{"paper_id":"862506b7-fbb0-4882-8076-1b3e6dae05d1","body_text":"Autoradiography of intracerebral tumours in the chick embryo model: A feasibility study using different PET tracers | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Autoradiography of intracerebral tumours in the chick embryo model: A feasibility study using different PET tracers Sandra Krause, Alexandru Florea, Chang-Hoon Choi, Wieland A. Worthoff, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5144367/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Jan, 2025 Read the published version in Molecular Imaging and Biology → Version 1 posted 5 You are reading this latest preprint version Abstract Purpose: In addition to rodent models, the chick embryo model has gained attention for radiotracer evaluation. Previous studies investigated tumours on the chorioallantoic membrane (CAM), but its value for radiotracer imaging of intracerebral tumours has not yet been demonstrated. Procedures: Human U87 glioblastoma cells and U87-IDH1 mutant glioma cells were implanted into the brains of chick embryos at developmental day 5. After 12-14 days of tumour growth, blood-brain-barrier integrity was evaluated using in vivo MRI contrast enhancement or ex vivo with Evans blue dye. The tracers O-(2-[ 18 F]fluoroethyl)-L-tyrosine ([ 18 F]FET) (n=5), 3,4-dihydroxy-6-[ 18 F]-fluoro-L-phenylalanine ([ 18 F]FDOPA) (n=3), or [ 68 Ga] labelled quinoline-based small molecule fibroblast activation protein inhibitor ([ 68 Ga]FAPI-46) (n=4) were injected intravenously if solid tumours were detectable in MRI. For time-activity curves for [ 18 F]FET, additional micro PET (µPET) was performed. The chick embryos were sacrificed 60 minutes post-injection, and cryosections of the tumour-bearing brains were produced and evaluated with autoradiography and immunohistochemistry. Results: Intracerebral tumours were produced with a 100 % success rate in viable chick embryos at experimental endpoint, however, 52% of chick embryos (n=85) did not survive the procedure on a long term. For the evaluated radiotracers, the tumour-to-brain ratios (TBR) derived from ex vivo autoradiography, as well as the tracer kinetics derived from µPET for intracerebral chick embryo tumours, were comparable to those previously reported in rodents and patients: TBRmean for [ 18 F]FET was 1.69 ± 0.54 (n=5), and 3.8 for one hypermetabolic tumour and < 2.0 for two isometabolic tumors using [ 18 F]FDOPA, with TBRmean of 1.92 ± 1,11 (n=3). The TBRmean of [ 68 Ga]FAPI-46 for intracerebral chick embryo tumours was 19.13 ± 0.64 (n=4). In one of U87-MG tumours (n=5), an intact blood-tumour barrier was observed. Conclusions: Radiotracer imaging of intracerebral tumours in the chick embryo offers a fast model for the evaluation of radiotracer uptake, accumulation, and kinetics. Our results indicate a high comparability of chick embryo intracerebral tumour imaging to xenograft rodent models or brain tumour patients. preclinical alternative chick embryo radiotracer glioma xenograft Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Rodent models have been widely used for in vivo oncology research because they mimic complex metabolic pathways and organ-specific challenges, such as the blood-brain barrier (BBB) ( 1 , 2 ). In recent years, the chick embryo has been demonstrated to be a comparable alternative for xenograft glioma invasion ( 3 – 5 ). Compared to rodent models, the utilisation of chick embryos is ethically preferential due to the phylogenetically lower stage of development. Legally, avian embryos are not considered in the directive on the protection of animals used for scientific purposes in Europe ( 6 ). In Germany, with the latest hints of nociception in the later developmental stages ( 7 – 9 ), the animal protection law was revised, however, killing of chick embryos is only prohibited by law during or after a gender reveal starting from embryonic development day (EDD) 13 ( 10 ). Thus, no animal application has to be filed for chick embryo experiments without gender acquisition. Furthermore, chick embryo research is economically preferable with lower costs and limited animal husbandry requirements. Fertilised chick eggs are easy to obtain and manipulate, enabling developmental synchronicity for the precise control of experiments. The chick chorioallantoic membrane (CAM), a highly vascularised, extra-embryonic membrane, is a widely-accepted research tool for tumour engraftments ( 11 – 15 ), allowing in vivo imaging, such as MRI and PET, for real-time monitoring of tumour growth and treatment effects ( 16 – 21 ). While the CAM tumour model provides a convenient and rapid way to study tumour growth, the investigation of the interactions between tumours and the surrounding brain tissue in vivo requires orthotopic brain tumour implantation models. One approach is to implant cells into the neural tube of chick embryos in early development, allowing the growth and interaction of implanted cells in a more sophisticated in vivo model ( 22 ). Another method uses pressure injection of brain tumour cells into the midbrain at EDD 6, which also leads to the growth of intraventricular tumours ( 3 ). However, both methods suffer from high failure rates or technically challenging injection procedures, and further optimisation of intracerebral tumour models in the chick embryo is required for widespread acceptance as an alternative in vivo model ( 16 ). In this study, we explored a modified orthotopic chick embryo brain tumour implantation model by injecting tumour cells with a micro syringe into the mesencephalon. Our approach is based on established methods for rodent studies (using a binocular microscope inside the stereotactical frame holder) and does not require additional technical equipment. As proof of feasibility, tumoral tracer uptake and distribution, as well as tracer kinetics of three clinically important radiotracers, were assessed in the chick embryo model and compared to results from mammalian models and brain tumour patients. Firstly, we applied the amino acid radiotracer O-(2-[ 18 F]fluoroethyl)-L-tyrosine ([ 18 F]FET), which has proven to be a powerful tracer in brain cancer diagnostics, providing additional information on tumour metabolism, tumour extent, treatment effects and response assessment ( 23 – 26 ). The extensive clinical and preclinical knowledge of [ 18 F]FET uptake in brain tumours makes it an ideal candidate to evaluate the use of the chick embryo model in brain tumour imaging ( 27 , 28 , 24 , 29 – 34 ). As a second radiotracer, which is increasingly becoming attractive in the diagnostics and theranostics of several cancer types, [ 68 Ga]-labelled quinoline-based small molecule fibroblast activation protein inhibitor ([ 68 Ga]FAPI-46) was used to assess FAP expression. This protein is over-expressed in a number of neoplasms, particularly in epithelial cancers ( 35 , 36 ) and gliomas ( 37 , 38 ). Even though this tracer does not pass the intact BBB, it was tested in intracerebral tumours with disrupted blood-tumour barriers, as FAP is knowingly involved in tissue remodelling in embryogenesis and reactive stromal cells ( 39 ). Thus, it is of special interest for the assessment of possible limitations to evaluate the utility in a model that does not resemble a mature system. Lastly, another amino acid analogue with high relevance for the identification of metabolically active brain tumour tissues is 3,4-dihydroxy-6-[ 18 F]-fluoro-L-phenylalanine ([ 18 F]FDOPA). This radiotracer shows low uptake in healthy brain tissue like [ 18 F]FET but additional uptake in the basal ganglia as a precursor of dopamine synthesis ( 40 , 41 ). Thus, the aim of this study was to establish a more convenient intracerebral tumour model in the chick embryo model and to provide first examples of the use of this preclinical model for radiotracer evaluation using autoradiography, PET, CT, and MRI. Materials and Methods Treatment of fertilised eggs Specific pathogen-free fertilised eggs for medical use (Valo BioMedia GmbH, Germany) were utilised for this study. Following arrival, the eggs were allowed to rest for at least 24 hours at 14–18°C. The eggs were then warmed at room temperature (22 ± 1°C) for 12 hours prior to incubation. Incubation was started on EDD 1 at 37.8°C and a humidity of 54% (Favorit-Olymp 192, Heka, Germany). On EDD 5, a window was cut in the eggshell to access the embryo as described elsewhere (supplemental material) and sealed with adhesive tape. The eggs were incubated on their sides and not turned so that the albumen would not be spilt until the final experiments on EDD 18–20. Cell culture Glioma with and without mutation of isocitrate dehydrogenase (IDH) were used due to another on-going study. U87 MG (ATCC® HTB-14™) and U87 IDH1 R132H (ATCC® HTB-14IG™) cells were cultured in Minimum Essential Medium Eagle containing 10% foetal calf serum, 1% L-glutamine, 1% MEM non-essential amino acids, and 1% penicillin/streptomycin, at 37°C and 5% CO 2 . The cells were resolved in the medium to a concentration of 5000/40 µl and cultured in a hanging drops culture under the lid of a petri dish for four days, resulting in cell pellets of approx. 50,000 cells, suitable for one injection. Just prior to implantation, one cell pellet was drawn under a binocular microscope into a neuro-syringe (Neuros™ 700/1700 Series, Hamilton, USA), connected to a stereotactical frame, with a volume ≤ 0.5 µl. Brain tumour implantation On EDD 5, the windowed egg was placed under the binocular microscope inside the stereotactical frame holding the neuro-syringe (Neuros™ 700/1700 Series, Hamilton, USA) to inject the tumour cells. With a thin, sharp glass capillary, the chorion was punctured and sliced next to the head of the embryo to gain access. Next, the spoon-end of a microspatula was positioned under the head to hold it in place while rupturing the amnion next to the head. For this, the tip of the glass capillary was used to press the amnion to the spatula and simultaneously pull it upwards the spatula until the amnion ruptured and the mesencephalon exposed. With the head still lying on the spatula, the mesencephalon was punctured with the glass capillary. Depending on what was more comfortable, the head of the embryo either rested on the spatula or was released into its bedding before the neuro-syringe was lowered into the hole made into the mesencephalon (Fig. 1 A). For this step, it is crucial that the amnion does not cover the hole, otherwise, the tip of the syringe will be blocked and cells cannot be inserted into the mesencephalon. After injecting the cells into the brain, the syringe was pulled out, extra-embryotic membranes (amnion and chorion) were placed back, and the window of the egg was covered with fresh tape. The membranes healed within the next 24–48 hours, and a solid tumour grew inside the ventricles, infiltrating healthy brain tissue (Fig. 1 B,C). Anaesthesia and tracer injection For all imaging modalities, the egg was placed in a plexiglass cylinder, which was designed and constructed in-house to hold the anaesthetic. 