Intrinsic Heart Regeneration in Adult Vertebrates May be Strictly Limited to Low-Metabolic Ectotherms.

The heart has a high-metabolic rate, and its "around-the-clock" vital role to sustain life sets it apart in a regenerative setting from other organs and appendages. The landscape of vertebrate species known to perform intrinsic heart regeneration is strongly biased toward ectotherms-for example, fish, salamanders, and embryonic/neonatal ectothermic mammals. It is hypothesized that intrinsic heart regeneration is exclusively limited to the low-metabolic hearts of ectotherms. The biomedical field of regenerative medicine seeks to devise biologically inspired regenerative therapies to diseased human hearts. Falsification of the ectothermy dependency for heart regeneration hypothesis may be a crucial prerequisite to meaningfully seek inspiration in established ectothermic regenerative animal models. Otherwise, engineering approaches to construct artificial heart components may constitute a more viable path toward regenerative therapies. A more strict definition of regenerative phenomena is generated and several testable sub-hypotheses and experimental avenues are put forward to elucidate the link between heart regeneration and metabolism. Also see the video abstract here https://youtu.be/fZcanaOT5z8.

Dual-tracer PET of viable tumor volume and hypoxia for identification of necrosis-containing radio-resistant Sub-volumes.

Introduction: Positron emission tomography (PET) using hypoxia-selective tracers like FAZA may guide radiation dose-escalation approaches. However, poor resolution combined with slow tracer retention in relatively inaccessible target cells and slow clearance of unbound tracer results in low-contrast images, and areas where viable hypoxic tracer retaining cells and necrosis (no tracer) are intermixed may pass unnoticed during image thresholding. Here we hypothesized that a clinical feasible one-day dual tracer approach that combines a short-lived (e.g., 11C labeled) metabolic tracer that provides voxel-wise information on viable tissue volume (preferably independently of tumor microenvironment) and a hypoxia marker, may limit threshold-based errors. Material and methods: 11C-acetate and 11C-methionine uptake was quantified in tumor cell lines under tumor microenvironment-mimicking conditions of high/low O2 (21%/0%) and pH (7.4/6.7). Next, tumor-bearing mice were administered FAZA and sacrificed 1 h (mimics a clinical low-contrast image scenario) or 4 h (high contrast) later. In addition, all mice were administered pimonidazole (hypoxia) and 14C-methionine 1 h prior to sacrifice. Tumor tissue sections were analyzed using dual-tracer autoradiography. Finally, FAZA, or FAZA normalized to 14C-methionine retention (to adjust for differences in viable tissue volume) was compared to hypoxic fraction (deduced from immune-histological analysis of pimonidazole; ground truth) in PET-mimicking macroscopic pixels with variable extent of necrosis/hypoxia. Results/conclusions: Low pH stimulated 11C-acetate retention in many cell lines, and uptake was further modified by anoxia, compromising its usefulness as a universal marker of viable tumor volume. In contrast, 11C-methionine was largely unaffected by the in vitro microenvironment and was further tested in mice. Necrosis increased the risk of missing hypoxia-containing pixels during thresholding and hypoxic fraction and FAZA signal correlated poorly in the low contrast-scenario. Voxel-based normalization to 14C-methionine increased the likelihood of detecting voxels harboring hypoxic cells profoundly, but did not consistently improve the correlation between the density of hypoxic cells and tracer signal.

Characterization and radiosensitivity of HPV-related oropharyngeal squamous cell carcinoma patient-derived xenografts.

