Novel Characterization in Radiopharmaceutical Preclinical Design: Comparison
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In this entry, the potential of a digital autoradiography system equipped with a super resolution screen has been evaluated to investigate the biodistribution of a 18F-PSMA inhibitor in a prostate cancer mouse model. Twelve double xenograft NOD/SCID mice (LNCAP and PC3 tumours) were divided into three groups according to post-injection time points of an 18F-PSMA inhibitor. Groups of 4 mice were used to evaluate the biodistribution of the radiopharmaceutical after 30-, 60- and 120-min post-injection. Data here reported demonstrated that the digital autoradiography system is suitable to analyse the biodistribution of an 18F-PSMA inhibitor in both whole small-animal bodies and in single organs. The exposure of both whole mouse bodies and organs on the super resolution screen surface allowed the radioactivity of the PSMA inhibitor distributed in the tissues to be detected and quantified. Data obtained by using a digital autoradiography system were in line with the values detected by the activity calibrator. In addition, the image obtained from the super resolution screen allowed a perfect overlap with the tumour images achieved under the optical microscope. In conclusion, biodistribution studies performed by the autoradiography system allow the microscopical modifications induced by therapeutic radiopharmaceuticals to be studied by comparing the molecular imaging and histopathological data at the sub-cellular level. 

  • nuclear imaging
  • pre-clinical model
  • radiopharmaceutical
  • digital autoradiography system

1. Introduction

In the era of 4P medicine (predictive, preventative, personalized, participatory), the development of new therapies and/or diagnostic procedures requires continuous and constant enhancement of the technological armamentarium available for researchers. In this context, multidisciplinary approaches offer the chance to identify and develop new molecules for personalized target therapies [1,2,3][1][2][3].
Molecular imaging investigations, both in pre-clinical models and clinical trials, in collaboration with other biomedical disciplines, such as histology, pathology and molecular biology, currently represent a scientific multidisciplinary platform for developing appropriate pre-clinical models more and more similar to the complex mechanisms of human diseases [4,5,6][4][5][6]. Molecular imaging procedures can be used to assess patients’ state of health earlier and to carefully choose the best clinical individual design for each single patient by translational applications to reach a novel methodological approach (multimodal, theragnostic, pre-targeting) that aims at a deep understanding of pathologies [7].
Macroscopic visualization of cellular mechanisms by dedicated positron emission tomography (PET) in pre-clinical research certainly represents a very appreciated upcoming imaging technology, even if its high cost limits its access to the research community. Thus, numerous promising molecules showing interesting in vitro data about their affinity to their ligands are not furtherly investigated for lack of funds or dedicated micro-molecular imaging devices [8].
Therefore, the development of appropriate and pivotal methodologies capable of supporting researchers in radiopharmaceutical pre-clinical studies and the possibility to combine imaging diagnostic data with histopathology and/or molecular biological analysis could provide a crucial incentive for developing biomedical research involved in the realization of tailored target therapies.
In this scenario, we adopted a digital autoradiography system comprised of a laser scanning device (Cyclone Plus PerkinElmer, Inc., Waltham, MA, USA) commonly used for radiopharmaceutical thin layer chromatography (TLC) quality control in nuclear pharmacy practice to characterize quantitative imaging of spatial radioactivity distribution on animal tissue sections to develop the best procedure for a new radiopharmaceutical scale-up while assuring the best safety route in a pre-clinical development package [9]. The use of this autoradiography system with a super resolution (SR) storage phosphor screen allows the distribution of the radioactivity to be examined as well as the amount of detected radioactivity in terms of Digital Light Units (DLU) to be quantified very quickly on both the whole animal (mouse, rat) and excised organs [9].
Moreover, the ability to closely associate biodistribution data with histopathological images in animal models could enable the characterization of investigated molecular and sub-molecular events crucial for the implementation of personalized medicine, especially in malignant neoplasms such as prostate cancer (PC).
In this research, a mouse model of PC was used to investigate the biodistribution of a new molecular compound labelled with Fluorine-18 (18F) capable of selectively binding the PSMA (prostate-specific membrane antigen) expressed by PC cells. 18F-PSMA inhibitor was studied within xenograft tumours and mouse organs by using a digital autoradiography system, an activity calibrator (Talete, Comecer, Castel Bolognese, Italy) and histopathological investigation to analyse aspects of pharmacodynamics, pharmacokinetics and toxicology at the sub-cellular level.