15 MBq ± 5 MBq of O-(2-[ 18 F]fluoroethyl)-L-tyrosine ([ 18 F]-FET) (n = 3), 3,4-dihydroxy-6-[ 18 F]-fluoro-L-phenylalanine ([ 18 F]-FDOPA) (n = 3), or [ 68 Ga]-labelled quinoline-based small molecule fibroblast activation protein inhibitor ([ 68 Ga]-FAPI-46) (n = 4) was injected intravenously in 100–150 µL in 20 seconds, including 20 µL Evans Blue dye (2% in 0.9% NaCl solution) for addressing BBB disruption as described elsewhere ( 24 ), and the chick embryos were sacrificed 60 minutes post-injection. Imaging was performed under constant 2% isoflurane anaesthesia in oxygen, initialized with 5 % ioflurane in oxygen for 5–10 min, to mitigate embryo movement. For the MR scans, 120 µL isoflurane was pipetted onto tissue paper placed inside the plexiglass cylinder holding the MRI radiofrequency coil and the egg to reach approximately 2 % ioflurane in air; the cylinder was then sealed immediately. To maintain anesthesia at 2 % ioflurane during the PET and CT scans, the plexiglass cylinder was connected to a small animal isoflurane vaporiser. Whenever possible, the temperature was maintained with an infrared lamp. To facilitate good access, the eggs were opened as wide as the spread of the CAM would allow before the commencement of the injections. All substances were injected intravenously into a middle-sized vein of the CAM, which are the light red vessels with blood flow from smaller to bigger vessels. Substances were either injected via a catheter (tip of a 30 G cannula connected to a PE tube) placed in the CAM vessel or directly using a 1 ml syringe with a 32 G or 33 G cannula. The rate of successful intravenous injection was increased to > 95% through the utilization of the following tools: more accurate injection was facilitated by employing a stereomicroscope (5–8 magnification) and a syringe. Prior to retracting the cannula, a few drops of policresulen (360 mg/g) were administered to the injection site to prevent heavy bleeding if a bigger cannula and vessel were used for injection. However, using 32 G or 33 G cannulas for veins not bigger than the cannula itself negates the need to use policresulen, as pinching the vessel for a few seconds stops potential bleeding. Images of the procedure are shown in Figure S1 . MR imaging MRI was used to verify brain tumour growth before tracer experiments and to evaluate BBB integrity after the injection of a paramagnetic contrast agent. Embryos with adequate-sized tumours in T1 and T2w images were chosen for PET and autoradiography the next day. All MR experiments were conducted at a 7 T clinical whole-body scanner (Siemens Healthineers, Erlangen, Germany) on EDD 18–19. In order to maximise the MR sensitivity and coverage, we designed, constructed, and used a low-pass birdcage radio-frequency coil and interface specifically for egg measurements. Further details are given in supplemental material and Figure S1 . PET/CT imaging and autoradiography Dynamic PET scans were performed with an INVEON scanner (Siemens, Erlangen, Germany) ( 42 ) or a Triumph II CT scanner (Northridge Tri-Modality Imaging, USA) for 65 min ([ 18 F]FET). If the chick embryos were injected under a stereomicroscope, emission scans started 3 min after the injection, followed by 10 min of transmission scan (INVEON) or a low dose CT (Triumph II) for attenuation correction. If the chick embryos were injected via a venous catheter (INVEON only), transmission scans were performed first, followed by emission scans with simultaneous tracer injection. Details on PET reconstruction are given in supplemental material. After the scan, the chick embryos were checked for vital signs ( i.e. , visual assessment of pulse in CAM arteries under the stereomicroscope) before further processing of the tissue and data. For autoradiography, organs were taken out, frozen in isopentane at -50°C, and cryo-cut in 20 µm slices. Every tenth slice and freshly prepared 20 µm 18 F or 68 Ga standards with known activity for a calibration curve were exposed to an imaging plate (Fuji Imaging Plate, Raytest) overnight, scanned (Fuji BAS Reader 5000, Raytest), and evaluated for tracer uptake with a pixel size of 25 µm (AIDA Version 4.50, Raytest). Immunohistochemistry The brains were removed from the chick embryos, frozen in isopentane at -50°C, and cryo-cut in 20 µm coronal slices. Slices were fixed with paraformaldehyde and subsequently stained for glial fibrillary acid protein ( i.e. , anti-GFAP, DAKO Z0334), Ki-67 (ab16667, Abcam), human nuclei (MAB1281, Merck Millipore) and nuclei ( i.e. , DAPI) using the standard protocols for fixed cryo-slices. Quantification of proliferative cells was performed on overview images of Ki-67 stained brain slices using ImageJ (National Institute of Health, Bethesda, MD, United States). Evans blue dye extravasation was acquired by fluorescence and pictured using an Aida Image Analyzer (AIDA Version 4.50; Raytest-Fuji). Statistics All statistical calculations were performed using GraphPad Prism 10.0.2 (GraphPad Software, Inc., La Jolla, CA, United States). Descriptive statistics are provided as mean and standard deviation (SD). A paired t-test and Pearson correlation (r) were chosen for methodology comparison. A p-value of less than 0.05 was considered to indicate significant statistical differences in all tests. Results Intracerebral tumours were produced with a success rate of 100% in viable chick embryos at experimental endpoint, however, 52% of embryos did not survive the procedure on EDD5 on a long term (n = 85 in general). Without intervention, the mortality rate was 13.3%. Sample sizes of each experiment are shown in Table 1 . Tumour cells were reliably found within one or both optic tecti growing from the ventricle into the brain tissue infiltratively (Fig. 1 C). The BBB integrity was tested either in ovo with MRI after injection of paramagnetic contrast agent (Fig. 2 ) or ex ovo using Evans blue extravasation (Fig. 3 ). In cases with BBB disruption, Evans blue dye extravasation could be observed in the tumour region, whereas Evans blue dye uptake was not seen in tumours with an intact blood-tumour barrier. An intact blood-tumour-barrier was observed in one of the U87-MG intracerebral tumours (n = 5) tested, which was enrolled in the [ 18 F]FET study. For our tested U87-IDH1 R132H tumours (n = 7), no intact blood-tumour barrier was found. Table 1 Sample sizes of each experiment, as well as related mortality rates (where applicable). Developmental failure refers to the loss of chick embryo viability during embryonic development. Windowing on EDD5 serves as baseline without intervention. Subexperiment Sample size n [-] Developmental failure n [-] Mortality rate [%] Tumour cell implantation in the chick embryo brain 85 44 51.76 Windowing on EDD5, based on survival until EDD10 226 30 13.3 [ 18 F]FET autoradiography 5 [ 18 F]FDOPA autoradiography 3 [ 68 Ga]FAPI autoradiography 4 [ 18 F]FET dynamic PET 5 The mean tumour-to-brain ratio (TBRmean) of [ 18 F]FET uptake in intracerebral U87 MG tumours in chick embryos was 1.69 ± 0.54, as determined by ex vivo autoradiography which showed high correlation efficiency to PET derived values (Figure S2 ). A representative overview of the histological DAPI staining with densely packed tumour cells, BBB disruption within the tumour via Evans blue dye extravasation, [ 18 F]FET accumulation within the tumour, as well as astrocyte activation in the vicinity of the tumour via immunostaining using anti-GFAP antibody, can be seen in Fig. 4 . The TBRs derived from ex vivo autoradiography were comparable to those previously reported in rodents and patients (Table 2 ). A detailed comparison of derived values for [ 18 F]FET compared to rodent xenografts and brain tumour patients is given in the supplementary Table S1 . Table 2 Tumour-to-Brain Ratios (TBRmean) of the evaluated tracers in different species, in mean with standard deviation. Chick Embryo Rodents Human [ 18 F]FET 1.69 ± 0.54 2.15 ± 0.37 ( 29 ) 2.02 ± 0.54 ( 28 ) [ 18 F]FDOPA 1.92 ± 1.11 2.41 to up to 3.36 ( 60 ) 1.76 ± 0.60 ( 61 ) [ 68 Ga]FAPI-46 19.13 ± 0.64 19.95 ± 13.22 ( 38 ) Dynamic [ 18 F]FET PET imaging using small animal PET allowed the assessment of the time-activity curves (TAC) of tracer accumulation in different organs ( i.e. , heart, liver, kidney, and brain). (Figure S3 ). The plateau of the TAC of [ 18 F]FET was reached at about 20 minutes after intravenous injection for all investigated organs. [ 18 F]FET showed the highest tracer uptake in the heart (SUV heart = 3.70 ± 0.35), followed by the liver (SUV liver = 3.51 ± 0.41 and kidneys (SUV kidney = 3.26 ± 0.43), indicating [ 18 F]FET clearance through hepatic/pancreatic and urinary pathway. The SUV within the brain was moderate, with an average of 1.09 ± 0.09. A representative image is shown in Figure S4 . An interesting example of ex vivo autoradiography using [ 18 F]FDOPA in a chick embryo bearing two U87-IDH1 R132H tumours in the right and left ventricle of each tectum is shown in Fig. 4 . The right-sided tumour showed high tracer uptake (TBR, 3.8), while the left showed isometabolism (TBR, 1.1). A chick-genetic origin of one of the tumours was excluded by immunostaining using a human nuclei marker (Fig. 5 C, red). Both tumours showed high proliferative indices (Fig. 5 C, green), with significant differences in proliferation density in the infiltration zone: 37% proliferative cells for the isometabolic tumour but 62% Ki-67 positive cells in the FDOPA positive tumour. Detailed images visualise the infiltrative growth pattern of the isometabolic tumour into the brain of the chick embryo (Fig. 5 D), while the hypermetabolic tumour shows a solid border of the tumour and brain (Fig. 5 E). For intracerebral U87-MG (n = 2), one chick brain tumour also showed isometabolism (TBR, 1.1), whereas the tumour of the other subject exhibited higher uptake (TBR, 1.7). An example of the distribution of [ 68 Ga]FAPI-46 in a chick embryo brain in a U87 IDH1 R132H intracerebral tumour at EDD 19 is shown in Fig. 6 . The TBR of [ 68 Ga]FAPI-46 uptake in U87-IDH1 R132H tumours with disrupted blood-tumour barriers (n = 4) was 19.13 ± 0.64. Discussion The presented chick embryo model for intracerebral tumours yielded solid brain tumours inside the ventricles of the optic tectum of the chick embryo, infiltrating healthy brain tissue. The growth and interaction of implanted cells with the surrounding developing brain tissue was similar to established rodent brain tumour models. Autoradiographic experiments with the amino acid tracer [ 18 F]FET showed low uptake in the normal brain and TBR values similar to those reported in the literature for both humans ( 28 ) and rats ( 24 , 43 ). The occurrence of one U87-MG tumour with intact blood-tumour barrier did not influence the tracer uptake due to the ability of [ 18 F]FET to pass an intact BBB. In addition, the time-activity curves (Figure S3 ) determined by the small animal PET yielded similar results compared with rodents and humans, i.e., decreasing [ 18 F]FET uptake after an early peak in most organs, while the brain showed SUVs in the range of 1 in rats, humans, and chick embryos. The whole-body distribution and [ 18 F]FET clearance from the organs of the chick embryo were also comparable to known elimination pathways in rodents and humans. In mice, urinary and pancreatic excretion has been shown ( 44 ), while human dynamic whole-body scans showed urinary excretion with high [ 18 F]FET accumulations in the urinary bladder and kidneys ( 45 ). Also, a noticeable amount of [ 18 F]FET activity was found in the human myocardium ( 45 ). In chick embryos, the highest [ 18 F]FET accumulation was found in the heart, followed by high liver and kidney uptakes shortly after distribution. This might indicate a [ 18 F]FET clearance through both hepatic and kidney / urinary excretion within the chick embryo on EDD 18–19. However, pancreatic excretion cannot be ruled out due to the anatomically close proximity of the liver and pancreas ( 46 ). Experiments with [ 18 F]FDOPA yielded brain tumours with different uptake behaviour in the same cell lines, which is a rather unusual finding (Fig. 5 ). Obviously, brain tumour cells in the chick embryo model may show variable differentiation, which allows further exploration of the mechanisms of tracer uptake. Low uptake of FET or FDOPA also occurs in up to 30% of brain tumour patients ( 47 ), resembling the isometabolic uptake in the chick embryo (Fig. 5 ). The proliferative index of the chick embryo isometabolic tumour was lower than the chick embryo hypermetabolic tumour, which is in line with the finding that patients with isometabolic brain tumours tend to have a more favourable prognosis ( 47 , 48 ). Finally, [ 68 Ga]FAPI-46 uptake within the chick embryo at the later EDDs was similar to the uptake reported in patients ( 38 ). In the regarded embryonic stage, FAPI uptake within the embryonic brain was as low as in the mouse model ( 37 ). Tissue remodelling during embryogenesis and reactive stromal cells showed high uptakes in the jaw and spine (data not shown), but did not increase FAPI uptake in the brain, thus, no limitation of utility arose due to the embryonic origin. Thus, all three radiotracers showed similar results compared with those in rodents and humans, indicating that the chick embryo model is an excellent preclinical model for tracer evaluation in intracerebral tumours. Compared with previously published techniques for brain tumour implantation ( 3 , 4 , 22 , 49 , 50 ), our method is based on established methods for rodent studies, is technically less challenging, and has comparably moderate chick embryo mortality rates. A recent study proposed a pneumatic pico-pump for glioblastoma cell injection in embryos at EDD 6 with viable brain tumours in 25–75 % of the experimens and evaluation of tumour growth up to EDD16 ( 5 ). Our approach yielded a similar rate of viable brain