Background: Oropharyngeal squamous cell carcinomas (OPSCC) are rising rapidly in incidence due to Human Papillomavirus (HPV) and/or tobacco smoking. Prognosis is better for patients with HPV-positive disease, but may also be influenced by tobacco smoking and other factors. There is a need to individualize treatment to minimize morbidity and improve prognosis. Patient-derived xenografts (PDX) is an emerging pre-clinical research model that may more accurately reflect the human disease, and is an attractive platform to study disease biology and develop treatments and biomarkers. In this study we describe the establishment of PDX models, compare PDX tumors to the human original, and assess the suitability of this model for radiotherapy research and biomarker development. Material and methods: Tumor biopsies from 34 patients with previously untreated OPSCC were implanted in immunodeficient mice, giving rise to 12 squamous cell carcinoma PDX models (7 HPV+, 5 HPV-). Primary and PDX tumors were characterized extensively, examining histology, immunohistochemistry, cancer gene sequencing and gene expression analysis. Radiosensitivity was assessed in vivo in a growth delay assay. Results: Established PDX models maintained histological and immunohistochemical characteristics as well as HPV-status of the primary tumor. Important cancer driver gene mutations, e.g., in TP53, PIK3CA and others, were preserved. Gene expression related to cancer stem cell markers and gene expression subtype were preserved, while gene expression related to hypoxia and immune response differed. Radiosensitivity studies showed high concordance with clinical observations. Conclusion: PDX from OPSCC preserves important molecular characteristics of the human primary tumor. Radiosensitivity were in accordance with clinically observed treatment response. The PDX model is a clinically relevant surrogate model of head and neck cancer. Perspectives include increased understanding of disease biology, which could lead to development of novel treatments and biomarkers.

Metformin targets brown adipose tissue in vivo and reduces oxygen consumption in vitro.

AIMS: To test the hypothesis that brown adipose tissue (BAT) is a metformin target tissue by investigating in vivo uptake of [11 C]-metformin tracer in mice and studying in vitro effects of metformin on cultured human brown adipocytes. MATERIALS AND METHODS: Tissue-specific uptake of metformin was assessed in mice by PET/CT imaging after injection of [11 C]-metformin. Human brown adipose tissue was obtained from elective neck surgery and metformin transporter expression levels in human and murine BAT were determined by qPCR. Oxygen consumption in metformin-treated human brown adipocyte cell models was assessed by Seahorse XF technology. RESULTS: Injected [11 C]-metformin showed avid uptake in the murine interscapular BAT depot. Metformin exposure in BAT was similar to hepatic exposure. Non-specific inhibition of the organic cation transporter (OCT) protein by cimetidine administration eliminated BAT exposure to metformin, demonstrating OCT-mediated uptake. Gene expression profiles of OCTs in BAT revealed ample OCT3 expression in both human and mouse BAT. Incubation of a human brown adipocyte cell models with metformin reduced cellular oxygen consumption in a dose-dependent manner. CONCLUSION: These results support BAT as a putative metformin target.

Hypoxia positron emission tomography imaging: combining information on perfusion and tracer retention to improve hypoxia specificity.

BACKGROUND: Static positron emission tomography (PET) allows mapping of tumor hypoxia, but low resolution and slow tracer retention/clearance results in poor image contrast and the risk of missing areas where hypoxic cells and necrosis are intermixed. Fully dynamic PET may improve accuracy but scan protocols suitable for routine clinical use are warranted. A modeling study proposed that hypoxia specificity can be improved by a clinically feasible blood-flow normalization procedure that only requires a 10- to 15-min dynamic scan (perfusion), followed by a short late static scan, but experimental validation is desired. METHODS: Tumor-bearing mice were administered pimonidazole (hypoxia marker) and the PET hypoxia-tracer 18F-azomycin arabinoside (FAZA) and scanned for 3h. Subsequently, the distributions of FAZA (autoradiography) and hypoxic cells (pimonidazole) were compared on tissue sections. PET images collected in 10-min time intervals between 60 and 90 min post-injection (PETearly), which mimics the image contrast seen in patients, were compared voxel-by-voxel to 3-h PET (PETlate). For comparison, PETearly was normalized to the perfusion peak area, deduced from the first 10 min of the scan (PETperf), and the resulting parameter PETearly/PETperf was compared with PETlate. RESULTS: Tissue analysis revealed a near-perfect spatial match between FAZA signal and hypoxic cell density (pimonidazole) 3 h post-injection, regardless of the tumor type. Only a weak inverse or no correlation between PETperf and PETlate was seen, and the correlation between PETearly/PETperf and PETlate proved inferior to the correlation between PETearly and PETlate. CONCLUSIONS: Late PET scans in rodents, unlike patients, provide an accurate map of hypoxia against which earlier time-point scans can be compared. PETearly and PETlate correlated to a variable extent but the correlation was lowered by normalization to perfusion (PETearly/PETperf). Our study challenges the validity/robustness of a perfusion normalization approach. This may reflect that the chaotic tumor vasculature uncouples microregional blood flow and oxygen extraction.

