor-blocking studies were further conducted with the simultaneous application of 3.7 MBq of [ 44 Sc]Sc-DOTA-c(RGD) 2 and 50 mg/kg (approximately 1 mg) of c(RGD) 2 to confirm receptor selectivity. During the competitive assays, continuously elevating amounts of cRGD, (cRGD) 2 , and DOTA-(cRGD) 2 were administered to 1 × 10 5 U87MG cells that were pre-incubated with 125 I-echistatin. As part of the ex vivo experiments, the weight and radioactivity of the harvested major tissues and organs, the blood, and the tumour were measured. The tracer-uptake values were obtained as mean %ID/g ± SD. Due to the significantly elevated tracer accumulation of the tumours, along with the negligible background activity, high-contrast PET images could be obtained at each investigated time point.
Quantitative in vivo analyses corresponding to the ex vivo data strengthened prominent [ 44 Sc]Sc-DOTA-c(RGD) 2 uptake in the tumourous lesions, with %ID/g values of 3.93 ± 1.19, 3.07 ± 1.17, and 3.00 ± 1.25 measured 0.5, 2, and 4 h after the injection, respectively. Similar to the PET data, insignificant ex vivo non-target activity was detected. Receptor-blocking studies showed the reduction of the radiotracer uptake of the tumours both in vivo and ex vivo. However, a (cRGD) 2 -derived decrease in the tracer concentration was recorded ex vivo in the background organs/tissues, which could be attributed to their physiological α v β 3 expression.
Assessing the adhering affinities of cRGD, (cRGD) 2 , and DOTA-(cRGD) 2 , Hernandez et al. noted that the administration of the investigated peptides induced the concentrationdependent disposition of 125 I-echistain. Although a binding competence 10 times stronger was depicted in the case of (cRGD) 2 relative to its monomeric counterpart, DOTA complexation exerted no meaningful effect on the peptide-targeting affinity of (cRGD) 2 . Based on current literature data, Hernandez et al. were the first to propose a peptide-based PET radiopharmaceutical labelled with cyclotron-derived 44 Sc. Therefore, they managed to strengthen the suitability of 44 Sc for PET investigations in addition to the alreadyestablished radiometals. A summary of their study can be seen in Table 1.
In  Table 1) [76]. In this study they also assessed 68 68 Ga-labelled compounds was compared, as well. The liver showed increased uptake of the 68 Ga-labelled DOTA-chelated peptides relative to the 44 Sc-appended DOTA matches. This finding was in accordance with the in vivo PET data. Regarding other organs, the uptake of the 68 Ga and 44 Sc peptides was mostly identical. Since the 44 Sc-appended molecules complexed with NODAGA demonstrated a largely identical tracer-accumulation pattern compared to the DOTA-chelated ones, NODAGA seems to be an attractive alternative chelator to more established DOTA for the development of 44 Sc-labelled PET radiotracers [76].