tumours (48 %) and allowed the evluation of intracerebral tumour growth and infiltration up to EDD20, which is advantageous for therapy monitoring studies. The formation of a BBB in chick embryos has been described in several studies ( 51 – 55 ). Impairment of the BBB within the area of the brain tumour could be detected in later EDDs, similar to that observed with established rat models with the same tumour cell line ( 24 ). As demonstrated in Fig. 3 , however, some intracerebral tumours in the chick embryo brains did not show signs of BBB disruption, which is similar to non-enhancing gliomas in humans. Since an intact BBB is rarely found in rodent brain tumour models, this observation indicates the potential of the chick embryo as a model for non-enhancing gliomas. Some limitations that restrict the applicability of the chick embryo model need to be addressed. First, the implantation site in the developing mesencephalon of the chick embryo brain varies from rodent models as the striatum is not developed at the time of implantation. However, the presence of intermediate filament protein GFAP expression in the vicinity of the solid brain tumour indicates reactive astrocytosis in response to tumour growth, which is comparable to that observed in mammals ( 56 – 59 ). Thus, the tumour microenvironment seems to be comparable to that seen in rodent xenografts. Due to the limited spatial resolution of the small animal PET, a clear delineation of tumours was not possible. Therefore, the tumour-bearing chick embryo brain was investigated using ex vivo autoradiography on EDD 18–19. Nevertheless, a significant correlation between mean SUV and TBR values derived using PET and autoradiography could be demonstrated (Figure S2 ). Another limitation is the restricted timeframe for experimental investigations. The fast development of chick embryos (20 days) excludes the investigation of slow-growing tumours or, in the case of drug testing, the use of drugs with longer administration times. Ethical considerations present a significant constraint in chick embryo research, requiring attention to ensure ethically correct treatment of these organisms during their development based on the newest hints of nociception starting from EDD13 ( 7 – 9 ). While the utilisation of chick embryos might be ethically preferable to working with mature animals, an ethical dilemma persists and researchers must apply the 3R principles to minimize potential pain or harm. A balance between pursuing scientific knowledge with an ethical imperative has to be found: As an example, all embryos sacrificed in this study were anaesthetized beforehand as refinement implementation. Research planning involved the aspect of reduction of needed embryo numbers. Based on knowledge from patients as well as observations in rodent xenograft models, the developed medium sized brain tumours do not cause pain. Thus, no pain, suffering or harm during the developmental stages with possible nociception are to be expected. Continuous reassessment and refinement of protocols based on newest findings are imperative to uphold the highest standards of animal welfare. In addition, a notable limitation arises from the inherent differences between avian and mammalian immunobiology, particularly in cases when host receptor-binding is required for specific radiotracers or where antibodies are used for immunohistochemical target control. Nonetheless, if the target binding is restricted to the derived tumour itself, it does not pose a limitation on the evaluation of tracer developments or neuroimaging, as the xenograft human-originated tumour still retains human physiological properties. In the context of host immunological processes, the extrapolation of findings from chick embryo studies to potential applications in mammalian systems might entail challenges, and conclusions must be drawn carefully. Thus, the translational relevance of immunological results obtained in chick embryos may be limited. Conclusions Radiotracer imaging of intracerebral tumours in the chick embryo offers a fast model for the evaluation of radiotracer uptake, accumulation, and kinetics. Our results indicate a high comparability of chick embryo intracerebral tumour imaging to xenograft rodent models or brain tumour patients. The results of multimodal imaging, as well as the tumour growth and micro-environment, indicate an excellent in vivo alternative to the standard rodent model in many preclinical aspects of neuro-oncology and non-invasive medical imaging. Furthermore, the occurrence of isometabolic tumours as well as intact blood-tumour barriers offer new options to study the role of blood-brain-barrier permeability for the uptake of radiotracers. Declarations Author Contributions: SK,CS, KJL, PL designed the study; SK, CS, SF, NB, AF, CC and WW performed the image acquisitions; SK performed the image analysis; SK, CS and KJL interpreted the results; SK wrote the initial manuscript; AH, BN, NJS and FM assisted with study design and provided strategic guidance; all authors contributed to and approved the final manuscript. Acknowledgements: The authors would like to thank Ms. Claire Rick for English proofreading. Funding : The open access publication of this work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – 491111487. A PhD position for Ms. Sandra Krause is funded by the DFG (Project no. 513201378). Conflicts of Interest : N. Burda, Dr. Choi, Prof. Neumaier, S. Fischer, Dr. Florea, Prof. Heinzel and Dr. Worthoff have nothing to disclose. S. Krause and Dr. Stegmayr report grants from Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) during the conduct of the study. Prof. Langen and Prof. Mottaghy report personal fees from Telix Radiopharmaceuticals, outside the submitted work. Dr. Lohmann reports personal fees from Blue Earth Diagnostics, outside the submitted work. Ethical Approval: This study followed all applicable institutional and/or national guidelines for the care and use of animals. Data Availability: All research data and computer codes are available from the corresponding author upon request. References Pardridge WM (2005) The blood-brain barrier: Bottleneck in brain drug development. NeuroRx: The Journal of the American Society for Experimental NeuroTherapeutics; 2. Day C-P, Merlino G, van Dyke T (2015) Preclinical mouse cancer models: a maze of opportunities and challenges. Cell; 163(1):39–53. DOI:10.1016/j.cell.2015.08.068. Cretu A, Fotos JS, Little BW, Galileo DS (2005) Human and rat glioma growth, invasion, and vascularization in a novel chick embryo brain tumor model. Clin Exp Metastasis; 22(3):225–36. 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(2005) Evaluation of F-18-labeled amino acid derivatives and 18FFDG as PET probes in a brain tumor-bearing animal model. Nucl Med Biol; 32(4):367–75. DOI:10.1016/j.nucmedbio.2005.01.005. Maurer GD, Brucker DP, Stoffels G et al. (2020) 18F-FET PET Imaging in Differentiating Glioma Progression from Treatment-Related Changes: A Single-Center Experience. J Nucl Med; 61(4):505–11. DOI:10.2967/jnumed.119.234757. Lohmann P, Herzog H, Rota Kops E et al. (2015) Dual-time-point O-(2-(18)Ffluoroethyl)-L-tyrosine PET for grading of cerebral gliomas. Eur Radiol; 25(10):3017–24. DOI:10.1007/s00330-015-3691-6. Rosenkrans ZT, Massey CF, Bernau K et al. (2022) 68 GaGa-FAPI-46 PET for non-invasive detection of pulmonary fibrosis disease activity. Eur J Nucl Med Mol Imaging; 49(11):3705–16. DOI:10.1007/s00259-022-05814-9. Chandekar KR, Prashanth A, Vinjamuri S, Kumar R (2023) FAPI PET/CT Imaging-An Updated Review. Diagnostics (Basel); 13(12). DOI:10.3390/diagnostics13122018. Röhrich M, Loktev A, Wefers AK et al. (2019) IDH-wildtype glioblastomas and grade III/IV IDH-mutant gliomas show elevated tracer uptake in fibroblast activation protein-specific PET/CT. Eur J Nucl Med Mol Imaging; 46(12):2569–80. DOI:10.1007/s00259-019-04444-y. Yao Y, Tan X, Yin W et al. (2022) Performance of 18 F-FAPI PET/CT in assessing glioblastoma before radiotherapy: a pilot study. BMC Med Imaging; 22(1):226. DOI:10.1186/s12880-022-00952-w. Jacob M, Chang L, Puré E (2012) Fibroblast activation protein in remodeling tissues. Curr Mol Med; 12(10):1220–43. DOI:10.2174/156652412803833607. Roach JR, Plaha P, McGowan DR, Higgins GS (2022) The role of 18Ffluorodopa positron emission tomography in grading of gliomas. J Neurooncol; 160(3):577–89. DOI:10.1007/s11060-022-04177-3. Walker MD, Dinelle K, Kornelsen R et al. (2013) In-vivo measurement of LDOPA uptake, dopamine reserve and turnover in the rat brain using 18FFDOPA PET. J Cereb Blood Flow Metab; 33(1):59–66. DOI:10.1038/jcbfm.2012.120. Bao Q, Newport D, Chen M, Stout DB, Chatziioannou AF (2009) Performance evaluation of the inveon dedicated PET preclinical tomograph based on the NEMA NU-4 standards. J Nucl Med; 50(3):401–8. DOI:10.2967/jnumed.108.056374. Stegmayr CEffect of pharmacological interventions on reproducibility of O-(2-[18F]fluoroethyl)-L-tyrosine (FET) uptake kinetics in rat glioma models. Forschungszentrum Jülich(2016). Heiss P, Mayer S, Herz M, Wester H-J, Schwaiger M, Senekowitsch-Schmidtke R (1999) Investigation of Transport Mechanism and Uptake Kinetics of O-(2-[18F]Fluoroethyl)-L-Tyrosine In Vitro and In Vivo. The Journal of Nuclear Medicine; (Vol. 40 No. 8):1368-1373. Pauleit D, Floeth F, Herzog H et al. (2003) Whole-body distribution and dosimetry of O-(2-18Ffluoroethyl)-L-tyrosine. Eur J Nucl Med Mol Imaging; 30(4):519–24. DOI:10.1007/s00259-003-1118-0. Bellairs R, Osmond MThe atlas of chick development. Third edition. Oxford: Academic Press. Galldiks N, Unterrainer M, Judov N et al. (2019) Photopenic defects on O-(2-18F-fluoroethyl)-L-tyrosine PET: clinical relevance in glioma patients. Neuro Oncol; 21(10):1331–8. DOI:10.1093/neuonc/noz083. Galldiks N, Verger A, Zaragori T et al. (2019) Comment on \"Hypometabolic gliomas on FET-PET-is there an inverted U-curve for survival?\". Neuro Oncol; 21(12):1612–3. DOI:10.1093/neuonc/noz173. Boulland J-L, Leung DSY, Thuen M et al. (2012) Evaluation of intracellular labeling with micron-sized particles of iron oxide (MPIOs) as a general tool for in vitro and in vivo tracking of human stem and progenitor cells. Cell Transplant; 21(8):1743–59. DOI:10.3727/096368911X627598. Cage TA, Louie JD, Liu SR, Alvarez-Buylla A, Gupta N, Hyer J (2012) Distinct patterns of human medulloblastoma dissemination in the developing chick embryo nervous system. Clin Exp Metastasis; 29(4):371–80. DOI:10.1007/s10585-012-9456-6. Wakai S, Hirokawa N (1978) Development of the blood-brain barrier to horseradish peroxidase in the chick embryo. Cell Tissue Res:195–203. Janzer RC, Raff MC (1987) Astrocytes induce blood-brain barrier properties in endothelial cells. Letters to Nature; (325):253–7. Möller W, Kummer W (2003) The blood-brain barrier of the chick glycogen body (corpus gelatinosum) and its functional implications. Cell Tissue Res; 313(1):71–80. DOI:10.1007/s00441-003-0742-0. Parvas M, Parada C, Bueno D (2008) A blood-CSF barrier function controls embryonic CSF protein composition and homeostasis during early CNS development. Dev Biol; 321(1):51–63. DOI:10.1016/j.ydbio.2008.05.552. Parvas M, Bueno D (2010) The embryonic blood-CSF barrier has molecular elements to control E-CSF osmolarity during early CNS development. J Neurosci Res; 88(6):1205–12. DOI:10.1002/jnr.22293. Lee J, Broboa AK, Baird A, Eliceiri BP (2011) Non-invasive quantification of brain tumor-induced astrogliosis. BMC Neuroscience. Piroth MD, Prasath J, Willuweit A et al. (2013) Uptake of O-(2-18Ffluoroethyl)-L-tyrosine in reactive astrocytosis in the vicinity of cerebral gliomas. Nucl Med Biol; 40(6):795–800. DOI:10.1016/j.nucmedbio.2013.05.001. Sofroniew MV, Vinters HV (2010) Astrocytes: biology and pathology. Acta Neuropathol; 119(1):7–35. DOI:10.1007/s00401-009-0619-8. Chekhonin VP, Baklaushev VP, Yusubalieva GM, Pavlov KA, Ukhova OV, Gurina OI (2007) Modeling and immunohistochemical analysis of C6 glioma In Vivo. Cell Technologies in Biology and Medicine; 2. Clément A, Zaragori T, Filosa R et al. (2022) Multi-tracer and multiparametric PET imaging to detect the IDH mutation in glioma: a preclinical translational in vitro, in vivo, and ex vivo study. Cancer Imaging; 22(1):16. DOI:10.1186/s40644-022-00454-6. Cicone F, Filss CP, Minniti G et al. (2015) Volumetric assessment of recurrent or progressive gliomas: comparison between F-DOPA PET and perfusion-weighted MRI. Eur J Nucl Med Mol Imaging; 42(6):905–15. DOI:10.1007/s00259-015-3018-5. Supplementary