FDG-PET reproducibility in tumor-bearing mice: comparing a traditional SUV approach with a tumor-to-brain tissue ratio approach.

BACKGROUND: Current [F-18]-fluorodeoxyglucose positron emission tomography (FDG-PET) procedures in tumor-bearing mice typically includes fasting, anesthesia, and standardized uptake value (SUV)-based quantification. Such procedures may be inappropriate for prolonged multiscan experiments. We hypothesize that normalization of tumor FDG retention relative to a suitable reference tissue may improve accuracy as this method may be less susceptible to uncontrollable day-to-day changes in blood glucose levels, physical activity, or unnoticed imperfect tail vein injections. MATERIAL AND METHODS: Fed non-anesthetized tumor-bearing mice were administered FDG intravenously (i.v.) or intraperitoneally (i.p.) and PET scanned on consecutive days using a Mediso nanoScan PET/magnetic resonance imaging (MRI). Reproducibility of various PET-deduced measures of tumor FDG retention, including normalization to FDG signal in reference organs and a conventional SUV approach, was evaluated. RESULTS: Day-to-day variability in i.v. injected mice was lower when tumor FDG retention was normalized to brain signal (T/B), compared to normalization to other tissues or when using SUV-based normalization. Assessment of tissue radioactivity in dissected tissues confirmed the validity of PET-derived T/B ratios. Mean T/B and SUV values were similar in i.v. and i.p. administered animals, but SUV normalization was more robust in the i.p. group than in the i.v. group. CONCLUSIONS: Multimodality scanners allow tissue delineation and normalization of tumor FDG uptake relative to reference tissues. Normalization to brain, but not liver or kidney, improved scan reproducibility considerably and was superior to traditional SUV quantification in i.v. tracer-injected animals. Day-to-day variability in SUV's was lower in i.p. than in i.v. injected animals, and i.p. injections may therefore be a valuable alternative in prolonged rodent studies, where repeated vein injections are undesirable.

The potential of hyperpolarized 13C magnetic resonance spectroscopy to monitor the effect of combretastatin based vascular disrupting agents.

BACKGROUND: Targeting tumor vasculature with vascular disrupting agents (VDAs) results in substantial cell death that precede tumor shrinkage. Here, we investigate the potential of hyperpolarized magnetic resonance spectroscopy (HPMRS) to monitor early metabolic changes associated with VDA treatment. METHODS: Mice bearing C3H mammary carcinomas were treated with the VDAs combretastatin-A4-phosphate (CA4P) or the analog OXi4503, and HPMRS was performed following [1-13C]pyruvate administration. Similarly, treated mice were positron emission tomography (PET) scanned following administration of the glucose analog FDG. Finally, metabolic imaging parameters were compared to tumor regrowth delay and measures of vascular damage, derived from dynamic contrast-agent enhanced magnetic resonance imaging (DCE-MRI) and histology. RESULTS: VDA-treatment impaired tumor perfusion (histology and DCE-MRI), reduced FDG uptake, increased necrosis, and slowed tumor growth. HPMRS, revealed that the [1-13C]pyruvate-to-[1-13C]lactate conversion remained unaltered, whereas [1-13C]lactate-to-[13C]bicarbonate (originating from respiratory CO2) ratios increased significantly following treatment. CONCLUSIONS: DCE-MRI and FDG-PET revealed loss of vessel functionality, impaired glucose delivery and reduced metabolic activity prior to cell death. [1-13C]lactate-to-[13C]bicarbonate ratios increased significantly during treatment, indicating a decline in respiratory activity driven by the onset of hypoxia. HPMRS is promising for early detection of metabolic stress inflicted by VDAs, which cannot easily be inferred based on blood flow measurements.

Hyperpolarized magnetic resonance spectroscopy for assessing tumor hypoxia.