In a study by Nagy et al., the applicability of mesocyclic chelating agent AAZTA (1,4bis(carboxymethyl)-6-[bis(carboxymethyl)]amino-6-methylperhydro-1,4-diazepine) for conjugation with 44 Sc-labelled PET radiometalated peptides was ratified [77]. The outstanding complex formation between Sc III and AAZTA laid the basis for the proposal of 44 Sc-labelled PET-imaging probes chelated by AAZTA. Tumour-naïve healthy control and 4T1 tumourbearing BALB/c mice were used for the evaluation of the in vivo biodistribution of 44 Sc 3+ , 44 Sc(AAZTA) − , and 44 Sc(CNAAZTA-c(RGDfK)) (c(RGDfK): cyclo(-Arg-Gly-Asp-d-Phe-Lys)). The choice of using 4T1 tumours for their experiments was reinforced by the integrin α v β 3 overexpression of breast cancers [93]. Positron emission tomography/magnetic resonance imaging (PET/MRI) examinations were performed 30 and 90 min post administration of the free 44 Sc 3+ , 44 Sc(AAZTA) − complex, and 44 Sc-labelled CNAAZTA-conjugate. Discrete hepatic, pulmonary, and lienal uptake of 44 Sc 3+ was registered on the PET scans of the control group 90 min post injection. In contrast, 44 Sc(AAZTA) − showed no accretion in either the thoracic or the abdominal organs. Investigating the distribution pattern of 44 Sc(CNAAZTA-c(RGDfK)), Nagy et al. revealed physiological accumulation in the liver, intestines, and urinary tract of the control BALB/c study mice. Based on the enhanced 4T1 tumour-tracer uptake, the tumour-homing capability of this newly produced α v β 3 integrinaffine, 44 Sc-labelled, AAZTA-conjugated imaging probe was confirmed. Comparing the radioactivity of the neoplasms with that of the non-target tissues and organs, a radiopharmaceutical concentration 25 times more elevated was encountered in the subcutaneously (sc.) growing 4T1 tumours than in the background tissues. Consequently, imaging with 44 Sc(CNAAZTA-c(RGDfK)) ensured high T/M ratios, which enable precise differentiation between the neoplastic alterations and the surrounding healthy tissues. It can be concluded that the application of a chelator other than the one generally used (e.g., DOTA) has no influence on the imaging properties of 44 Sc. Furthermore, accurate lesion detection and anatomical localization provided by the appropriate T/M ratios are of crucial significance in terms of PET-image reporting. These findings also support the feasibility of 44 Sc-labelled PET probes in tumour diagnostics. The details of their study are presented in Table 1. In a recent study by Ghiani et al., the potential of an AAZTA-chelated PSMA inhibitor (B28110) radiolabelled with 44 Sc ([ 44 Sc]Sc-B28110) was confirmed for the PET imaging of prostate cancer [94]. For the accomplishment of their study, LNCaP tumour-bearing mice were generated by the sc. transplantation of 5 × 10 6 PSMA-positive LNCaP (prostate cancer derived from a metastatic lymph-node lesion of a human with prostate cancer) tumour cells into the right-shoulder and the right-thigh region of CB17 SCID male mice. Among others, they investigated the in vivo and ex vivo biodistribution pattern and the tumour-targeting capability of 44  showed the highest concentration in the LNCaP tumours, although no remarkable difference was noted between the accumulation of these two compounds. The ex vivo results also revealed an uptake two times higher of the labelled B28110 derivatives relative to that of the [ 44 Sc]Sc-PSMA-617. Therefore, the findings of Ghiani et al. also strengthened the suitability of AAZTA for the conjugation of 44 Sc PET probes in diagnostic settings [94].
Aminobenzoyl-bombesin analogue (4-aminobenzoyl-Q-W-A-V-G-H-L-M-NH 2 , AMBA) is regarded as a highly valuable imaging agent in the nuclear medical diagnostics of GRPRupregulated tumours [115]. A former preclinical study proved the GRPR-binding ability of 18 F-and 64 Cu-labelled AMBA derivatives [116]. Furthermore, the targeting potential of DOTA-conjugated AMBA labelled with 68 Ga or 177 Lu was also reported at the preclinical level [115,117]. Table 1) [75]. The application of PC-3 cell lines was suitable for these studies, as previous research had confirmed their GRPR positivity [111,118]. Standardized uptake values (SUV) and T/M ratios were registered to quantify the radiopharmaceutical uptake. Both qualitative analyses and quantitative SUV data confirmed the GRPRtargeting adequacy of the investigated radiolabelled AMBA derivatives. Although 60 min post administration of [ 44 Sc]Sc-NODAGA-AMBA higher SUV mean (0.90 ± 0.17), SUV max (1.54 ± 0.18), T/M SUV mean (6.16 ± 1.24), and T/M SUV max (6.71 ± 1.08) values of the sc. growing PC-3 tumours were registered compared to the 68 Ga-labelled counterpart, this was not statistically significant (p ≤ 0.05). Moreover, 120 min after the injection of the radiotracers, decreased non-target activity was observed, which led to excellent T/M ratios.

