Introduction

Prostate specific membrane antigen (PSMA) is a type II transmembrane glycoprotein that exhibits glutamate carboxypeptidase activity in the proximal small intestines (folate hydrolase) and the brain (NAALADase)1. Furthermore, PSMA expression can be found on several tissues including prostate tissue, renal tubules, liver, spleen, and salivary and lacrimal glands2. In prostate cancer (PCa) cells, PSMA is expressed 100–1000 fold higher. This overexpression is positively correlated with tumor grade, pathological stage and metastatic castration-resistant prostate cancer (mCRPC), making it an attractive target for both diagnostic and therapeutic purposes3,4,5.

For molecular imaging, radiopharmaceuticals are preferably produced with a high molar activity (MA) to ensure that the injected solution contains only a small amount (pico to microgram range) of the tracer substance to avoid pharmacological activity and potential toxic effects. With competitive binding of the radioligand to a target with a saturable receptor density, the chemical mass may affect receptor binding and tracer pharmacokinetics, leading to impaired imaging quality6. This mass effect has already been described in the central nervous system, where sub-nanomolar binding affinity ranges and high molar activities are required to distinguish specific from non-specific binding7,8. Noguchi et al. demonstrated a positive impact of a high molar activity of [11C]raclopride for characterization of the D2-receptor in low density regions in the rat brain9. For neuroendocrine tumors containing somatostatin receptors, multiple somatostatin-targeting radioligands have shown a dependency between peptide mass and radioligand uptake. Peptide mass-dependent uptake of [68Ga]DOTATOC, a radiolabelled somatostatin receptor ligand, was observed both in organs and tumor tissue10. 111In[DOTA0,TYR3]octreotide showed an organ-dependent relationship between radioligand uptake and peptide mass, which was suggested to be a balance of the amount of peptide positively influencing receptor clustering while negatively impacting receptor saturation11. For [111In]pentreotide, a bell-shaped relationship between specific uptake in octreotide receptor-positive tissues and carrier dose was found. This introduced an additional parameter to ameliorate the target-to-background contrast. Attributing factors to this phenomenon could be receptor accessibility (blood perfusion, presence of endogenous ligand) and receptor binding properties (dissociation constant, rate of internalization and re-expression rate of the receptor)12. Furthermore, a simulation study using PBPK modelling investigated the effect of ligand amount, affinity and internalization rate on PSMA imaging and RLT. Overall, the influence of the amount of ligand was more pronounced for radiotracers with a dissociation constant < 1 nM as well as for therapy compared to imaging. Increasing ligand amount significantly reduced absorbed doses in highly perfused or high affinity tissues13. These studies show that the amount of peptide administered could be optimized to increase absolute tumor uptake and tumor-to-background ratios.

The impact of the molar activity as a parameter for PET imaging has been investigated for several imaging targets, and it was reported to be highly pronounced for radioligands targeting PSMA. Increasing the amount of peptide had predominantly an effect on uptake in PSMA expressing organs compared to tumors which seemed to be tissue-dependent14. Soeda et al. also demonstrated a higher sensitivity of the salivary glands to increasing amounts of peptide compared to tumor tissue using [18F]PSMA-1007, potentially minimizing xerostomia during PSMA radioligand therapy (RLT)15. In this two-part preclinical study, we aim to validate PSMA as a target for expression-based imaging by determining the relationship between differences in PSMA expression levels and tumor uptake. Furthermore, changes in the molar activity/amount of peptide of [18F]AlF-PSMA-11 on uptake parameters and tumor-to-organ ratios will be evaluated.