INTRODUCTION: Hypoxic tumor cells are radioresistant, therefore, identification of hypoxia is crucial. Hyperpolarized magnetic resonance spectroscopy (HPMRS) allows real time measurements of the conversion of pyruvate to lactate, the final step of anaerobic energy production, and may thus allow non-invasive identification of hypoxia or treatment-induced changes in oxygenation. The aim of the study was to investigate the usefulness of HPMRS as a means to assess tumor hypoxia and its dynamics during intervention. MATERIAL AND METHODS: C3H mammary carcinomas grown in CDF1 mice were used. To manipulate with tumor oxygenation, non-anaesthetized mice were gassed with air, 10% or 100% oxygen prior to administration of hyperpolarized [1-¹³C]pyruvate and HPMRS analysis. A direct assessment of tumor oxygen partial pressure (pO2) distributions were made using the Eppendorf oxygen electrode in a separate, but similarly treated, group of mice. RESULTS: Even though breathing 100% oxygen improved tumor oxygenation as evidenced by pO2 measurements, the mean (with 1 S.E.) [1-¹³C]lactate/[1-¹³C]pyruvate ratio was unaffected by this intervention, being 34 (30-37) in mice breathing air and 37 (33-42) in mice breathing 100% oxygen. In contrast, and in accordance with pO2 measurements, a significant increase in the [1-¹³C]lactate/[1-¹³C]pyruvate ratio was seen in 10% oxygen-breathing mice with a ratio of 46 (42-50). CONCLUSIONS: Although, no metabolic change was observed during 100% O2 breathing using HPMRS, the significant increase in the [1-¹³C]lactate/[1-¹³C]pyruvate ratio during 10% oxygen breathing suggests, that HPMRS can detect hypoxia-driven changes in the metabolic fate of pyruvate. To what extent and for what purposes HPMRS may best supplement or complement established techniques like hypoxia PET needs to be unraveled in future research.

Targeting tumour hypoxia to prevent cancer metastasis. From biology, biosensing and technology to drug development: the METOXIA consortium.

The hypoxic areas of solid cancers represent a negative prognostic factor irrespective of which treatment modality is chosen for the patient. Still, after almost 80 years of focus on the problems created by hypoxia in solid tumours, we still largely lack methods to deal efficiently with these treatment-resistant cells. The consequences of this lack may be serious for many patients: Not only is there a negative correlation between the hypoxic fraction in tumours and the outcome of radiotherapy as well as many types of chemotherapy, a correlation has been shown between the hypoxic fraction in tumours and cancer metastasis. Thus, on a fundamental basis the great variety of problems related to hypoxia in cancer treatment has to do with the broad range of functions oxygen (and lack of oxygen) have in cells and tissues. Therefore, activation-deactivation of oxygen-regulated cascades related to metabolism or external signalling are important areas for the identification of mechanisms as potential targets for hypoxia-specific treatment. Also the chemistry related to reactive oxygen radicals (ROS) and the biological handling of ROS are part of the problem complex. The problem is further complicated by the great variety in oxygen concentrations found in tissues. For tumour hypoxia to be used as a marker for individualisation of treatment there is a need for non-invasive methods to measure oxygen routinely in patient tumours. A large-scale collaborative EU-financed project 2009-2014 denoted METOXIA has studied all the mentioned aspects of hypoxia with the aim of selecting potential targets for new hypoxia-specific therapy and develop the first stage of tests for this therapy. A new non-invasive PET-imaging method based on the 2-nitroimidazole [(18)F]-HX4 was found to be promising in a clinical trial on NSCLC patients. New preclinical models for testing of the metastatic potential of cells were developed, both in vitro (2D as well as 3D models) and in mice (orthotopic grafting). Low density quantitative real-time polymerase chain reaction (qPCR)-based assays were developed measuring multiple hypoxia-responsive markers in parallel to identify tumour hypoxia-related patterns of gene expression. As possible targets for new therapy two main regulatory cascades were prioritised: The hypoxia-inducible-factor (HIF)-regulated cascades operating at moderate to weak hypoxia (<1% O(2)), and the unfolded protein response (UPR) activated by endoplasmatic reticulum (ER) stress and operating at more severe hypoxia (<0.2%). The prioritised targets were the HIF-regulated proteins carbonic anhydrase IX (CAIX), the lactate transporter MCT4 and the PERK/eIF2α/ATF4-arm of the UPR. The METOXIA project has developed patented compounds targeting CAIX with a preclinical documented effect. Since hypoxia-specific treatments alone are not curative they will have to be combined with traditional anti-cancer therapy to eradicate the aerobic cancer cell population as well.

Hypo- and hyperactivated Notch signaling induce a glycolytic switch through distinct mechanisms.