Investigating the GRPR selectivity of [ 44 Sc]Sc-NODAGA-AMBA, Kálmán-Szabó et al. performed in vitro receptor binding and in vivo examinations using a GRPRpos. PCa PC-3 (prostate cancer) xenograft (displayed in
Ex vivo biodistribution studies of both the healthy control and the tumour-bearing mice were accomplished 30, 60, 120, and 180 min after the administration of 11.3 ± 1.4 MBq of [ 44 Sc]Sc-NODAGA-AMBA or [ 68 Ga]Ga-NODAGA-AMBA. The radioactivity of the explanted tissues and organs was registered with a calibrated gamma counter, and the counts-per-minute (CPM) values of all samples were converted to the percentage of administered dose per gram of tissue. The radiotracer concentration was presented as %ID/g. The biodistribution pattern of the control small animals displayed lower accumulation of [ 44 Sc]Sc-NODAGA-AMBA relative to [ 68 Ga]Ga-NODAGA-AMBA; however, this difference was not statistically significant (p < 0.05). After analysing the tracer uptake of the organs separately, the blood, liver, spleen, small and large intestines, stomach, heart, and lungs were depicted with moderate radioactivity, whereas the kidneys, urine, adrenal glands, and pancreas demonstrated significant tracer accretion. The outstanding urinary activity could have been due to the renal method of elimination or the physiological presence of GRPR in the urogenital smooth muscle [119]. The natural appearance of GRPR in the pancreas and in the adrenal glands underlies the remarkable radiotracer uptake of these organs [105,120]. Discrete receptor expression of the neuroendocrine gastrointestinal cells and the pulmonary existence of the GRPR gene may explain tracer accumulation in the lungs and in the intestines [121,122]. In line with the in vivo findings, the [ 44 Sc]Sc-NODAGA-AMBA uptake of the PC-3 tumours was more prominent in comparison to the 68 Ga-labelled derivative.
Receptor-supressing in vivo and ex vivo studies were carried out with the injection of 15 mg/kg of BBN into the tumorous study mice 30 min before radiotracer application to further ratify the GRPR selectivity of the labelled BBN analogues. Correspondingly to the ex vivo blocking figures, both qualitative and quantitative in vivo analyses revealed considerably mitigated tracer uptakes in the PC-3 tumours 60 and 120 min post injection.
Given the GRPR selectivity of [ 44 Sc]Sc-NODAGA-AMBA established by Kálmán-Szabó et al., along with the better imaging characteristics of 44 Sc over 68 Ga, 44 Sc-labelled BBN-based radiotracers, will hopefully be embraced in the diagnostic armamentarium of GRPRpos. tumours.