Materials and methods

Synthesis of [18F]AlF-PSMA-11

[18F]AlF-PSMA-11 was synthesized on a modified SynthraFCHOL synthesis module (Synthra GmbH, Hamburg, Germany) as previously reported with some minor modifications16. The radiochemical purity was 95.9–97.1% as determined by thin layer chromatography (Alugram RP18-W/UV254 plates (Machery Nagel, Düren, Germany)) using 3:1 v/v acetonitrile in water as mobile phase (Supplementary Fig. 1). Molar activities between 76 and 538 MBq/nmol were achieved as analysed by high performance liquid chromatography (Prevail C18 reversed-phase column, 4.6 × 250 mm, 5 µm, Lokeren, Belgium) and a calibrated dose calibrator (Biodex medical systems, USA) (Supplementary Fig. 2). To achieve differences in apparent molar activity (MAapp) levels, varying amounts of a 0.1 µg/µL PSMA-11 (ABX) solution were added to a stock vial to obtain solutions of 1.92 ± 0.27 MBq/nmol (low MAapp), 20.06 ± 3.22 MBq/nmol (medium MAapp) and 211.2 ± 38.9 MBq/nmol (high MAapp).

Preparation of tumor models

The study was approved by the Ghent University Ethical Committee on animal experiments (ECD 18/116). All animals were kept and handled according to the European guidelines (Directive 2010/63/EU). PCa cell lines C4-2 (ATCC; CRL-3314), 22Rv1 (ATCC; CRL-2505) and PC-3 (ATCC; CRL-1435) were cultured using RPMI 1640 medium supplemented with 10% FBS, 1% streptomycin/penicillin (10,000 U/mL) and 1% glutamine 200 mM, and maintained at 37 °C in 5% CO2 in humidified air. For inoculation, PCa cells were rinsed with FBS-free RPMI 1640 medium and cell suspensions of 5 × 106 cells/100 µL were prepared. Four-to-six-week-old male NOD/SCID mice (Janvier, France) were subcutaneously injected at shoulder height with 200 µL 1:1 cell suspension:Matrigel on either side of each mouse (C4-2, n = 8; 22Rv1, n = 6; PC-3, n = 5). Tumor growth was monitored weekly for 5–6 weeks until tumors reached a diameter between 5 and 10 mm.

Small animal PET/CT imaging

To determine the impact of varying molar activities, each C4-2 xenograft bearing mouse underwent three PET/CT scans within a timeframe of 14 days. On day 1, 7 and 14, mice received either high/low/medium MAapp (n = 2) or medium/low/high MAapp (n = 4), respectively. The mean administered [18F]AlF-PSMA-11 activity was 9.29 ± 0.37 MBq with a mean MAapp of either 196.8 ± 32.4 MBq/nmol (high MAapp), 19.10 ± 1.69 MBq/nmol (medium MAapp) or 1.94 ± 0.274 MBq/nmol (low MAapp). To evaluate the activity uptake in PCa tumors with different PSMA expression levels, the images of the medium MAapp of C4-2 were compared to PC-3 and 22Rv1. The mean administered activity was 9.30 ± 0.62 MBq [18F]AlF-PSMA-11 with a mean medium MAapp of 20.06 ± 3.22 MBq/µg. To evaluate whether the influence of the MAapp on low expression tumors, 22Rv1 xenograft bearing mice underwent one additional PET/CT scan on day 7. Each mouse was administered 9.47 ± 0.24 MBq [18F]AlF-PSMA-11 with a high MAapp of 243.8 ± 32.8 MBq/nmol.

The images were acquired in list mode using a small animal PET scanner (β-cube, Molecubes, Ghent, Belgium) with a spatial resolution of 0.85 mm and an axial field-of-view of 13 cm. All PET scans were reconstructed into a 192 × 192 × 384 matrix by an ordered subsets maximization expectation (OSEM) algorithm using 30 iterations and a voxel size of 400 × 400 × 400 µm3.

Immunohistochemical evaluation

After the last scan, two mice/cell line xenograft were sacrificed and tumors were collected for immunohistochemical (IHC) evaluation. Sections were taken from the centre of the tumor sample and stained using Haematoxylin/Eosin, incubated with a primary PSMA antibody (1:400, 2 h, ab133579, Abcam) and counterstained using Haematoxylin (Mayer). Sections were digitally scanned with a virtual scanning microscope (Olympus BX51, Olympus Belgium SA/NV, Berchem, Belgium) at high resolution (20 × magnification).