Files ElectronicSuppKrausefinalVersionCLEAN.docx Cite Share Download PDF Status: Published Journal Publication published 21 Jan, 2025 Read the published version in Molecular Imaging and Biology → Version 1 posted Reviewers agreed at journal 13 Nov, 2024 Reviewers invited by journal 13 Nov, 2024 Editor assigned by journal 07 Nov, 2024 First submitted to journal 07 Nov, 2024 Editorial decision: Minor revisions 24 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-5144367\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":377631158,\"identity\":\"045c5ce7-6df1-4d82-81e5-46ebf239086e\",\"order_by\":0,\"name\":\"Sandra Krause\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABC0lEQVRIie2QP2sCMRyGfyFwXUJnoVi/woUbBT/LBaEuVhwLCuYQzCLcGqf7CnHqIhg5sMu53+gtnW5QXDqVxn+gQ8qNheYhkATy5H0TAIfjj4L4acJ6B+ABPCCOza6K4jF5UnB1hVB5TrsOCw2x0Wi2aLFETPfRFzyyGOPo0Ifms03xs16I1GebqWwz5zXTbjZG4ycJncCqQNdHW42Zqr2+b+m3x1SKJphAyritWFwelRFLkrLgoUlZXpSRTYHcpCht7swJ4tooCp+V0FosL/2V1B+Byl5oxMELZGreQvwOtRfr0mKqB/VEpIX5sXU9FqI4kLdmw5ZyRN+s19f034R7htWPOhwOx7/hBz4OWSlq9Gk7AAAAAElFTkSuQmCC\",\"orcid\":\"https://orcid.org/0009-0009-3694-8489\",\"institution\":\"Institute of Neurosciences and Medicine: Forschungszentrum Julich Institut fur Neurowissenschaften und Medizin\",\"correspondingAuthor\":true,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Sandra\",\"middleName\":\"\",\"lastName\":\"Krause\",\"suffix\":\"\"},{\"id\":377631159,\"identity\":\"13d871b5-b098-4dad-b37c-a9253e132c2c\",\"order_by\":1,\"name\":\"Alexandru Florea\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University Hospital Aachen: Universitatsklinikum Aachen\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Alexandru\",\"middleName\":\"\",\"lastName\":\"Florea\",\"suffix\":\"\"},{\"id\":377631160,\"identity\":\"274eb942-e82d-41a4-bfe0-036c93043477\",\"order_by\":2,\"name\":\"Chang-Hoon Choi\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Institute of Neurosciences and Medicine: Forschungszentrum Julich Institut fur Neurowissenschaften und Medizin\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Chang-Hoon\",\"middleName\":\"\",\"lastName\":\"Choi\",\"suffix\":\"\"},{\"id\":377631161,\"identity\":\"d4f24b99-3152-4442-8918-2f6b42bc2ee8\",\"order_by\":3,\"name\":\"Wieland A. 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On EDD 19, brains were removed and cryosliced \\u003cstrong\\u003e(B)\\u003c/strong\\u003e. Tumors were usually present on one or both optic tecti (grey plane in B), growing from the ventricle infiltratively into the brain tissue in a DAPI staining \\u003cstrong\\u003e(C)\\u003c/strong\\u003e.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5144367/v1/70144f3e63188b17c9b00d95.png\"},{\"id\":69058278,\"identity\":\"6db5c8c0-0a0f-4c7e-ad24-d53bc4b827da\",\"added_by\":\"auto\",\"created_at\":\"2024-11-15 06:53:56\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":341181,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eMR images of an EDD 19 chick embryo. A U87-MG tumour can be seen in the optic tectum (crosshair), presented in T2 \\u003cstrong\\u003e(A)\\u003c/strong\\u003e and T1 with \\u003cstrong\\u003e(B)\\u003c/strong\\u003e and without \\u003cstrong\\u003e(C)\\u003c/strong\\u003e contrast agent. Contrast agent enhancement is most profound in the tumour-rim region.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5144367/v1/3dff86d3424446d9f7e9f0e0.png\"},{\"id\":69058275,\"identity\":\"af4ed3ed-5aed-467b-8ab5-623b76f6514b\",\"added_by\":\"auto\",\"created_at\":\"2024-11-15 06:53:56\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":273445,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eTwo representative EDD 19 chick embryo brains with U87-MG tumours showing a disrupted \\u003cstrong\\u003e(A,B)\\u003c/strong\\u003e and an intact \\u003cstrong\\u003e(C,D)\\u003c/strong\\u003e blood-tumour-barrier. \\u003cstrong\\u003eA,C:\\u003c/strong\\u003e 4’,6-diamidino-2-phenylindole (DAPI) staining shows densely packed tumour cells in the tectum opticum. \\u003cstrong\\u003eB,D\\u003c/strong\\u003e\\u003cem\\u003e:\\u003c/em\\u003e Consecutive slices of A and C, respectively, with \\u003cstrong\\u003e(B)\\u003c/strong\\u003e and without \\u003cstrong\\u003e(D)\\u003c/strong\\u003e Evans blue dye extravasation in the tumour region, thus indicating a disrupted \\u003cstrong\\u003e(B)\\u003c/strong\\u003eand an intact \\u003cstrong\\u003e(D)\\u003c/strong\\u003e blood-tumour-barrier.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5144367/v1/dcc745d48a2ee47283eb036e.png\"},{\"id\":69058253,\"identity\":\"b8f790f5-e8ca-463b-a854-055aa16b9ac5\",\"added_by\":\"auto\",\"created_at\":\"2024-11-15 06:53:55\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":310817,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eConsecutive 20 µm slices of an EDD 19 chick embryo brain with a U87-MG tumour using [\\u003csup\\u003e18\\u003c/sup\\u003eF]FET. 4’,6-diamidino-2-phenylindole (DAPI) staining shows densely packed tumour cells in the ventricle and adjacent brain regions forming a solid tumour \\u003cstrong\\u003e(A)\\u003c/strong\\u003e. The blood-brain barrier is disrupted within the tumour, as shown by Evans blue dye extravasation, especially in the tumour rim region \\u003cstrong\\u003e(B)\\u003c/strong\\u003e. The corresponding [\\u003csup\\u003e18\\u003c/sup\\u003eF]FET autoradiography shows a profound tracer accumulation in the tumour that is independent of the degree of blood-brain-barrier disruption \\u003cstrong\\u003e(C)\\u003c/strong\\u003e. Astrocyte staining using an anti-GFAP antibody (Z0334, Dako) reveals astrocyte activation in the vicinity of the tumour \\u003cstrong\\u003e(D)\\u003c/strong\\u003e.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5144367/v1/d03022481a72e52cff7c4a53.png\"},{\"id\":69058282,\"identity\":\"b3b4178f-ad94-4dc7-ad95-f868f536646d\",\"added_by\":\"auto\",\"created_at\":\"2024-11-15 06:53:57\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":996973,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eConsecutive 20 µm slices of an EDD 19 chick brain with a U87-IDH1 R132H tumour. 4’,6-diamidino-2-phenylindole (DAPI) staining shows densely packed tumour cells in both ventricles and adjacent brain regions that form two solid tumours \\u003cstrong\\u003e(A)\\u003c/strong\\u003e. \\u003cem\\u003eEx vivo \\u003c/em\\u003eautoradiography with [\\u003csup\\u003e18\\u003c/sup\\u003eF]FDOPA shows a profound tracer accumulation in the right tumour, while the left tumour visualises isometabolism \\u003cstrong\\u003e(B)\\u003c/strong\\u003e. Immunostaining with a human nuclei marker (MAB1281, Merck Millipore) demonstrates the human origin of the tumours in red, whereas proliferating cells are visualised in green with the proliferation marker Ki-67 (ab16667, Abcam). All cell nuclei can be seen in blue. Detailed images of the border region of both the isometabolic left tumour \\u003cstrong\\u003e(D)\\u003c/strong\\u003e and hypermetabolic right tumour \\u003cstrong\\u003e(E)\\u003c/strong\\u003e are shown in higher magnification.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5144367/v1/329155d89b6d7c0e1090b3c7.png\"},{\"id\":69058284,\"identity\":\"1b1d61d0-b166-47d8-b72b-cb35b2c91d03\",\"added_by\":\"auto\",\"created_at\":\"2024-11-15 06:53:59\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":174489,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e[\\u003csup\\u003e68\\u003c/sup\\u003eGa]FAPI-46 of a chick embryo on EDD 19. \\u003cstrong\\u003e(A)\\u003c/strong\\u003e 4’,6-diamidino-2-phenylindole (DAPI) image of U87 IDH1\\u003csup\\u003eR132H\\u003c/sup\\u003e intracerebral tumour showing densely packed solid tumour cells in the ventricles \\u003cstrong\\u003e(B)\\u003c/strong\\u003e autoradiography of consecutive slice of A, showing profound tracer accumulation in the tumours.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5144367/v1/5d44101e7a0c113e305d4bc0.png\"},{\"id\":74858674,\"identity\":\"e1b1a0bb-c3e1-43b7-bec3-b42eeade873d\",\"added_by\":\"auto\",\"created_at\":\"2025-01-27 16:12:47\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":3701704,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5144367/v1/cee53d0a-176b-4df0-82a3-ca88ba8d3612.pdf\"},{\"id\":69058287,\"identity\":\"b7114142-e72e-4044-ba75-878114c09e0f\",\"added_by\":\"auto\",\"created_at\":\"2024-11-15 06:53:59\",\"extension\":\"docx\",\"order_by\":14,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1787326,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"ElectronicSuppKrausefinalVersionCLEAN.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5144367/v1/afa082bd7a485de62ac43997.docx\"}],\"financialInterests\":\"\",\"formattedTitle\":\"Autoradiography of intracerebral tumours in the chick embryo model: A feasibility study using different PET tracers\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eRodent models have been widely used for \\u003cem\\u003ein vivo\\u003c/em\\u003e oncology research because they mimic complex metabolic pathways and organ-specific challenges, such as the blood-brain barrier (BBB) (\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e). In recent years, the chick embryo has been demonstrated to be a comparable alternative for xenograft glioma invasion (\\u003cspan additionalcitationids=\\\"CR4\\\" citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e). Compared to rodent models, the utilisation of chick embryos is ethically preferential due to the phylogenetically lower stage of development. Legally, avian embryos are not considered in the directive on the protection of animals used for scientific purposes in Europe (\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e). In Germany, with the latest hints of nociception in the later developmental stages (\\u003cspan additionalcitationids=\\\"CR8\\\" citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e), the animal protection law was revised, however, killing of chick embryos is only prohibited by law during or after a gender reveal starting from embryonic development day (EDD) 13 (\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e). Thus, no animal application has to be filed for chick embryo experiments without gender acquisition. Furthermore, chick embryo research is economically preferable with lower costs and limited animal husbandry requirements. Fertilised chick eggs are easy to obtain and manipulate, enabling developmental synchronicity for the precise control of experiments.\\u003c/p\\u003e \\u003cp\\u003eThe chick chorioallantoic membrane (CAM), a highly vascularised, extra-embryonic membrane, is a widely-accepted research tool for tumour engraftments (\\u003cspan additionalcitationids=\\\"CR12 CR13 CR14\\\" citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e), allowing \\u003cem\\u003ein vivo\\u003c/em\\u003e imaging, such as MRI and PET, for real-time monitoring of tumour growth and treatment effects (\\u003cspan additionalcitationids=\\\"CR17 CR18 CR19 CR20\\\" citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e). While the CAM tumour model provides a convenient and rapid way to study tumour growth, the investigation of the interactions between tumours and the surrounding brain tissue \\u003cem\\u003ein vivo\\u003c/em\\u003e requires orthotopic brain tumour implantation models. One approach is to implant cells into the neural tube of chick embryos in early development, allowing the growth and interaction of implanted cells in a more sophisticated \\u003cem\\u003ein vivo\\u003c/em\\u003e model (\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e). Another method uses pressure injection of brain tumour cells into the midbrain at EDD 6, which also leads to the growth of intraventricular tumours (\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e). However, both methods suffer from high failure rates or technically challenging injection procedures, and further optimisation of intracerebral tumour models in the chick embryo is required for widespread acceptance as an alternative \\u003cem\\u003ein vivo\\u003c/em\\u003e model (\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eIn this study, we explored a modified orthotopic chick embryo brain tumour implantation model by injecting tumour cells with a micro syringe into the mesencephalon. Our approach is based on established methods for rodent studies (using a binocular microscope inside the stereotactical frame holder) and does not require additional technical equipment. As proof of feasibility, tumoral tracer uptake and distribution, as well as tracer kinetics of three clinically important radiotracers, were assessed in the chick embryo model and compared to results from mammalian models and brain tumour patients.