A switch from oxidative phosphorylation to glycolysis is frequently observed in cancer cells and is linked to tumor growth and invasion, but the underpinning molecular mechanisms controlling the switch are poorly understood. In this report we show that Notch signaling is a key regulator of cellular metabolism. Both hyper- and hypoactivated Notch induce a glycolytic phenotype in breast tumor cells, although by distinct mechanisms: hyperactivated Notch signaling leads to increased glycolysis through activation of the phosphatidylinositol 3-kinase/AKT serine/threonine kinase pathway, whereas hypoactivated Notch signaling attenuates mitochondrial activity and induces glycolysis in a p53-dependent manner. Despite the fact that cells with both hyper- and hypoactivated Notch signaling showed enhanced glycolysis, only cells with hyperactivated Notch promoted aggressive tumor growth in a xenograft mouse model. This phenomenon may be explained by that only Notch-hyperactivated, but not -hypoactivated, cells retained the capacity to switch back to oxidative phosphorylation. In conclusion, our data reveal a role for Notch in cellular energy homeostasis, and show that Notch signaling is required for metabolic flexibility.

Correlation between local instantaneous dose rate and oxygen pressure reduction during proton pencil beam scanning irradiation.

Background and purpose: Oxygen dynamics may be important for the tissue-sparing effect observed at ultra-high dose rates (FLASH sparing effect). This study investigated the correlation between local instantaneous dose rate and radiation-induced oxygen pressure reduction during proton pencil beam scanning (PBS) irradiations of a sample and quantified the oxygen consumption g-value. Materials and methods: A 0.2 ml phosphorescent sample (1 µM PtG4 Oxyphor probe in saline) was irradiated with a 244 MeV proton PBS beam. Four irradiations were performed with variations of a PBS spot pattern with 5 × 7 spots. During irradiation, the partial oxygen pressure (pO2) was measured with 4.5 Hz temporal resolution with a phosphorometer (Oxyled) that optically excited the probe and recorded the subsequently emitted light. A calibration was performed to calculate the pO2 level from the measured phosphorescence lifetime. A fiber-coupled scintillator simultaneously measured the instantaneous dose rate in the sample with 50 kHz sampling rate. The oxygen consumption g-value was determined on a spot-by-spot level and using the total pO2 change for full spot pattern irradiation. Results: A high correlation was found between the local instantaneous dose rate and pO2 reduction rate, with a correlation coefficient of 0.96-0.99. The g-vales were 0.18 ± 0.01 mmHg/Gy on a spot-by-spot level and 0.17 ± 0.01 mmHg/Gy for full spot pattern irradiation. Conclusions: The pO2 reduction rate was directly related to the local instantaneous dose rate per delivered spot in PBS deliveries. The methodology presented here can be applied to irradiation at ultra-high dose rates with modifications in the experimental setup.

Dynamic whole-body [18F]FES PET/CT increases lesion visibility in patients with metastatic breast cancer.

BACKGROUND: Correct classification of estrogen receptor (ER) status is essential for prognosis and treatment planning in patients with breast cancer (BC). Therefore, it is recommended to sample tumor tissue from an accessible metastasis. However, ER expression can show intra- and intertumoral heterogeneity. 16α-[18F]fluoroestradiol ([18F]FES) Positron Emission Tomography/Computed Tomography (PET/CT) allows noninvasive whole-body (WB) identification of ER distribution and is usually performed as a single static image 60 min after radiotracer injection. Using dynamic whole-body (D-WB) PET imaging, we examine [18F]FES kinetics and explore whether Patlak parametric images ( K i ) are quantitative and improve lesion visibility. RESULTS: This prospective study included eight patients with metastatic ER-positive BC scanned using a D-WB PET acquisition protocol. The kinetics of [18F]FES were best characterized by the irreversible two-tissue compartment model in tumor lesions and in the majority of organ tissues. K i values from Patlak parametric images correlated with K i values from the full kinetic analysis, r2 = 0.77, and with the semiquantitative mean standardized uptake value (SUVmean), r2 = 0.91. Furthermore, parametric K i images had the highest target-to-background ratio (TBR) in 162/164 metastatic lesions and the highest contrast-to-noise ratio (CNR) in 99/164 lesions compared to conventional SUV images. TBR was 2.45 (95% confidence interval (CI): 2.25-2.68) and CNR 1.17 (95% CI: 1.08-1.26) times higher in K i images compared to SUV images. These quantitative differences were seen as reduced background activity in the K i images. CONCLUSION: [18F]FES uptake is best described by an irreversible two-tissue compartment model. D-WB [18F]FES PET/CT scans can be used for direct reconstruction of parametric K i images, with superior lesion visibility and K i values comparable to K i values found from full kinetic analyses. This may aid correct ER classification and treatment decisions. Trial registration ClinicalTrials.gov: NCT04150731, https://clinicaltrials.gov/study/NCT04150731.