2. Analysis on Results

2.1. Cell Cultures Characterization

Immunofluorescence analysis was performed to characterize the cell cultures before the xenograft development. Specifically, after confluence, each cell line was tested for the expression of PSMA and Ki67 (proliferation mark). As one aspect, the LNCAP cell line was characterized by more than 90% of PSMA-positive cells. Conversely, no/rare PSMA positive cells were observed in PC3 cells. Similar expression of Ki67 was observed in both LNCAP and PC3 cell lines.

2.2. Measurement Evaluation by Activity Calibrator

A significant increase in radioactivity was detected in LNCAP tumours (PSMA positive) (0.637 ± 0.11 MBq) as compared to both PC3 tumours (PSMA negative) (0.234 ± 0.08 MBq) (Figure 21A) and all other examined organs after 30 min (Figure 21B).
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Figure 21. Measurement by activity calibrator. (A) Graph shows the radioactivity value detected by the activity calibrator in terms of MBq in LNCAP and PC3 tumours after 30, 60 and 90 min. (B) Graph displays the radioactivity value detected by the activity calibrator in terms of MBq in LNCAP, PC3, bowel, heart, liver and kidney tumours after 30, 60 and 90 min.
High values of radioactivity, though significantly lower with respect to LNCAP tumours, were observed in kidneys 0.351 ± 0.15 MBq). Of note, a constant increase of the uptake of the radiopharmaceutical was observed in tumours expressing the biological target (LNCAP) (60 min 0.703 ± 0.57 MBq; 120 min 0.772 ± 0.98 MBq). Otherwise, a constant decrease of the radioactivity was observed in the remaining organs after both 60 and 120 min (see Table 1). This condition is in line with the decay of the radioisotope (18F). All measurements were corrected for radioactive decay.
Table 1. Measurement by activity calibrator.
Measurement by activity calibrator.
 30 Min60 Min120 Min
 mean (MBq)SDmean (MBq)SDmean (MBq)mean (DLU)SD
SDmean (DLU)SDmean (DLU)SDLNCAP0.6370.110.7030.570.772
LNCAP0.98
32,866104039,780304764,0339623PC30.2340.080.1850.120.1120.10
PC33036164149.393.7510.32Bowel0.2770.120.1970.090.1110.16
Bowel1655472.115214529.07Heart0.2370.130.1790.090.1180.13
Heart245577.715132400.330.04Liver0.2670.080.1860.120.1700.11
Liver228558.6275.768.090.320.04Kidney0.3510.150.3000.110.1100.12

2.3. Radioactivity Detection by Digital Autoradiography System

The analysis performed by the autoradiography system allowed the biodistribution study of 18F-PSMA inhibitor (Figure 32A–D) to be performed. In particular, after 30 min, 18F-PSMA inhibitor was widespread throughout the animal (Figure 32D), although the analysis of individual organs showed a significant increase in the uptake of the radiopharmaceutical in LNCAP tumours (Figure 32D).
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Figure 32. Evaluation of radioactivity detection by a digital autoradiography system and histological analysis. (A) Graph shows the radioactivity value detected by a digital autoradiography system in terms of DLU in LNCAP and PC3 tumours after 30, 60 and 90 min. (B) Graph displays the radioactivity value detected by a digital autoradiography system in terms of DLU in LNCAP, PC3, bowel, heart, liver and kidney tumours after 30, 60 and 90 min. (C) Whole body and excised organs. (D) Autoradiographic image shows 18F-PSMA inhibitor uptake in both the whole body and excised organs. (E) Morphological and immunohistochemical images of LNCAP tumours reveal an association among 18F-PSMA inhibitor uptake, mitosis and ki67 expression.
It is noteworthy that, 60 min after the injection, the evaluation of radioactivity in the mouse displayed uptake for LNCAP tumours and bladder (Table 2). At 120 min, the autoradiographic analysis was able to show the uptake of the radiolabelled PSMA inhibitor only in the LNCAP tumours (Table 2). DLU data showed the same trend of radioactivity value detected by the activity calibrator (Table 2 and Figure 32A,B). The proportional relationship between DLU and MBq was assayed in our previous in vitro experiments. In a complex biological contest like this, DLU data, however, demonstrated results in line with the activity calibrator measurement.
Table 2. Radioactivity detection by digital autoradiography system.
Radioactivity detection by digital autoradiography system.
 30 Min60 Min120 Min
 
Kidney
9856721108040.90.230.04
Both investigations showed a progressive increase of the radioactivity in LNCAP tumours as well as a constant reduction of the radioactivity in all other investigated organs, including PC3 tumours.