\\u003c/p\\u003e \\u003cp\\u003eFirstly, we applied the amino acid radiotracer O-(2-[\\u003csup\\u003e18\\u003c/sup\\u003eF]fluoroethyl)-L-tyrosine ([\\u003csup\\u003e18\\u003c/sup\\u003eF]FET), which has proven to be a powerful tracer in brain cancer diagnostics, providing additional information on tumour metabolism, tumour extent, treatment effects and response assessment (\\u003cspan additionalcitationids=\\\"CR24 CR25\\\" citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e). The extensive clinical and preclinical knowledge of [\\u003csup\\u003e18\\u003c/sup\\u003eF]FET uptake in brain tumours makes it an ideal candidate to evaluate the use of the chick embryo model in brain tumour imaging (\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e, \\u003cspan additionalcitationids=\\\"CR30 CR31 CR32 CR33\\\" citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e). As a second radiotracer, which is increasingly becoming attractive in the diagnostics and theranostics of several cancer types, [\\u003csup\\u003e68\\u003c/sup\\u003eGa]-labelled quinoline-based small molecule fibroblast activation protein inhibitor ([\\u003csup\\u003e68\\u003c/sup\\u003eGa]FAPI-46) was used to assess FAP expression. This protein is over-expressed in a number of neoplasms, particularly in epithelial cancers (\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e) and gliomas (\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e). Even though this tracer does not pass the intact BBB, it was tested in intracerebral tumours with disrupted blood-tumour barriers, as FAP is knowingly involved in tissue remodelling in embryogenesis and reactive stromal cells (\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e). Thus, it is of special interest for the assessment of possible limitations to evaluate the utility in a model that does not resemble a mature system. Lastly, another amino acid analogue with high relevance for the identification of metabolically active brain tumour tissues is 3,4-dihydroxy-6-[\\u003csup\\u003e18\\u003c/sup\\u003eF]-fluoro-L-phenylalanine ([\\u003csup\\u003e18\\u003c/sup\\u003eF]FDOPA). This radiotracer shows low uptake in healthy brain tissue like [\\u003csup\\u003e18\\u003c/sup\\u003eF]FET but additional uptake in the basal ganglia as a precursor of dopamine synthesis (\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eThus, the aim of this study was to establish a more convenient intracerebral tumour model in the chick embryo model and to provide first examples of the use of this preclinical model for radiotracer evaluation using autoradiography, PET, CT, and MRI.\\u003c/p\\u003e\"},{\"header\":\"Materials and Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eTreatment of fertilised eggs\\u003c/h2\\u003e \\u003cp\\u003eSpecific pathogen-free fertilised eggs for medical use (Valo BioMedia GmbH, Germany) were utilised for this study. Following arrival, the eggs were allowed to rest for at least 24 hours at 14\\u0026ndash;18\\u0026deg;C. The eggs were then warmed at room temperature (22\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1\\u0026deg;C) for 12 hours prior to incubation. Incubation was started on EDD 1 at 37.8\\u0026deg;C and a humidity of 54% (Favorit-Olymp 192, Heka, Germany). On EDD 5, a window was cut in the eggshell to access the embryo as described elsewhere (supplemental material) and sealed with adhesive tape. The eggs were incubated on their sides and not turned so that the albumen would not be spilt until the final experiments on EDD 18\\u0026ndash;20.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eCell culture\\u003c/h3\\u003e\\n\\u003cp\\u003eGlioma with and without mutation of isocitrate dehydrogenase (IDH) were used due to another on-going study. U87 MG (ATCC\\u0026reg; HTB-14\\u0026trade;) and U87 IDH1\\u003csup\\u003eR132H\\u003c/sup\\u003e (ATCC\\u0026reg; HTB-14IG\\u0026trade;) cells were cultured in Minimum Essential Medium Eagle containing 10% foetal calf serum, 1% L-glutamine, 1% MEM non-essential amino acids, and 1% penicillin/streptomycin, at 37\\u0026deg;C and 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e.\\u003c/p\\u003e \\u003cp\\u003eThe cells were resolved in the medium to a concentration of 5000/40 \\u0026micro;l and cultured in a hanging drops culture under the lid of a petri dish for four days, resulting in cell pellets of approx. 50,000 cells, suitable for one injection. Just prior to implantation, one cell pellet was drawn under a binocular microscope into a neuro-syringe (Neuros\\u0026trade; 700/1700 Series, Hamilton, USA), connected to a stereotactical frame, with a volume\\u0026thinsp;\\u0026le;\\u0026thinsp;0.5 \\u0026micro;l.\\u003c/p\\u003e\\n\\u003ch3\\u003eBrain tumour implantation\\u003c/h3\\u003e\\n\\u003cp\\u003eOn EDD 5, the windowed egg was placed under the binocular microscope inside the stereotactical frame holding the neuro-syringe (Neuros\\u0026trade; 700/1700 Series, Hamilton, USA) to inject the tumour cells. With a thin, sharp glass capillary, the chorion was punctured and sliced next to the head of the embryo to gain access. Next, the spoon-end of a microspatula was positioned under the head to hold it in place while rupturing the amnion next to the head. For this, the tip of the glass capillary was used to press the amnion to the spatula and simultaneously pull it upwards the spatula until the amnion ruptured and the mesencephalon exposed. With the head still lying on the spatula, the mesencephalon was punctured with the glass capillary. Depending on what was more comfortable, the head of the embryo either rested on the spatula or was released into its bedding before the neuro-syringe was lowered into the hole made into the mesencephalon (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA). For this step, it is crucial that the amnion does not cover the hole, otherwise, the tip of the syringe will be blocked and cells cannot be inserted into the mesencephalon. After injecting the cells into the brain, the syringe was pulled out, extra-embryotic membranes (amnion and chorion) were placed back, and the window of the egg was covered with fresh tape. The membranes healed within the next 24\\u0026ndash;48 hours, and a solid tumour grew inside the ventricles, infiltrating healthy brain tissue (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB,C).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\\n\\u003ch3\\u003eAnaesthesia and tracer injection\\u003c/h3\\u003e\\n\\u003cp\\u003eFor all imaging modalities, the egg was placed in a plexiglass cylinder, which was designed and constructed in-house to hold the anaesthetic. 15 MBq\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;5 MBq of O-(2-[\\u003csup\\u003e18\\u003c/sup\\u003eF]fluoroethyl)-L-tyrosine ([\\u003csup\\u003e18\\u003c/sup\\u003eF]-FET) (n\\u0026thinsp;=\\u0026thinsp;3), 3,4-dihydroxy-6-[\\u003csup\\u003e18\\u003c/sup\\u003eF]-fluoro-L-phenylalanine ([\\u003csup\\u003e18\\u003c/sup\\u003eF]-FDOPA) (n\\u0026thinsp;=\\u0026thinsp;3), or [\\u003csup\\u003e68\\u003c/sup\\u003eGa]-labelled quinoline-based small molecule fibroblast activation protein inhibitor ([\\u003csup\\u003e68\\u003c/sup\\u003eGa]-FAPI-46) (n\\u0026thinsp;=\\u0026thinsp;4) was injected intravenously in 100\\u0026ndash;150 \\u0026micro;L in 20 seconds, including 20 \\u0026micro;L Evans Blue dye (2% in 0.9% NaCl solution) for addressing BBB disruption as described elsewhere (\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e), and the chick embryos were sacrificed 60 minutes post-injection. Imaging was performed under constant 2% isoflurane anaesthesia in oxygen, initialized with 5 % ioflurane in oxygen for 5\\u0026ndash;10 min, to mitigate embryo movement. For the MR scans, 120 \\u0026micro;L isoflurane was pipetted onto tissue paper placed inside the plexiglass cylinder holding the MRI radiofrequency coil and the egg to reach approximately 2 % ioflurane in air; the cylinder was then sealed immediately. To maintain anesthesia at 2 % ioflurane during the PET and CT scans, the plexiglass cylinder was connected to a small animal isoflurane vaporiser. Whenever possible, the temperature was maintained with an infrared lamp.\\u003c/p\\u003e \\u003cp\\u003eTo facilitate good access, the eggs were opened as wide as the spread of the CAM would allow before the commencement of the injections. All substances were injected intravenously into a middle-sized vein of the CAM, which are the light red vessels with blood flow from smaller to bigger vessels. Substances were either injected \\u003cem\\u003evia\\u003c/em\\u003e a catheter (tip of a 30 G cannula connected to a PE tube) placed in the CAM vessel or directly using a 1 ml syringe with a 32 G or 33 G cannula. The rate of successful intravenous injection was increased to \\u0026gt;\\u0026thinsp;95% through the utilization of the following tools: more accurate injection was facilitated by employing a stereomicroscope (5\\u0026ndash;8 magnification) and a syringe. Prior to retracting the cannula, a few drops of policresulen (360 mg/g) were administered to the injection site to prevent heavy bleeding if a bigger cannula and vessel were used for injection. However, using 32 G or 33 G cannulas for veins not bigger than the cannula itself negates the need to use policresulen, as pinching the vessel for a few seconds stops potential bleeding. Images of the procedure are shown in Figure \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e.\\u003c/p\\u003e\\n\\u003ch3\\u003eMR imaging\\u003c/h3\\u003e\\n\\u003cp\\u003eMRI was used to verify brain tumour growth before tracer experiments and to evaluate BBB integrity after the injection of a paramagnetic contrast agent. Embryos with adequate-sized tumours in T1 and T2w images were chosen for PET and autoradiography the next day. All MR experiments were conducted at a 7 T clinical whole-body scanner (Siemens Healthineers, Erlangen, Germany) on EDD 18\\u0026ndash;19. In order to maximise the MR sensitivity and coverage, we designed, constructed, and used a low-pass birdcage radio-frequency coil and interface specifically for egg measurements. Further details are given in supplemental material and Figure \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003ePET/CT imaging and autoradiography\\u003c/h2\\u003e \\u003cp\\u003eDynamic PET scans were performed with an INVEON scanner (Siemens, Erlangen, Germany) (\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e) or a Triumph II CT scanner (Northridge Tri-Modality Imaging, USA) for 65 min ([\\u003csup\\u003e18\\u003c/sup\\u003eF]FET). If the chick embryos were injected under a stereomicroscope, emission scans started 3 min after the injection, followed by 10 min of transmission scan (INVEON) or a low dose CT (Triumph II) for attenuation correction. If the chick embryos were injected via a venous catheter (INVEON only), transmission scans were performed first, followed by emission scans with simultaneous tracer injection. Details on PET reconstruction are given in supplemental material.\\u003c/p\\u003e \\u003cp\\u003eAfter the scan, the chick embryos were checked for vital signs (\\u003cem\\u003ei.e.\\u003c/em\\u003e, visual assessment of pulse in CAM arteries under the stereomicroscope) before further processing of the tissue and data. For autoradiography, organs were taken out, frozen in isopentane at -50\\u0026deg;C, and cryo-cut in 20 \\u0026micro;m slices. Every tenth slice and freshly prepared 20 \\u0026micro;m \\u003csup\\u003e18\\u003c/sup\\u003eF or \\u003csup\\u003e68\\u003c/sup\\u003eGa standards with known activity for a calibration curve were exposed to an imaging plate (Fuji Imaging Plate, Raytest) overnight, scanned (Fuji BAS Reader 5000, Raytest), and evaluated for tracer uptake with a pixel size of 25 \\u0026micro;m (AIDA Version 4.50, Raytest).\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eImmunohistochemistry\\u003c/h3\\u003e\\n\\u003cp\\u003eThe brains were removed from the chick embryos, frozen in isopentane at -50\\u0026deg;C, and cryo-cut in 20 \\u0026micro;m coronal slices. Slices were fixed with paraformaldehyde and subsequently stained for glial fibrillary acid protein (\\u003cem\\u003ei.e.\\u003c/em\\u003e, anti-GFAP, DAKO Z0334), Ki-67 (ab16667, Abcam), human nuclei (MAB1281, Merck Millipore) and nuclei (\\u003cem\\u003ei.e.