Another investigation, executed by Koumarianou et al., focused on the comparison of 44 Sc-and 68 Ga-labelled BBN analogues in in vitro and in vivo biological medium [33]. In a bid to evaluate the potential impact of 44 Sc on the binding ability of BBN analogue to GRPR, GRPR-affine DOTA-conjugated BBN analogue was labelled with 68 Ga and 44 Sc, and the in vitro receptor-adhering capacity, the ex vivo organ distribution, and the in vivo behaviour of both 44  The organ-distribution pattern of the control cohort-solely receiving the radiolabelled analogue-and that of the blocked group was compared. Androgen-independent Dunning R-3327-AT-1 prostate-cancer tumour-bearing small animals were generated by the subcutaneous (sc.) transplantation of GRPR-overexpressing R-3327-AT-1 cells (≈0.4 mL, 10 4 cells/µL) into the dorsal aspect of the hind foot of male Copenhagen rats [33,123]. Dynamic microPET acquisition was executed approximately 10-14 days post tumour induction by applying 30-50 MBq of either the 68 Ga-or the 44 Sc-appended imaging probes. nat Ga-DOTA-BN [2][3][4][5][6][7][8][9][10][11][12][13][14]NH 2 showed higher specificity towards GRPR receptors than the nat Sc-complexed counterpart. The pancreas was presented with considerable uptake using both labelled compounds with ID/g values of 0.64 ± 0.00% and 0.58 ± 0.05% for 68 Ga-DOTA-BN [2][3][4][5][6][7][8][9][10][11][12][13][14]NH2 1 and 2 h post injection, respectively, whereas the subsequent data were obtained in the case of the 44 Sc-labelled probe: 2.67 ± 0.53% ID/g and 1.51 ± 1.19% ID/g one and two hours after tracer administration, respectively. The physiological pancreatic GRPR expression could explain these results. Upon visual assessment of the in vivo PET images, the peripheral tumour areas demonstrated more enhanced tracer uptake compared to the central neoplastic regions. The heterogeneity of the receptor expression within the tumour and the unevenness regarding the radiopharmaceutical binding ability of GRPR could underpin the difference between the tracer accretion of the external and inner tumour regions. Followed by prompt radiotracer uptake post injection, Koumarinaou et al. noted a gradual decline in the accumulation kinetics of the 68 Ga-labelled compound throughout the entire examination period, whereas they observed a stable albeit lower tracer uptake in the case of the 44 Sc-appended match. Of note, the relative tumour radioactivity-based kinetics (normalized to the testis as a reference tissue) did not reveal any meaningful differences between the two tracers. Overall, the GRPR selectivity of 68 Ga and 44 Sc differed. However, given the similar distribution pattern, uptake, and excretion times of both the 68 Ga-and the 44 Sc-labelled radiopharmaceuticals, as well as their comparable accumulation in neoplastic tissues, these radiometals seemed equally effective in the detection of GRPRpos. tumours [33].

Hypoxia-Associated 2-Nitroimidazole (NI) Derivatives
Beyond the above-mentioned radiopharmaceuticals, hypoxia-associated PET tracers are also embraced in tumour diagnostics [124][125][126]. Since cancer-related neoangiogenesis, induced by oxygen insufficiency, leads to morphologically and functionally impaired bloodvessel development, which directly affects the efficiency of anti-tumour treatment, the early identification of hypoxic tumorous regions is of paramount importance [127][128][129]. In this respect, hypoxia-directed imaging probes could be potential weapons in the timely diagnostic assessment of tumour-related hypoxia and the achievement of therapeutic successes.
Given the fact that hypoxia-associated 2-nitroimidazole (NI) derivatives are intracellularly trapped under hypoxic conditions while they are reoxidised and excreted from normoxic cells, by applying NI-based imaging probes, healthy and hypoxic tumorous cells could be definitively differentiated from each other [130]. Therefore, NI compounds labelled with radioisotopes seem to be promising indicators of tumour-associated hypoxia. Previous successes were attained in clinical trials by applying 18 [124][125][126]. However, the low T/M ratios generated by the prolonged elimination of these lipophilic tracers may hamper their widespread application in diagnostic fields. In 2011, Hoigebazar et al. verified the feasibility of 68 Ga-labelled DO3AM-NI in hypoxia PET imaging [133].