Induction of hypoxia by vascular disrupting agents and the significance for their combination with radiation therapy.

PURPOSE: This pre-clinical study was designed to investigate the effect of various vascular disrupting agents (VDAs) that have undergone or are in clinical evaluation, had on the oxygenation status of tumours and what effects that could have on the combination with radiation. MATERIAL AND METHODS: The tumour model was a C3H mammary carcinoma grown in the right rear foot of female CDF1 mice and treated when at 200 mm(3) in size. The VDAs were the flavenoid compounds flavone acetic acid (FAA) and its more recent derivative 5,6-dimethylxanthenone-4-acetic acid (DMXAA), and the leading tubulin binding agent combretastatin A-4 phosphate (CA4P) and the A-1 analogue OXi4503. Oxygenation status was estimated using the Eppendorf oxygen electrode three hours after drug injection. Radiation response was determined following single or fractionated (10 fractions in 12 days) irradiations with a 240 kV x-ray machine using either a tumour re-growth or local tumour control assay. RESULTS: All VDAs significantly reduced the oxygenation status of the tumours. They also influenced radiation response, but the affect was time and sequence dependent using single radiation schedules; an enhanced effect when the VDAs were injected at the same time or after irradiating, but no or even a reduced effect when given prior to irradiation. Only OXi4503 showed an increased response when given before the radiation. CA4P and OXi4503 also enhanced a fractionated radiation treatment if the drugs were administered after fractions 5 and 10. CONCLUSIONS: VDAs clearly induced tumour hypoxia. This had the potential to decrease the efficacy of radiation. However, if the appropriate timing and scheduling were used an enhanced effect was observed using both single and fractionated radiation treatments.

Ultra-high field 1H magnetic resonance imaging approaches for acute hypoxia.

BACKGROUND: Currently, radiation treatments are being optimised based on in vivo imaging of radioresistant, hypoxic tumour areas. This study aimed at detecting nicotinamide's reduction of acute hypoxia in a mouse tumour model by two clinically relevant magnetic resonance imaging (MRI) methods at ultra-high magnetic field strength. MATERIAL AND METHODS: The C3H mammary carcinoma was grown to 200 mm(3) in the right rear foot of CDF1 mice. The mice were anaesthetised with ketamine and xylazine prior to imaging. A treatment group received nicotinamide intraperitoneally (i.p.) at the dose 1000 mg/kg, and a control group received saline. MRI was performed at 16.4 T with a spatial resolution of 0.156 × 0.156 × 0.5 mm(3). The imaging protocol included BOLD imaging and two DCE-MRI scans. Initial area under the curve (IAUC) and the parameters from the extended Toft's model were estimated from the DCE-MRI data. Tumour median values of 1) T2* mean, 2) T2* standard deviation, 3) DCE-MRI parameters, and 4) DCE-MRI parameter differences between scans were compared between the treatment groups using Student's t-test (significance level p < 0.05). RESULTS: Parametric maps showed intra- and inter-tumour heterogeneity. Blood volume was significantly larger in the nicotinamide-treated group, and also the blood volume difference between the two DCE-MRI scans was significantly larger in the treatment group. CONCLUSION: Higher blood volume and blood volume variation was observed by DCE-MRI in the treatment group. Other DCE-MRI parameters showed no significant differences, and the higher blood volume was not detected by BOLD MRI. The higher blood volume variation seen with DCE-MRI may be influenced by the drug effect reducing over time, and furthermore the anaesthesia may play an important role.

PET imaging of tumor hypoxia using 18F-labeled pimonidazole.