\\u003c/em\\u003e, DAPI) using the standard protocols for fixed cryo-slices. Quantification of proliferative cells was performed on overview images of Ki-67 stained brain slices using ImageJ (National Institute of Health, Bethesda, MD, United States). Evans blue dye extravasation was acquired by fluorescence and pictured using an Aida Image Analyzer (AIDA Version 4.50; Raytest-Fuji).\\u003c/p\\u003e\\n\\u003ch3\\u003eStatistics\\u003c/h3\\u003e\\n\\u003cp\\u003eAll statistical calculations were performed using GraphPad Prism 10.0.2 (GraphPad Software, Inc., La Jolla, CA, United States). Descriptive statistics are provided as mean and standard deviation (SD). A paired t-test and Pearson correlation (r) were chosen for methodology comparison. A p-value of less than 0.05 was considered to indicate significant statistical differences in all tests.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003eIntracerebral tumours were produced with a success rate of 100% in viable chick embryos at experimental endpoint, however, 52% of embryos did not survive the procedure on EDD5 on a long term (n\\u0026thinsp;=\\u0026thinsp;85 in general). Without intervention, the mortality rate was 13.3%. Sample sizes of each experiment are shown in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e. Tumour cells were reliably found within one or both optic tecti growing from the ventricle into the brain tissue infiltratively (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC). The BBB integrity was tested either \\u003cem\\u003ein ovo\\u003c/em\\u003e with MRI after injection of paramagnetic contrast agent (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e) or \\u003cem\\u003eex ovo\\u003c/em\\u003e using Evans blue extravasation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). In cases with BBB disruption, Evans blue dye extravasation could be observed in the tumour region, whereas Evans blue dye uptake was not seen in tumours with an intact blood-tumour barrier. An intact blood-tumour-barrier was observed in one of the U87-MG intracerebral tumours (n\\u0026thinsp;=\\u0026thinsp;5) tested, which was enrolled in the [\\u003csup\\u003e18\\u003c/sup\\u003eF]FET study. For our tested U87-IDH1\\u003csup\\u003eR132H\\u003c/sup\\u003e tumours (n\\u0026thinsp;=\\u0026thinsp;7), no intact blood-tumour barrier was found.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eSample sizes of each experiment, as well as related mortality rates (where applicable). Developmental failure refers to the loss of chick embryo viability during embryonic development. Windowing on EDD5 serves as baseline without intervention.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"4\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eSubexperiment\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eSample size n [-]\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eDevelopmental failure n [-]\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eMortality rate [%]\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eTumour cell implantation in the chick embryo brain\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e85\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e44\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e51.76\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eWindowing on EDD5, based on survival until EDD10\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e226\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e30\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e13.3\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003e[\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003e18\\u003c/b\\u003e\\u003c/sup\\u003e\\u003cb\\u003eF]FET autoradiography\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e5\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" morerows=\\\"3\\\" nameend=\\\"c4\\\" namest=\\\"c3\\\" rowspan=\\\"4\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003e[\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003e18\\u003c/b\\u003e\\u003c/sup\\u003e\\u003cb\\u003eF]FDOPA autoradiography\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e3\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003e[\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003e68\\u003c/b\\u003e\\u003c/sup\\u003e\\u003cb\\u003eGa]FAPI autoradiography\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e4\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003e[\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003e18\\u003c/b\\u003e\\u003c/sup\\u003e\\u003cb\\u003eF]FET dynamic PET\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e5\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe mean tumour-to-brain ratio (TBRmean) of [\\u003csup\\u003e18\\u003c/sup\\u003eF]FET uptake in intracerebral U87 MG tumours in chick embryos was 1.69\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.54, as determined by \\u003cem\\u003eex vivo\\u003c/em\\u003e autoradiography which showed high correlation efficiency to PET derived values (Figure \\u003cspan refid=\\\"MOESM2\\\" class=\\\"InternalRef\\\"\\u003eS2\\u003c/span\\u003e). A representative overview of the histological DAPI staining with densely packed tumour cells, BBB disruption within the tumour via Evans blue dye extravasation, [\\u003csup\\u003e18\\u003c/sup\\u003eF]FET accumulation within the tumour, as well as astrocyte activation in the vicinity of the tumour via immunostaining using anti-GFAP antibody, can be seen in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e. The TBRs derived from \\u003cem\\u003eex vivo\\u003c/em\\u003e autoradiography were comparable to those previously reported in rodents and patients (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). A detailed comparison of derived values for [\\u003csup\\u003e18\\u003c/sup\\u003eF]FET compared to rodent xenografts and brain tumour patients is given in the supplementary Table \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab2\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eTumour-to-Brain Ratios (TBRmean) of the evaluated tracers in different species, in mean with standard deviation.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"4\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eChick Embryo\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eRodents\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eHuman\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003e[\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003e18\\u003c/b\\u003e\\u003c/sup\\u003e\\u003cb\\u003eF]FET\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1.69\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.54\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e2.15\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.37 (\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e2.02\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.54 (\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e)\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003e[\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003e18\\u003c/b\\u003e\\u003c/sup\\u003e\\u003cb\\u003eF]FDOPA\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1.92\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.11\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e2.41 to up to 3.36 (\\u003cspan citationid=\\\"CR60\\\" class=\\\"CitationRef\\\"\\u003e60\\u003c/span\\u003e)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1.76\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.60 (\\u003cspan citationid=\\\"CR61\\\" class=\\\"CitationRef\\\"\\u003e61\\u003c/span\\u003e)\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003e[\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003e68\\u003c/b\\u003e\\u003c/sup\\u003e\\u003cb\\u003eGa]FAPI-46\\u003c/b\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e19.13\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.64\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e19.95\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;13.22 (\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e)\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003eDynamic [\\u003csup\\u003e18\\u003c/sup\\u003eF]FET PET imaging using small animal PET allowed the assessment of the time-activity curves (TAC) of tracer accumulation in different organs (\\u003cem\\u003ei.e.\\u003c/em\\u003e, heart, liver, kidney, and brain). (Figure \\u003cspan refid=\\\"MOESM3\\\" class=\\\"InternalRef\\\"\\u003eS3\\u003c/span\\u003e). The plateau of the TAC of [\\u003csup\\u003e18\\u003c/sup\\u003eF]FET was reached at about 20 minutes after intravenous injection for all investigated organs. [\\u003csup\\u003e18\\u003c/sup\\u003eF]FET showed the highest tracer uptake in the heart (SUV\\u003csub\\u003eheart\\u003c/sub\\u003e = 3.70\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.35), followed by the liver (SUV\\u003csub\\u003eliver\\u003c/sub\\u003e = 3.51\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.41 and kidneys (SUV\\u003csub\\u003ekidney\\u003c/sub\\u003e = 3.26\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.43), indicating [\\u003csup\\u003e18\\u003c/sup\\u003eF]FET clearance through hepatic/pancreatic and urinary pathway. The SUV within the brain was moderate, with an average of 1.09\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.09. A representative image is shown in Figure \\u003cspan refid=\\\"MOESM4\\\" class=\\\"InternalRef\\\"\\u003eS4\\u003c/span\\u003e.\\u003c/p\\u003e \\u003cp\\u003eAn interesting example of \\u003cem\\u003eex vivo\\u003c/em\\u003e autoradiography using [\\u003csup\\u003e18\\u003c/sup\\u003eF]FDOPA in a chick embryo bearing two U87-IDH1\\u003csup\\u003eR132H\\u003c/sup\\u003e tumours in the right and left ventricle of each tectum is shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e. The right-sided tumour showed high tracer uptake (TBR, 3.8), while the left showed isometabolism (TBR, 1.1). A chick-genetic origin of one of the tumours was excluded by immunostaining using a human nuclei marker (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eC, red). Both tumours showed high proliferative indices (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eC, green), with significant differences in proliferation density in the infiltration zone: 37% proliferative cells for the isometabolic tumour but 62% Ki-67 positive cells in the FDOPA positive tumour. Detailed images visualise the infiltrative growth pattern of the isometabolic tumour into the brain of the chick embryo (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eD), while the hypermetabolic tumour shows a solid border of the tumour and brain (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eE). For intracerebral U87-MG (n\\u0026thinsp;=\\u0026thinsp;2), one chick brain tumour also showed isometabolism (TBR, 1.1), whereas the tumour of the other subject exhibited higher uptake (TBR, 1.7).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eAn example of the distribution of [\\u003csup\\u003e68\\u003c/sup\\u003eGa]FAPI-46 in a chick embryo brain in a U87 IDH1\\u003csup\\u003eR132H\\u003c/sup\\u003e intracerebral tumour at EDD 19 is shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e. The TBR of [\\u003csup\\u003e68\\u003c/sup\\u003eGa]FAPI-46 uptake in U87-IDH1\\u003csup\\u003eR132H\\u003c/sup\\u003e tumours with disrupted blood-tumour barriers (n\\u0026thinsp;=\\u0026thinsp;4) was 19.13\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;0.64.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eThe presented chick embryo model for intracerebral tumours yielded solid brain tumours inside the ventricles of the optic tectum of the chick embryo, infiltrating healthy brain tissue. The growth and interaction of implanted cells with the surrounding developing brain tissue was similar to established rodent brain tumour models.