2.4. Histological and Immunohistochemical Analysis

The histological investigations showed no significant morphological alterations both in tumours and other organs. Comparative analysis between histological and autoradiographic images of LNCAP-positive tumours displayed a strictly spatio-temporal association between the uptake of 18F-PSMA inhibitor and the presence of mitotic figures (Figure 32E). Specifically, areas with higher radiopharmaceutical uptake were characterized by the presence of several mitoses (Figure 32E). Similarly, a spatio-temporal association was observed comparing the autoradiographic images with the expression of Ki67 (Figure 32E). Immunohistochemical evaluation of PSMA confirmed the expression of this molecule only in LNCAP xenografts (Figure 43A,B). High numbers of Ki67 positive cells were observed in both PC3 and LNCAP xenografts (Figure 43C,D). Additionally, the number of vimentin positive cells was ≥50% in each xenograft tissue, thus demonstrating a similar level of tumour differentiation (Figure 43E,F). Indeed, vimentin filaments are expressed by undifferentiated prostate cancer cells.
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Figure 43. Immunohistochemical investigation of xenograft tumours. (A) LNCAP xenograft tumour mass characterized by numerous PSMA-positive cells. (B) No/rare PSMA-positive cells in PC3 tumour mass. (C,D) Images show numerous Ki67 positive cells in both LNCAP (C) and PC3 (D) xenografts. (E) Moderate expression of vimentin in a LNCAP xenograft tumour. (F) Image displays numerous vimentin-positive cells in a PC3 xenograft. Scale bar 100 µm for all images.
These preliminary data support the idea that the high-resolution filmless autoradiography phosphor imager can be useful to perform comparative studies in which biodistribution of a radiopharmaceutical is associated with histological images at the sub-cellular level.

2.5. Electron Microscopy

Transmission electron microscopy analysis of xenograft tumours (both LNCAP and PC3) displayed heterogeneous epithelial cancer populations (Figure 54). Specifically, both well-differentiated and mesenchymal-like cells were observed (Figure 54A–D). However, PC3 tumour mass (Figure 54C,D) was characterized by a higher number of mesenchymal-like cells with respect to LNCAP (Figure 54A,B). In addition, mitotic figures were often detected. No/rare apoptotic cells were found. After biodistribution studies, a moderate increase in apoptotic cells were noted in LNCAP xenograft tumours with respect to PC3.
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Figure 54. Electron microscopy investigation of xenograft tumours. (A,B) LNCAP xenograft tumour mass shows a heterogenic cell population characterized by well-differentiated prostate cancer cells and some mesenchymal-like cells (asterisks). (C,D) Images show a PC3 xenograft tumour characterized by numerous mesenchymal-like cells (asterisks). Scale bars (A) 5 µm, (B) 10 µm, (C) 5 µm, (D) 5 µm.