Western blot analysis

The tumors of the other mice were harvested for western blot (WB) analysis to quantify the PSMA expression levels of the tumor tissues. After thorough rinsing of the tumors, tissue lysates were prepared using RIPA sample buffer. Protein concentrations were determined using the Pierce BCA Protein assay (ThermoFisher Scientific). Samples containing 50 µg of total protein were loaded on 8.5% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) gels. After the transfer, the nitrocellulose membranes were blocked using Odyssey Blocking Buffer (Li-cor Biosciences, Bad Homburg, Germany). Membranes were incubated overnight using a monoclonal PSMA Rabbit anti-Human primary antibody (1:2000, MA533086, Invitrogen, Fischer Scientific, Belgium) and polyclonal Rabbit anti-Human beta actin primary antibody (1:1000, PA1-183, Invitrogen, Fischer Scientific, Belgium), followed by 1 h incubation with IRDye® 800CW Goat anti-Rabbit IgG secondary antibody (1:15,000, Li-cor Biosciences, Bad Homburg, Germany) (Supplementary Fig. 3). The relative densities of both PSMA and β-actin bands were determined using ImageJ17.

Data analysis

Co-registration and analysis of the PET/CT images was performed using PMOD (PMOD Technologies®, Zürich, Switzerland). Images were visually analysed using the PROMISE criteria. This standardizes image interpretation based on activity uptake in reference tissues (blood, liver and salivary gland) and subdivides suspicious lesions into no, low, intermediate or high PSMA expression18. Volumes of interest (VOIs) were drawn manually for delineating the tumor, kidneys, bladder, salivary and lacrimal glands, heart (blood), liver, spleen, muscle and bone. Tissue uptake in each VOI was corrected for residual activity in the syringe and radioactive decay and expressed as SUVmean and SUVmax. Furthermore, tumor-to-organ ratios of tissues with a high likelihood for metastases including tumor-to-liver (TLR), tumor-to-muscle (TMR) and tumor-to-bone ratio (TBoR) were determined.

Statistical analysis

All uptake parameters (SUVmean, SUVmax, TLR, TMR and TBoR) were reported as mean ± SD. The statistical analysis was performed in R19 using the Mann–Whitney U test for comparison of uptake between different PSMA expressing xenografts and the Kruskal Wallis test for comparing radioligand uptake of varying MAapp, followed by post-hoc pairwise comparison using the Wilcoxon-signed rank test with holm correction for multiple testing. The significance level was set on p ≤ 0.05.

Ethics approval and consent to participate

The study was approved by the Ghent University Ethical Committee on animal experiments (ECD 18/116). All animals were kept and handled according to the European guidelines (Directive 2010/63/EU). The manuscript complies with the ARRIVE guidelines.

Results

Semi-quantitative analysis of PSMA expression levels in prostate cancer cell lines

The PSMA expression levels were determined by both western blot and immunohistochemical assessment (Fig. 1). WB analysis detected the highest presence of PSMA expression in C4-2 tissue, while only moderate to low expression levels could be observed for 22Rv1. Semi-quantification of blots revealed a β-actin normalized density for PSMA expression of 16.36 ± 3.05 for C4-2 and 0.94 ± 0.21 for 22Rv1 tumors. PC-3 tumors did not show PSMA expression. Similar results were demonstrated by IHC staining, where all C4-2 cells were clearly positively stained for PSMA, while 22Rv1 showed weaker and more heterogeneous staining of the tumor tissue.

Figure 1
figure 1

Analysis of PSMA expression levels. WB of PCa tumor tissues and a positive control, all samples were run on the same blot. The non-relevant bands were cropped from the image, the full-length blot is presented in Supplementary Fig. 1 (a). Semi-quantification of WB, the relative density of PSMA was normalized using β-actin as loading control. Quantification was only performed using bands from the same blot (b). Data for PC-3, 22Rv1 and C4-2 (n = 5) is presented as mean ± SD. IHC of PSMA expression of PC-3 (c), 22Rv1 (d) and C4-2 (e) tumor tissue.