Later in 2022, Szücs et al. published the synthesis of 44 Sc-appended hypoxia-specific DO3AM-NI ([ 44 Sc]Sc-DO3AM-NI) and its comparison to the 68 Ga-labelled diagnostic match [78]. To study the in vivo and the ex vivo biodistribution of the 44 Sc and 68 Ga hypoxia PET probes, healthy and CB17 SCID adult male mice bearing KB (human epidermal carcinoma) tumours in their left-shoulder area were applied (presented in Table 1). In vivo PET/MRI studies were conducted 13 ± 1 days post implantation of 5 × 10 6 KB tumour cells at an average tumour bulk of 110 mm 3 . Both the control and the tumourous study mice were iv. administered with 8.42 ± 0.38 MBq of [ 44 Sc]Sc-DO3AM-NI and [ 68 Ga]Ga-DO3AM-NI. Then, whole-body T1-weighted MRI and 20 min-long static whole-body PET acquisition was performed 90 and 240 min post tracer application. SUV and T/M ratios (ratios of tumour to skeletal muscles of the right-shoulder region) were defined as quantitative PET parameters. Ninety minutes and 4 h (240 min) post injection, organtissue specimens were harvested, weighted wet, and measured for radiopharmaceutical uptake by applying a gamma counter to obtain ex vivo data. Values were provided as mean %ID/g ± SD. Szücs et al. managed to label the NI derivative DO3AM-NI with 44 Sc with a high-labelling yield and RCP. Except for the kidneys and the urinary bladder, the organs of the abdomen and thorax displayed moderate tracer accretion upon visual assessment of the PET scans. The high uptake of both probes in the kidney underpinned its important role in radiopharmaceutical elimination. Intriguingly, enhanced hepatic [ 68 44 Sc in the targeted PET imaging of hypoxia-related tumour diagnostics. Unlike these findings, previous studies dealing with 18 F, iodine-131 ( 131 I), technetium-99m ( 99m Tc), and 64 Cu-appended NI imaging probes reported substantial tracer accumulation in the abdominal organs, mainly in the intestines, along with hepatic metabolization, which could lead to difficulties regarding the detection of abdominal malignancies [134][135][136][137].
In line with the in vivo findings, the ex vivo figures revealed that the KB tumour uptake of the two investigated probes did not differ significantly between the two assessed time points; however, the accumulation of the 44 68 Ga were in accordance with those of Szücs et al. [138,139]. In addition to the urinary method of excretion, they registered discrete radioactivity in the abdominal and thoracic organs [138,139]. As for the visualization of the PET scans, due to prompt urinary clearance and favourable T/M ratios, [ 44 Sc]Sc-DO3AM-NI seemed to be superior to the 99m Tc-, 64 Cu-, 18 F-, 131 I-, and 68 Ga-linked derivatives. Based on the exceptional tumour-homing capability, appropriate contrast and favourable quantitative parameters of the 44 Sc-appended molecular agent [ 44 Sc]Sc-DO3AM-NI could be a highly valuable hypoxia-targeting PET probe in the diagnostic algorithm of tumour identification.
Although no previous in vivo experiments that investigated 44 Sc-labelled VEGF-VEGFR-NRP-1-directed radiopharmaceuticals have been published so far, Masłowska et al. examined the potential diagnostic and therapeutic feasibility of such molecules under in vitro circumstances [79].

Conclusions
The in vivo tumour-targeting capability of 44 Sc-labelled, angiogenesis-targeting imaging probes, along with the favourable physicochemical properties of the radiometal, capitalize on the relevance of this isotope for the development of highly specific PET radiotracers. Their transition into clinical routine will inevitably herald a new era in personalized cancer diagnostics. Furthermore, the non-invasive assessment of angiogenesis-/neo-angiogenesisrelated molecules is of outstanding importance to identifying patients who may benefit from targeted antiangiogenic therapy. Together with its therapeutic surrogate, the 44 Sc/ 47 Sc isotopic pair could be effectively employed in dosimetry estimations, cancer treatment, and therapeutic follow-up.
However, the future progression of 44 Sc-labelled molecular vectors from preclinical level to human application could be hampered by some constraints regarding the usage of 44 Sc. First, the limited accessibility of the radiometal is a meaningful hurdle to its centralized distribution. In addition, the high-energy concomitant gamma rays (1157 keV) of 44 Sc could lead to reduced image quality; therefore, future studies to optimize PETimaging parameters are required [32]. Furthermore, 43 Sc lacking in accompanying gamma co-emission may serve as a valuable substitute for 44 Sc in imaging settings [46]. Global steps should be taken to overcome these drawbacks, with the ultimate goal of exploiting both the diagnostic and therapeutic value of 44 Sc. Data Availability Statement: The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.