\\u003c/p\\u003e \\u003cp\\u003eAutoradiographic experiments with the amino acid tracer [\\u003csup\\u003e18\\u003c/sup\\u003eF]FET showed low uptake in the normal brain and TBR values similar to those reported in the literature for both humans (\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e) and rats (\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e). The occurrence of one U87-MG tumour with intact blood-tumour barrier did not influence the tracer uptake due to the ability of [\\u003csup\\u003e18\\u003c/sup\\u003eF]FET to pass an intact BBB. In addition, the time-activity curves (Figure \\u003cspan refid=\\\"MOESM3\\\" class=\\\"InternalRef\\\"\\u003eS3\\u003c/span\\u003e) determined by the small animal PET yielded similar results compared with rodents and humans, i.e., decreasing [\\u003csup\\u003e18\\u003c/sup\\u003eF]FET uptake after an early peak in most organs, while the brain showed SUVs in the range of 1 in rats, humans, and chick embryos. The whole-body distribution and [\\u003csup\\u003e18\\u003c/sup\\u003eF]FET clearance from the organs of the chick embryo were also comparable to known elimination pathways in rodents and humans. In mice, urinary and pancreatic excretion has been shown (\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e), while human dynamic whole-body scans showed urinary excretion with high [\\u003csup\\u003e18\\u003c/sup\\u003eF]FET accumulations in the urinary bladder and kidneys (\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e). Also, a noticeable amount of [\\u003csup\\u003e18\\u003c/sup\\u003eF]FET activity was found in the human myocardium (\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e). In chick embryos, the highest [\\u003csup\\u003e18\\u003c/sup\\u003eF]FET accumulation was found in the heart, followed by high liver and kidney uptakes shortly after distribution. This might indicate a [\\u003csup\\u003e18\\u003c/sup\\u003eF]FET clearance through both hepatic and kidney / urinary excretion within the chick embryo on EDD 18\\u0026ndash;19. However, pancreatic excretion cannot be ruled out due to the anatomically close proximity of the liver and pancreas (\\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eExperiments with [\\u003csup\\u003e18\\u003c/sup\\u003eF]FDOPA yielded brain tumours with different uptake behaviour in the same cell lines, which is a rather unusual finding (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e). Obviously, brain tumour cells in the chick embryo model may show variable differentiation, which allows further exploration of the mechanisms of tracer uptake. Low uptake of FET or FDOPA also occurs in up to 30% of brain tumour patients (\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e), resembling the isometabolic uptake in the chick embryo (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e). The proliferative index of the chick embryo isometabolic tumour was lower than the chick embryo hypermetabolic tumour, which is in line with the finding that patients with isometabolic brain tumours tend to have a more favourable prognosis (\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eFinally, [\\u003csup\\u003e68\\u003c/sup\\u003eGa]FAPI-46 uptake within the chick embryo at the later EDDs was similar to the uptake reported in patients (\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e). In the regarded embryonic stage, FAPI uptake within the embryonic brain was as low as in the mouse model (\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e). Tissue remodelling during embryogenesis and reactive stromal cells showed high uptakes in the jaw and spine (data not shown), but did not increase FAPI uptake in the brain, thus, no limitation of utility arose due to the embryonic origin.\\u003c/p\\u003e \\u003cp\\u003eThus, all three radiotracers showed similar results compared with those in rodents and humans, indicating that the chick embryo model is an excellent preclinical model for tracer evaluation in intracerebral tumours.\\u003c/p\\u003e \\u003cp\\u003eCompared with previously published techniques for brain tumour implantation (\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e49\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e), our method is based on established methods for rodent studies, is technically less challenging, and has comparably moderate chick embryo mortality rates. A recent study proposed a pneumatic pico-pump for glioblastoma cell injection in embryos at EDD 6 with viable brain tumours in 25\\u0026ndash;75 % of the experimens and evaluation of tumour growth up to EDD16 (\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e). Our approach yielded a similar rate of viable brain tumours (48 %) and allowed the evluation of intracerebral tumour growth and infiltration up to EDD20, which is advantageous for therapy monitoring studies.\\u003c/p\\u003e \\u003cp\\u003eThe formation of a BBB in chick embryos has been described in several studies (\\u003cspan additionalcitationids=\\\"CR52 CR53 CR54\\\" citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e51\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e55\\u003c/span\\u003e). Impairment of the BBB within the area of the brain tumour could be detected in later EDDs, similar to that observed with established rat models with the same tumour cell line (\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e). As demonstrated in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e, however, some intracerebral tumours in the chick embryo brains did not show signs of BBB disruption, which is similar to non-enhancing gliomas in humans. Since an intact BBB is rarely found in rodent brain tumour models, this observation indicates the potential of the chick embryo as a model for non-enhancing gliomas.\\u003c/p\\u003e \\u003cp\\u003eSome limitations that restrict the applicability of the chick embryo model need to be addressed. First, the implantation site in the developing mesencephalon of the chick embryo brain varies from rodent models as the striatum is not developed at the time of implantation. However, the presence of intermediate filament protein GFAP expression in the vicinity of the solid brain tumour indicates reactive astrocytosis in response to tumour growth, which is comparable to that observed in mammals (\\u003cspan additionalcitationids=\\\"CR57 CR58\\\" citationid=\\\"CR56\\\" class=\\\"CitationRef\\\"\\u003e56\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR59\\\" class=\\\"CitationRef\\\"\\u003e59\\u003c/span\\u003e). Thus, the tumour microenvironment seems to be comparable to that seen in rodent xenografts. Due to the limited spatial resolution of the small animal PET, a clear delineation of tumours was not possible. Therefore, the tumour-bearing chick embryo brain was investigated using \\u003cem\\u003eex vivo\\u003c/em\\u003e autoradiography on EDD 18\\u0026ndash;19. Nevertheless, a significant correlation between mean SUV and TBR values derived using PET and autoradiography could be demonstrated (Figure \\u003cspan refid=\\\"MOESM2\\\" class=\\\"InternalRef\\\"\\u003eS2\\u003c/span\\u003e). Another limitation is the restricted timeframe for experimental investigations. The fast development of chick embryos (20 days) excludes the investigation of slow-growing tumours or, in the case of drug testing, the use of drugs with longer administration times. Ethical considerations present a significant constraint in chick embryo research, requiring attention to ensure ethically correct treatment of these organisms during their development based on the newest hints of nociception starting from EDD13 (\\u003cspan additionalcitationids=\\\"CR8\\\" citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e). While the utilisation of chick embryos might be ethically preferable to working with mature animals, an ethical dilemma persists and researchers must apply the 3R principles to minimize potential pain or harm. A balance between pursuing scientific knowledge with an ethical imperative has to be found: As an example, all embryos sacrificed in this study were anaesthetized beforehand as refinement implementation. Research planning involved the aspect of reduction of needed embryo numbers. Based on knowledge from patients as well as observations in rodent xenograft models, the developed medium sized brain tumours do not cause pain. Thus, no pain, suffering or harm during the developmental stages with possible nociception are to be expected. Continuous reassessment and refinement of protocols based on newest findings are imperative to uphold the highest standards of animal welfare. In addition, a notable limitation arises from the inherent differences between avian and mammalian immunobiology, particularly in cases when host receptor-binding is required for specific radiotracers or where antibodies are used for immunohistochemical target control. Nonetheless, if the target binding is restricted to the derived tumour itself, it does not pose a limitation on the evaluation of tracer developments or neuroimaging, as the xenograft human-originated tumour still retains human physiological properties. In the context of host immunological processes, the extrapolation of findings from chick embryo studies to potential applications in mammalian systems might entail challenges, and conclusions must be drawn carefully. Thus, the translational relevance of immunological results obtained in chick embryos may be limited.\\u003c/p\\u003e\"},{\"header\":\"Conclusions\",\"content\":\"\\u003cp\\u003eRadiotracer imaging of intracerebral tumours in the chick embryo offers a fast model for the evaluation of radiotracer uptake, accumulation, and kinetics. Our results indicate a high comparability of chick embryo intracerebral tumour imaging to xenograft rodent models or brain tumour patients. The results of multimodal imaging, as well as the tumour growth and micro-environment, indicate an excellent \\u003cem\\u003ein vivo\\u003c/em\\u003e alternative to the standard rodent model in many preclinical aspects of neuro-oncology and non-invasive medical imaging. Furthermore, the occurrence of isometabolic tumours as well as intact blood-tumour barriers offer new options to study the role of blood-brain-barrier permeability for the uptake of radiotracers.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAuthor Contributions:\\u0026nbsp;\\u003c/strong\\u003eSK,CS, KJL, PL designed the study; SK, CS, SF, NB, AF, CC and WW performed the image acquisitions; SK performed the image analysis; SK, CS and KJL interpreted the results; SK wrote the initial manuscript; AH, BN, NJS and FM assisted with study design and provided strategic guidance; all authors contributed to and approved the final manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements:\\u003c/strong\\u003e The authors would like to thank Ms. Claire Rick for English proofreading.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e: The open access publication of this work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – 491111487. A PhD position for Ms. Sandra Krause is funded by the DFG (Project no. 513201378).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflicts of Interest\\u003c/strong\\u003e: N. Burda, Dr. Choi, Prof. Neumaier, S. Fischer, Dr. Florea, Prof. Heinzel and Dr. Worthoff have nothing to disclose. S. Krause and Dr. Stegmayr report grants from Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) during the conduct of the study. Prof. Langen and Prof. Mottaghy report personal fees from Telix Radiopharmaceuticals, outside the submitted work. Dr. Lohmann reports personal fees from Blue Earth Diagnostics, outside the submitted work.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthical Approval:\\u003c/strong\\u003e This study followed all applicable institutional and/or national guidelines for the care and use of animals.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData Availability:\\u0026nbsp;\\u003c/strong\\u003e All research data and computer codes are available from the corresponding author upon request.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003ePardridge WM (2005) The blood-brain barrier: Bottleneck in brain drug development. NeuroRx: The Journal of the American Society for Experimental NeuroTherapeutics; 2.\\u003c/li\\u003e\\n\\u003cli\\u003eDay C-P, Merlino G, van Dyke T (2015) Preclinical mouse cancer models: a maze of opportunities and challenges. Cell; 163(1):39\\u0026ndash;53. 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(2023) Modern Photodynamic Glioblastoma Therapy Using Curcumin- or Parietin-Loaded Lipid Nanoparticles in a CAM Model Study. ACS Appl Bio Mater; 6(12):5502\\u0026ndash;14. DOI:10.1021/acsabm.3c00695.\\u003c/li\\u003e\\n\\u003cli\\u003eBoulland J-L, Halasi G, Kasumacic N, Glover JC (2010) Xenotransplantation of human stem cells into the chicken embryo. J Vis Exp; (41). DOI:10.3791/2071.\\u003c/li\\u003e\\n\\u003cli\\u003eGalldiks N, Langen K-J (2015) Applications of PET imaging of neurological tumors with radiolabeled amino acids. Q J NUCL MED MOL IMAGING; 59:70\\u0026ndash;82.