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Relationship between PSMA expression levels and [18F]AlF-PSMA-11 tumor uptake

Comparative images of [18F]AlF-PSMA-11 uptake (medium MAapp) in different PSMA expressing tumors are presented in Fig. 2. Tumor uptake was positively correlated with higher expression levels. According to the PROMISE criteria, PC-3 tumors were PSMA negative (score 0) as tumor uptake was comparable with activity in the blood (heart). 22Rv1 tumors were visually detectable and the tumor activity was higher than the liver and salivary glands, which corresponds to high PSMA expression (score 3). C4-2 tumors showed good image contrast with tumor uptake above activity in the salivary glands (score 3) (Supplementary Table 1).

Figure 2
figure 2

Representative PET images of PC-3 (left), s22Rv1 (middle) and C4-2 (right) xenografts 60 min p.i. of [18F]AlF-PSMA-11 with medium MAapp. Arrows indicate tumors. There was no activity uptake detectable in PC-3 tumors, C4-2 and 22Rv1 tumors were visible, although the latter showed less tumor contrast. Color maps were generated using Horos v4.0.0, https://horosproject.org/.

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Quantitative VOI analysis demonstrated significant differences for SUVmean and SUVmax as well as tumor-to-organ ratios including tumor-to-liver, tumor-to-blood and tumor-to-muscle in function of PSMA expression levels (Table 1). When comparing C4-2 (high PSMA expression) to 22Rv1 (low PSMA expression), SUVmean and SUVmax values were approximately 3.22 and 3.17 times higher, respectively (p < 0.0001). Similar ratios of 2.77, 2.23 and 2.96 were found for tumor-to-liver, tumor-to-blood and tumor-to-muscle, respectively (p < 0.0001). SUV values were significantly lower for PC-3 tumors compared to both C4-2 (p < 0.01) and 22Rv1 tumors (p < 0.001).

Table 1 SUV values and tumor-to-organ ratios (liver, blood and muscle) for different PCa tumor cell lines with varying PSMA expression levels: PC-3 (no PSMA expression), 22Rv1 (low PSMA expression) and C4-2 (high PSMA expression).
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Impact of the molar activity on tumor uptake

To evaluate the impact of the MAapp on tumor uptake, all C4-2 xenograft bearing mice underwent three PET/CT scans with different MAapp levels (high MAapp = 194.8 ± 32.1 MBq/nmol, medium MAapp = 18.91 ± 1.67 MBq/nmol and low MAapp = 1.92 ± 0.27 MBq/nmol). Images show high uptake in C4-2 tumors after injection of a high MAapp activity dose, which decreased with lower MAapp (Fig. 3). Kidney uptake decreased as well with lower MAapp while activity in de bladder increased. PSMA expressing tissues such as salivary glands, lacrimal glands and spleen were clearly visible on high MAapp PET images but were barely visible on medium MAapp images and not detectable on low MAapp images (Supplementary Table 2).

Figure 3
figure 3

Representative PET images after [18F]AlF-PSMA-11 injection of C4-2 (top) and 22Rv1 (bottom) xenografts of low (only C4-2), medium and high MAapp images in the same animal. Arrows indicate tumors. Increasing MAapp resulted in higher activity uptake in tumors as well as the kidneys, while activity in the bladder decreased. Color maps were generated using Horos v4.0.0, https://horosproject.org/.

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When comparing intra-individual tumor uptake of the high, medium and low MAapp, SUVmean (2.14 ± 0.54, 1.48 ± 0.47 and 0.58 ± 0.10, respectively) and SUVmax (4.44 ± 1.25, 3.27 ± 1.07 and 1.15 ± 0.19, respectively) showed a significantly decreasing trend (both p < 0.001). Post-hoc pairwise comparison revealed statistically significant changes between the high and low MAapp (p < 0.05) as well as between the medium and low MAapp (p < 0.05), but not between the high and medium MAapp (p = 0.055 and 0.25, respectively) (Fig. 4a). Additionally, 22Rv1 xenograft bearing mice (n = 3) received an additional PET/CT scan with high MAapp (246.3 ± 33.1 MBq/µg). Comparison of SUVmean and SUVmax values demonstrated a statistically significant difference between the high and medium MAapp (both p = 0.031).