3. Current Insights

Small-animal imaging has become a fundamental technique for the development of new diagnostic or therapeutical radiopharmaceuticals. Indeed, currently, pre-clinical imaging of animal models represents an invaluable tool in studying the etiopathogenesis of and therapeutic responses in various human pathologies such as neurological, cardiovascular and oncological diseases [14][10]. Molecular imaging techniques can be used to assess biological processes at the cellular and molecular levels, enabling the detection of disease in very early or pre-symptomatic stages, and to estimate the efficacy of novel therapies in individual patients [15,16,17,18][11][12][13][14]. The assessment of biological properties of tumours, such as metabolism, proliferation, hypoxia, angiogenesis, apoptosis, and gene and receptor expression, contributes to the realization of precision medicine [19[15][16],20], owing to the possibility of monitoring physio-pathological processes in vivo, detecting therapeutic responses, identifying non-responders at an early stage, and enabling the switch to novel therapeutic approaches [21,22][17][18]. In this context, PC represents a unique model for the realization of new protocols of personalized medicine. Indeed, PC is a very heterogeneous disease, and contemporary management is focused on identification and treatment of the prognostically adverse high-risk tumours while minimizing overtreatment of indolent, low-risk ones [23][19]. In recent years, imaging has gained increasing importance in the detection, staging, posttreatment assessment and detection of recurrence of PC [24,25,26][20][21][22]. Several imaging modalities, including conventional and functional methods, are used in different clinical scenarios with their very own advantages and limitations. Thus, several groups are involved in the development of new radiopharmaceuticals for both the diagnosis and therapy of PC. To these aims, some laboratories now have a combination of different small-animal imaging systems, which are being used by biologists, pharmacists, physicians and physicists.
Unfortunately, the number of laboratories equipped with innovative small-animal imaging systems are currently very few, due to the high costs of these scientific devices. This fact often precludes the development of several promising radiopharmaceuticals. Thus, the enhancement of an instrumental armamentarium available for researchers could significantly increase the chance of success of pre-clinical investigations based on the identification of new radiolabelled molecules.
For several years, the in situ detection of radiolabeled molecules has been performed by using film or film emulsion (conventional autoradiographic analysis). Despite the fact that the spatial resolution obtained with these devices is very good, the sensitivity of film for low activity levels is poor, due to the low x-ray/β particle detection efficiency. According to this, film autoradiographs frequently must take several days to produce a satisfactory image. In addition, the limited dynamic range of film can cause under- or over-exposure of parts of the image. Therefore, better autoradiography systems based on digital position-sensitive detectors have been developed. Among these, the most sensitive are phosphor imaging plates [27][23], multiwire proportional chambers [28][24], scintillating optical fibres [29][25], microchannel plates [30][26], silicon strip detectors [31][27], and silicon or gallium arsenide pixel detectors [32][28]. Moreover, in the last years, extremely sensitive digital autoradiographs have been developed both for quality control and in vivo research.
Starting from these considerations, in this study, the potential of a digital autoradiography system equipped with an SR screen has been evaluated to characterize 18F-PSMA inhibitor biodistribution in a PC mouse model within xenograft tumours and mouse organs. In addition, a multidisciplinary investigation including histopathological analysis was performed to study radiopharmaceutical behavior at the sub-cellular level.
A digital autoradiography system is a very versatile and sensitive device for radioisotope imaging, replacing film autoradiography [33][29]. This system has been designed for a great variety of applications, such as the analysis of purity for radiopharmaceuticals, nucleotide metabolism studies, in vitro imaging of tissue sections and also gene and protein expression studies [33][29]. In fact, it can image and quantify activity distribution of different radionuclides (photon-, β- and α-particles emitting).
An SR phosphor screen is a flexible support film formulated with the finest grade of barium fluorobromide and containing traces of bivalent europium (BaFBr/Eu2+) phosphor crystals, which acts as a bioluminescence center to provide the best resolution. When the screen is exposed to a radioactive sample, the energy of the radioisotope ionizes the Eu+ 3 to Eu2+, liberating electrons which are trapped in the bromine vacancies [34][30]. Subsequently, the exposed SR screen, wrapped around the carousel of the photometer reading device, is scanned by a focused red light laser beam (633 nm); the laser-stimulated luminescence releases blue light photons (390 nm) which are detected by a photo-multiplier tube (PMT) and converted to electrical signals expressed as DLU. The SR screen was scanned in a few minutes to create a high-resolution digitized image of the locations and intensity of the radioactivity in the sample, which is quantified by OptiQuantTM image analysis software and stored for future reference.
The data here reported showed that the digital autoradiography system is suitable to analyse the biodistribution of an 18F-PSMA inhibitor in both whole small-animal bodies (mice) and in single organs. Specifically, the exposure of both whole mouse bodies and organs on the SR screen surface allowed the radioactivity of the PSMA inhibitor distributed in the tissues to be detected and quantified. It is noteworthy that data obtained by using the digital autoradiography system were in line with the value of measurement detected by the activity calibrator, thus highlighting the high sensitivity of the digital autoradiography system. As expected, a significant and constant increase in the uptake of PSMA inhibitor was observed only in PSMA-positive tumours (LNCAP), while a decrease in the value of radioactivity was noted in other investigated organs as well as in the PSMA negative-tumours (PC3). These data were supported by the immunophenotypical characterization performed on both prostate cancer cell cultures and xenograft tumours. Indeed, no/rare PSMA-positive prostate cancer cells were observed.
Even though the distribution of radioactivity evaluated on whole mouse bodies by the digital autoradiography system cannot have the same sensitivity of micro-PET investigation, it allows an excellent space-time assessment of the biodistribution of a radiopharmaceutical. In particular, in this study, it was possible to follow the biodistribution of a PSMA inhibitor at three different time points, observing a progressive increase of radioactivity in the PSMA-positive tumour area.

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