\\u003c/li\\u003e\\n\\u003cli\\u003eStegmayr C, Oliveira D, Niemietz N et al. (2017) Influence of Bevacizumab on Blood-Brain Barrier Permeability and O-(2-18F-Fluoroethyl)-l-Tyrosine Uptake in Rat Gliomas. J Nucl Med; 58(5):700\\u0026ndash;5. DOI:10.2967/jnumed.116.187047.\\u003c/li\\u003e\\n\\u003cli\\u003eLangen K-J, Stoffels G, Filss C et al. (2017) Imaging of amino acid transport in brain tumours: Positron emission tomography with O-(2-18Ffluoroethyl)-L-tyrosine (FET). Methods; 130:124\\u0026ndash;34. DOI:10.1016/j.ymeth.2017.05.019.\\u003c/li\\u003e\\n\\u003cli\\u003eSingnurkar A, Poon R, Detsky J (2023) 18F-FET-PET imaging in high-grade gliomas and brain metastases: a systematic review and meta-analysis. J Neurooncol; 161(1):1\\u0026ndash;12. DOI:10.1007/s11060-022-04201-6.\\u003c/li\\u003e\\n\\u003cli\\u003eStegmayr C, Willuweit A, Lohmann P, Langen K-J (2019) O-(2-18F-Fluoroethyl)-L-Tyrosine (FET) in Neurooncology: A Review of Experimental Results. Curr Radiopharm; 12(3):201\\u0026ndash;10. DOI:10.2174/1874471012666190111111046.\\u003c/li\\u003e\\n\\u003cli\\u003eStegmayr C, Stoffels G, Kops ER et al. (2019) Influence of Dexamethasone on O-(2-18F-Fluoroethyl)-L-Tyrosine Uptake in the Human Brain and Quantification of Tumor Uptake. Mol Imaging Biol; 21(1):168\\u0026ndash;74. 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(2005) Evaluation of F-18-labeled amino acid derivatives and 18FFDG as PET probes in a brain tumor-bearing animal model. Nucl Med Biol; 32(4):367\\u0026ndash;75. DOI:10.1016/j.nucmedbio.2005.01.005.\\u003c/li\\u003e\\n\\u003cli\\u003eMaurer GD, Brucker DP, Stoffels G et al. (2020) 18F-FET PET Imaging in Differentiating Glioma Progression from Treatment-Related Changes: A Single-Center Experience. J Nucl Med; 61(4):505\\u0026ndash;11. DOI:10.2967/jnumed.119.234757.\\u003c/li\\u003e\\n\\u003cli\\u003eLohmann P, Herzog H, Rota Kops E et al. (2015) Dual-time-point O-(2-(18)Ffluoroethyl)-L-tyrosine PET for grading of cerebral gliomas. Eur Radiol; 25(10):3017\\u0026ndash;24. DOI:10.1007/s00330-015-3691-6.\\u003c/li\\u003e\\n\\u003cli\\u003eRosenkrans ZT, Massey CF, Bernau K et al. (2022) 68 GaGa-FAPI-46 PET for non-invasive detection of pulmonary fibrosis disease activity. Eur J Nucl Med Mol Imaging; 49(11):3705\\u0026ndash;16. 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Forschungszentrum J\\u0026uuml;lich(2016).\\u003c/li\\u003e\\n\\u003cli\\u003eHeiss P, Mayer S, Herz M, Wester H-J, Schwaiger M, Senekowitsch-Schmidtke R (1999) Investigation of Transport Mechanism and Uptake Kinetics of O-(2-[18F]Fluoroethyl)-L-Tyrosine In Vitro and In Vivo. The Journal of Nuclear Medicine; (Vol. 40 No. 8):1368-1373.\\u003c/li\\u003e\\n\\u003cli\\u003ePauleit D, Floeth F, Herzog H et al. (2003) Whole-body distribution and dosimetry of O-(2-18Ffluoroethyl)-L-tyrosine. Eur J Nucl Med Mol Imaging; 30(4):519\\u0026ndash;24. DOI:10.1007/s00259-003-1118-0.\\u003c/li\\u003e\\n\\u003cli\\u003eBellairs R, Osmond MThe atlas of chick development. Third edition. Oxford: Academic Press.\\u003c/li\\u003e\\n\\u003cli\\u003eGalldiks N, Unterrainer M, Judov N et al. (2019) Photopenic defects on O-(2-18F-fluoroethyl)-L-tyrosine PET: clinical relevance in glioma patients. Neuro Oncol; 21(10):1331\\u0026ndash;8. DOI:10.1093/neuonc/noz083.\\u003c/li\\u003e\\n\\u003cli\\u003eGalldiks N, Verger A, Zaragori T et al. (2019) Comment on \\u0026quot;Hypometabolic gliomas on FET-PET-is there an inverted U-curve for survival?\\u0026quot;. Neuro Oncol; 21(12):1612\\u0026ndash;3. DOI:10.1093/neuonc/noz173.\\u003c/li\\u003e\\n\\u003cli\\u003eBoulland J-L, Leung DSY, Thuen M et al. (2012) Evaluation of intracellular labeling with micron-sized particles of iron oxide (MPIOs) as a general tool for in vitro and in vivo tracking of human stem and progenitor cells. Cell Transplant; 21(8):1743\\u0026ndash;59. DOI:10.3727/096368911X627598.\\u003c/li\\u003e\\n\\u003cli\\u003eCage TA, Louie JD, Liu SR, Alvarez-Buylla A, Gupta N, Hyer J (2012) Distinct patterns of human medulloblastoma dissemination in the developing chick embryo nervous system. Clin Exp Metastasis; 29(4):371\\u0026ndash;80. DOI:10.1007/s10585-012-9456-6.\\u003c/li\\u003e\\n\\u003cli\\u003eWakai S, Hirokawa N (1978) Development of the blood-brain barrier to horseradish peroxidase in the chick embryo. Cell Tissue Res:195\\u0026ndash;203.\\u003c/li\\u003e\\n\\u003cli\\u003eJanzer RC, Raff MC (1987) Astrocytes induce blood-brain barrier properties in endothelial cells. Letters to Nature; (325):253\\u0026ndash;7.\\u003c/li\\u003e\\n\\u003cli\\u003eM\\u0026ouml;ller W, Kummer W (2003) The blood-brain barrier of the chick glycogen body (corpus gelatinosum) and its functional implications. Cell Tissue Res; 313(1):71\\u0026ndash;80. DOI:10.1007/s00441-003-0742-0.\\u003c/li\\u003e\\n\\u003cli\\u003eParvas M, Parada C, Bueno D (2008) A blood-CSF barrier function controls embryonic CSF protein composition and homeostasis during early CNS development. Dev Biol; 321(1):51\\u0026ndash;63. DOI:10.1016/j.ydbio.2008.05.552.\\u003c/li\\u003e\\n\\u003cli\\u003eParvas M, Bueno D (2010) The embryonic blood-CSF barrier has molecular elements to control E-CSF osmolarity during early CNS development. J Neurosci Res; 88(6):1205\\u0026ndash;12. DOI:10.1002/jnr.22293.\\u003c/li\\u003e\\n\\u003cli\\u003eLee J, Broboa AK, Baird A, Eliceiri BP (2011) Non-invasive quantification of brain tumor-induced astrogliosis. BMC Neuroscience.\\u003c/li\\u003e\\n\\u003cli\\u003ePiroth MD, Prasath J, Willuweit A et al. (2013) Uptake of O-(2-18Ffluoroethyl)-L-tyrosine in reactive astrocytosis in the vicinity of cerebral gliomas. Nucl Med Biol; 40(6):795\\u0026ndash;800. DOI:10.1016/j.nucmedbio.2013.05.001.\\u003c/li\\u003e\\n\\u003cli\\u003eSofroniew MV, Vinters HV (2010) Astrocytes: biology and pathology. Acta Neuropathol; 119(1):7\\u0026ndash;35. DOI:10.1007/s00401-009-0619-8.\\u003c/li\\u003e\\n\\u003cli\\u003eChekhonin VP, Baklaushev VP, Yusubalieva GM, Pavlov KA, Ukhova OV, Gurina OI (2007) Modeling and immunohistochemical analysis of C6 glioma In Vivo. Cell Technologies in Biology and Medicine; 2.\\u003c/li\\u003e\\n\\u003cli\\u003eCl\\u0026eacute;ment A, Zaragori T, Filosa R et al. (2022) Multi-tracer and multiparametric PET imaging to detect the IDH mutation in glioma: a preclinical translational in vitro, in vivo, and ex vivo study. Cancer Imaging; 22(1):16. DOI:10.1186/s40644-022-00454-6.\\u003c/li\\u003e\\n\\u003cli\\u003eCicone F, Filss CP, Minniti G et al. (2015) Volumetric assessment of recurrent or progressive gliomas: comparison between F-DOPA PET and perfusion-weighted MRI. Eur J Nucl Med Mol Imaging; 42(6):905\\u0026ndash;15. DOI:10.1007/s00259-015-3018-5.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"molecular-imaging-and-biology\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"mibi\",\"sideBox\":\"Learn more about [Molecular Imaging and Biology](http://link.springer.com/journal/11307)\",\"snPcode\":\"11307\",\"submissionUrl\":\"https://www.editorialmanager.com/mibi/default2.aspx\",\"title\":\"Molecular Imaging and Biology\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"preclinical, alternative, chick embryo, radiotracer, glioma xenograft\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-5144367/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-5144367/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003e\\u003cstrong\\u003ePurpose:\\u003c/strong\\u003e In addition to rodent models, the chick embryo model has gained attention for radiotracer evaluation. Previous studies investigated tumours on the chorioallantoic membrane (CAM), but its value for radiotracer imaging of intracerebral tumours has not yet been demonstrated.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eProcedures:\\u003c/strong\\u003e Human U87 glioblastoma cells and U87-IDH1 mutant glioma cells were implanted into the brains of chick embryos at developmental day 5. After 12-14 days of tumour growth, blood-brain-barrier integrity was evaluated using \\u003cem\\u003ein vivo\\u003c/em\\u003e MRI contrast enhancement or \\u003cem\\u003eex vivo\\u003c/em\\u003e with Evans blue dye. The tracers O-(2-[\\u003csup\\u003e18\\u003c/sup\\u003eF]fluoroethyl)-L-tyrosine ([\\u003csup\\u003e18\\u003c/sup\\u003eF]FET) (n=5), 3,4-dihydroxy-6-[\\u003csup\\u003e18\\u003c/sup\\u003eF]-fluoro-L-phenylalanine ([\\u003csup\\u003e18\\u003c/sup\\u003eF]FDOPA) (n=3), or [\\u003csup\\u003e68\\u003c/sup\\u003eGa] labelled quinoline-based small molecule fibroblast activation protein inhibitor ([\\u003csup\\u003e68\\u003c/sup\\u003eGa]FAPI-46) (n=4) were injected intravenously if solid tumours were detectable in MRI. For time-activity curves for [\\u003csup\\u003e18\\u003c/sup\\u003eF]FET, additional micro PET (µPET) was performed. The chick embryos were sacrificed 60 minutes post-injection, and cryosections of the tumour-bearing brains were produced and evaluated with autoradiography and immunohistochemistry.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eResults:\\u003c/strong\\u003e Intracerebral tumours were produced with a 100 % success rate in viable chick embryos at experimental endpoint, however, 52% of chick embryos (n=85) did not survive the procedure on a long term. For the evaluated radiotracers, the tumour-to-brain ratios (TBR) derived from \\u003cem\\u003eex vivo\\u003c/em\\u003e autoradiography, as well as the tracer kinetics derived from µPET for intracerebral chick embryo tumours, were comparable to those previously reported in rodents and patients: TBRmean for [\\u003csup\\u003e18\\u003c/sup\\u003eF]FET was 1.69 ± 0.54 (n=5), and 3.8 for one hypermetabolic tumour and \\u0026lt; 2.0 for two isometabolic tumors using [\\u003csup\\u003e18\\u003c/sup\\u003eF]FDOPA, with TBRmean of 1.92 ± 1,11 (n=3). The TBRmean of [\\u003csup\\u003e68\\u003c/sup\\u003eGa]FAPI-46 for intracerebral chick embryo tumours was 19.13 ± 0.64 (n=4). In one of U87-MG tumours (n=5), an intact blood-tumour barrier was observed. \\u003cstrong\\u003eConclusions: \\u003c/strong\\u003eRadiotracer imaging of intracerebral tumours in the chick embryo offers a fast model for the evaluation of radiotracer uptake, accumulation, and kinetics. Our results indicate a high comparability of chick embryo intracerebral tumour imaging to xenograft rodent models or brain tumour patients.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Autoradiography of intracerebral tumours in the chick embryo model: A feasibility study using different PET tracers\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-11-15 06:53:32\",\"doi\":\"10.21203/rs.3.rs-5144367/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"reviewerAgreed\",\"content\":\"\",\"date\":\"2024-11-13T11:11:07+00:00\",\"index\":0,\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2024-11-13T11:06:53+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2024-11-07T20:40:46+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Molecular Imaging and Biology\",\"date\":\"2024-11-07T05:30:33+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"decision\",\"content\":\"Minor revisions\",\"date\":\"2024-10-24T05:31:22+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"molecular-imaging-and-biology\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"mibi\",\"sideBox\":\"Learn more about [Molecular Imaging and Biology](http://link.springer.com/journal/11307)\",\"snPcode\":\"11307\",\"submissionUrl\":\"https://www.editorialmanager.com/mibi/default2.aspx\",\"title\":\"Molecular Imaging and Biology\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"9196523e-975d-441b-899f-97b8479dbc81\",\"owner\":[],\"postedDate\":\"November 15th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-01-27T16:07:35+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-5144367\",\"link\":\"https://doi.org/10.1007/s11307-025-01983-9\",\"journal\":{\"identity\":\"molecular-imaging-and-biology\",\"isVorOnly\":false,\"title\":\"Molecular Imaging and Biology\"},\"publishedOn\":\"2025-01-21 15:58:23\",\"publishedOnDateReadable\":\"January 21st, 2025\"},\"versionCreatedAt\":\"2024-11-15 06:53:32\",\"video\":\"\",\"vorDoi\":\"10.1007/s11307-025-01983-9\",\"vorDoiUrl\":\"https://doi.org/10.1007/s11307-025-01983-9\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-5144367\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-5144367\",\"identity\":\"rs-5144367\",\"version\":[\"v1\"]},\"buildId\":\"re_ckhLnmML6MCF96OHNJ\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}