Figure 4
figure 4

SUVmean (left) and SUVmax (right) (a) and tumor-to-liver, tumor-to-muscle, tumor-to-bone and tumor-to-salivary gland ratio (b) of 22Rv1 and C4-2 tumors after imaging with either the low MAapp (0.94 ± 0.27 MBq/µg, only C4-2), medium MAapp (20.26 ± 3.25 MBq/µg) or high MAapp (213.3 ± 39.3 MBq/µg) [18F]AlF-PSMA-11 60 min p.i.. The mean values were added as dots to the boxplot. The significance level was set at p = 0.05. Ns = not significant, *p < 0.05.

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Tumor-to-organ ratios were determined for tissues with a high likelihood for metastases including liver (TLR), muscle (lymph nodes) (TMR) and bone (TBoR). In C4-2 xenografts, tumor-to-organ ratios remained constant between the high and medium MAapp for TLR (15.71 ± 1.99 vs 17.32 ± 3.55, p = 0.46), TMR (20.69 ± 3.82 vs 19.64 ± 5.37, p = 0.74) and TBoR (3.77 ± 0.78 vs 3.33 ± 1.03, p = 0.31) while a statistically significant difference could be observed for high versus low MAapp and medium versus low MAapp (Fig. 4b). A similar trend between the high and medium MAapp was found for 22Rv1 xenografts, however, the tumor-to-liver ratio remained constant. On the other hand, SUVmean values of PSMA expressing tissues such as salivary and lacrimal glands, spleen, and kidneys significantly decreased between the high and medium MAapp (p < 0.01) (Fig. 5). Tumor-to-salivary gland ratios increased from 2.30 ± 0.37 for the high MAapp to 5.35 ± 0.59 for the medium MAapp. While kidney uptake significantly decreased, uptake in the bladder rose for the medium and low MAapp.

Figure 5
figure 5

C4-2 xenograft bearing mice were injected with [18F]AlF-PSMA-11 with either a low MAapp (1.92 ± 0.27 MBq/µg), medium MAapp (18.91 ± 1.67 MBq/µg) or high MAapp (194.8 ± 32.1 MBq/µg). Boxplots represent SUVmean values 60 min p.i. of PSMA expressing tissues including salivary glands, lacrimal glands, spleen and excretory organs (kidneys and bladder). The high MAapp was set as reference and the significance level was set at p = 0.05. Ns = not significant, **p < 0.01.

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Discussion

Although the diagnostic applications of PSMA as a target for imaging have been the subject of many clinical trials, preclinical studies are essential for a comprehensive investigation of several important aspects of PSMA PET imaging. In this paper, [18F]AlF-PSMA-11 was used for imaging experiments to further understand PSMA PET imaging. First, PSMA was validated as a valuable target for expression-based applications by determining the relationship between PSMA expression levels and [18F]AlF-PSMA-11 tumor uptake. This was examined using three human PCa cell lines with high (C4-2), low (22Rv1) and no detectable (PC-3) PSMA expression. Second, changes in target binding of PSMA positive tumors and tissues using varying amounts of cold PSMA-11 peptide were evaluated.

The relationship between target expression levels and [18F]AlF-PSMA-11 uptake was determined by PET/CT imaging of mice bearing PCa tumors with varying PSMA expression levels. The use of a medium MAapp for the expression level experiments was selected based on several practical considerations. A larger group of mice could be imaged on the same day, while the use of the high MAapp would significantly limit the size of the study population. Also, using the high MAapp would introduce a larger variation in the MAapp between animals, as the amount of PSMA-11 precursor is so small, that minor differences in amount of precursor added to the stock solution would lead to large differences in MAapp. Semi-quantitative western blot analysis showed that PSMA expression levels of C4-2 tumors were approximately 16 times higher compared to 22Rv1 tumors, while SUV values were only 3 times higher. A positive association between PSMA expression and SUV values could be found. Although 22Rv1 tumors had only low PSMA expression levels on western blot, activity uptake was above the liver, scoring positively (score 3) according to the PROMISE criteria. This suggests the ability of [18F]AlF-PSMA-11 to detect PCa lesions, even with low PSMA receptor abundancy. Increasing the MAapp of [18F]AlF-PSMA-11 increased absolute SUVmean and SUVmax values (Fig. 4), but due to higher salivary gland uptake, the PROMISE score did not increase. However, TMR and TBoR did increase significantly, which could be beneficial for the detection of lymph nodes and bone metastases with low PSMA expression levels, although this should be confirmed in a murine model of bone metastases. Lückerath et al. determined the in vivo correlation between [68Ga]PSMA-11 PET/CT and PSMA expression using four murine PCa cell lines. [68Ga]PSMA-11 PET was able to detect changes in PSMA expression at low levels, but this sensitivity was lost at higher PSMA levels, which is in agreement with the reported non-linear correlation20.

PSMA expression was shown to be an important prognosticator for overall survival of metastatic castrate resistant prostate cancer (mCRPC) patients under [177Lu]PSMA treatment. The presence of low average-PSMA expressing tumors was found to be a negative prognostic factor in these patients21. The threshold for determining the PSMA expression level was based on the criterium set by Hofman et al. who defined high PSMA expression as any metastatic lesion with a SUVmax of 1.5 times the liver SUV. Here, [177Lu]PSMA therapy resulted in 50% decline in PSA for 17/30 mCRPC patients presenting with high PSMA expression metastases22. The liver seems to be a solid choice as reference organ as our results show that changes in the MAapp did not significantly alter tumor-to-liver ratios (between the high and medium MAapp). It should however be noted that the difference in chemical structure between [177Lu]PSMA-617 and [18F]AlF-PSMA-11 could also have an influence on biodistribution and clearance pathways. Additional research should determine to which extent PSMA expression levels could be used as an exclusion criterium for [177Lu]PSMA therapy. In our study, the difference between the medium and high MAapp was more pronounced in low expression 22Rv1 tumors. SUV values, TMR and TBoR increased significantly when administering the high MAapp. This can be attributed to the lower amount of receptors available to bind radiolabelled PSMA molecules and more rapid saturation in the presence of a larger peptide mass.

Overall, the highest mean absolute SUVmean and SUVmax values were obtained after administration of [18F]AlF-PSMA-11 with high MAapp, but the difference between the medium and high MAapp was not statistically significant, both for SUV values as for tumor-to-organ ratios (TLR, TMR and TBoR). There was however a substantial decrease of activity uptake in PSMA expressing tissues (salivary and lacrimal glands, spleen and kidneys) between the high and medium MAapp. The use of medium MAapp [18F]AlF-PSMA-11 could therefore reduce activity uptake in PSMA positive healthy tissues while maintaining image contrast of tumor metastases compared to the surrounding tissue. This would be beneficial for both diagnostic purposes and radioligand therapy. Prostate cancer lesions could be more reliably detected due to the constant tumor-to-organ ratio while less non-specific uptake could facilitate scan interpretation by limiting the potential pitfalls of PSMA PET caused by aspecific physiological uptake23. In radioligand therapy, healthy tissue would be less exposed to the radiation while absolute tumor uptake would remain approximately constant.

During PSMA radioligand therapy, radiation exposure caused by activity uptake in PSMA-expressing non-tumor tissues can cause dose-limiting toxic side effects. Radioligand uptake in the salivary glands causes xerostomia and can be a reason for treatment discontinuation24,25. Lowering the MAapp for RLT ligands could be a useful tool for reducing radiation exposure and should be further investigated as an alternative for botulinum toxin injections26 or sialendoscopy and dilatation combination with saline irrigation and steroid injections27. A preclinical study by Fendler et al. also showed that administration of [177Lu]PSMA-617 with a molar activity of 62 MBq/nmol achieved the highest tumor-to-salivary gland ratio compared to lower molar activities (31 and 15 MBq/nmol)28. When correcting for the administered dose of 60 MBq, the highest molar activity corresponds to 0.97 nmol PSMA-617, which is in the same range as our medium MAapp group (0.49 nmol PSMA-11) that also showed the highest tumor-to-salivary gland ratio. However, it was reported that the low to moderate PSMA staining on IHC staining does not correlate with the high PSMA radioligand accumulation. It was hypothesized that tracer accumulation in the salivary glands is therefore a combination of both specific and non-specific uptake29. The latter mechanism is not yet elucidated and our results show a large impact of a higher amount of peptide on activity uptake in salivary glands, suggesting that this as a promising direction for further research. The difference in PSMA expression between murine and human salivary glands must also be taken into consideration. As the binding affinity and PSMA concentration in murine salivary glands is lower compared to human salivary glands, it would be expected that the influence of molar activities will be more pronounced in humans30. Another organ-at-risk are the kidneys. Renal clearance can lead to accumulation of radiolabelled peptides in the tubular cells where it irradiates the kidneys, potentially leading to renal dysfunction, particularly in patients with a known history of nephropathy. Although no major nephrotoxicity was reported for [177Lu]PSMA31, chronic kidney disease was reported in two patients for [225Ac]PSMA therapy. Limiting kidney exposure to [255Ac]PSMA may decrease toxicity and maintain its therapeutic potential32. A study by Kratochwil et al. investigated the influence of additional 2-PMPA administration on tumor and kidney uptake using [125I]MIP-1095. Injection of low doses 2-PMPA (0.2–1 mg/kg) 16 h after tracer administration significantly increased the tumor-to-kidney ratio33. These results indicate potential benefits of the use of cold precursor or PSMA inhibitory small molecules such as 2-PMPA for reducing radiation exposure to the kidneys. Additionally, a high tumor load in patients can also lead to reduced activity uptake in dose-limiting organs34. [18F]AlF-PSMA-11 might therefore be applied as a diagnostic tool for individual optimization of RLT protocols considering PET-based tumor load and radioligand MAapp.

These results show the importance of the amount of carrier on tumor targeting strategies. The optimal amount should be low to avoid competitive binding of unlabelled molecules at the target site but not too low as the tracer molecules could be trapped in non-specific binding places. For diagnostic purposed, using a high MAapp will increase the detection of low expression tumors while also increasing specific uptake in PSMA expressing tissues, possibly leading to false positive findings. For radioligand therapy, further research should focus on finding a balance between administration of a high MAapp for high absolute uptake in the tumor tissue while minimizing activity uptake in healthy organs in order to reduce dose-limiting toxicity. Also, although the considerations mentioned before are valid for high PSMA expressing PCa lesions, decreasing the MAapp to limit non-specific uptake could also affect the detectability of low PSMA expressing lesions, which could lead to an underestimation of the tumor burden.

A major limitation of this study is the translation from mouse to human. Although the differences in the administered MAapp in mice gave well defined results, the activity dose and amount of peptide that is administered compared to total body weight for PET/CT is different for mice and humans, which makes translation into clinical applications more challenging. However, as the molar activity of a PSMA PET tracer decreases over time, it would be beneficial to evaluate the effect on both tumor uptake and non-target uptake in humans. In a retrospective analysis of patients who underwent [18F]rhPSMA-7.3 PET/CT, only minor effects on biodistribution were found for a tenfold decrease of MAapp, except for the salivary glands and spleen, which showed significantly lower uptake for the lower molar activity35. Overall, these results encourage a prospective clinical trial using [18F]AlF-PSMA-11 comparing a high to medium MAapp.

Due to the intra-individual comparison of MAapp levels, most mice were imaged multiple times and tumor sizes differed slightly between scans. However, the tumor size was many times higher than the PET resolution and mice were randomized for MAapp sequence, therefore minimizing this limitation as much as possible.

Conclusion

This paper evaluated [18F]AlF-PSMA-11 PET for expression-based imaging of prostate cancer and the effect of the amount of carrier administered on tumor uptake and tumor-to-organ ratios. A positive association was found between PSMA expression and tumor uptake. The highest absolute tumor uptake was obtained by the high MAapp. And although activity uptake in PSMA expressing organs decreased significantly with lower MAapp, there was no statistically significant difference in tumor SUV between the high and medium MAapp. These results suggest that administration of a high MAapp increases the detection of low expression tumors while also increasing specific uptake in PSMA expressing tissues, possibly leading to false positive findings. In radioligand therapy, a medium MAapp could reduce radiation exposure to dose-limiting organs with only limited effect on radionuclide accumulation in the tumor. Overall, it is of utmost importance to validate the association between both PSMA expression and molar activity relative to radioligand activity uptake.