BACKGROUND: Tumor hypoxia contributes to loco-regional failure, and for optimal treatment planning, knowledge about tumor hypoxia in individual patients is required. Nitroimidazole-based tracers, which are retained in hypoxic cells, allow PET-based assessment of tumor hypoxia, but current tracers are characterized by slow tracer retention and clearance, resulting in low inter-tissue contrast. Pimonidazole is an immune detectable hypoxia marker widely used for detection of hypoxia in tumor samples. Pimonidazole has excellent chemical properties for hypoxia imaging, but labeling for non- invasive assay has not been attempted. Here we labeled pimonidazole with (18)F ([(18)F]FPIMO). MATERIAL AND METHODS: [(18)F]FPIMO was produced by fluorination of 1-[2-O-tosyl-3-(2-nitroimidazole-1-yl)-propyl]-piperidine, which resulted in two isomeric interchangeable forms (named "5" and "6") with a radiochemical purity of 91-100%. [(18)F]FPIMO was tested by incubation of two different tumor cell lines at high and low oxygen levels. [(18)F]FPIMO was also administered to tumor-bearing mice and tracer retention in tumors, non-hypoxic reference tissues and tissues involved in drug metabolism/clearance was evaluated by various techniques. RESULTS AND CONCLUSIONS: Retention of [(18)F]FPIMO was strongly hypoxia-driven in vitro, but isomeric form "5" was particularly promising and reached impressive anoxic-to-oxic retention ratios of 36 and 102, in FaDuDD and SiHa cells, respectively, following three hours of tracer incubation. This was equal to or higher than ratios measured using the established hypoxia tracer [(18)F]FAZA. [(18)F]FPIMO also accumulated in tumors grown in mice, and reached tumor levels that were two to six-fold higher than in muscle three hours post-administration. Furthermore, the intra-tumoral distribution of [(18)F]FPIMO (autoradiography) and unlabeled pimonidazole (immunohistochemistry) was largely identical. Nonetheless, [(18)F]FPIMO proved inferior to [(18)F]FAZA, since absolute tumor signal and intra-tumoral contrast was low, thus compromising PET imaging. Low tumor signal was coupled to extensive tracer accumulation in liver and kidneys, and analysis of blood metabolites revealed that [(18)F]FPIMO was metabolized rapidly, with little parent compound remaining 15 minutes post-administration. Ongoing work focuses on the possibility of labeling pimonidazole in different positions with (18)F to improve tracer stability in vivo.

Pre-Clinical Models to Study Human Prostate Cancer.

Prostate cancer is a common cancer among men and typically progresses slowly for several decades before becoming aggressive and spreading to other organs, leaving few treatment options. While large animals have been studied, the dog's prostate is anatomically similar to humans and has been used to study spontaneous prostate cancer. However, most research currently focuses on the mouse as a model organism due to the ability to genetically modify their prostatic tissues for molecular analysis. One milestone in this research was the identification of the prostate-specific promoter Probasin, which allowed for the prostate-specific expression of transgenes. This has led to the generation of mice with aggressive prostatic tumors through overexpression of the SV40 oncogene. The Probasin promoter is also used to drive Cre expression and has allowed researchers to generate prostate-specific loss-of-function studies. Another landmark moment in the process of modeling prostate cancer in mice was the orthoptic delivery of viral particles. This technology allows the selective overexpression of oncogenes from lentivirus or the use of CRISPR to generate complex loss-of-function studies. These genetically modified models are complemented by classical xenografts of human prostate tumor cells in immune-deficient mice. Overall, pre-clinical models have provided a portfolio of model systems to study and address complex mechanisms in prostate cancer for improved treatment options. This review will focus on the advances in each technique.

The effect of single bout and prolonged aerobic exercise on tumor hypoxia in mice.

The objectives of this study were to investigate 1) the effect of acute aerobic exercise on tumor hypoxia and blood perfusion, 2) the impact of exercise intensity, 3) the duration of the effect, and 4) the effect of prolonged training on tumor hypoxia and tumor growth. Female CDF1 mice were inoculated with the C3H mammary carcinoma either in the mammary fat pad or subcutaneously in the back. For experiments on the effect of different intensities in a single exercise bout, mice were randomized to 30-min treadmill running at low-, moderate-, or high-intensity speeds or no exercise. To investigate the prolonged effect on hypoxia and tumor growth, tumor-bearing mice were randomized to no exercise (CON) or daily 30-min high-intensity exercise averaging 2 wk (EX). Tumor hypoxic fraction was quantified using the hypoxia marker Pimonidazole. Initially, high-intensity exercise reduced tumor hypoxic fraction by 37% compared with CON [P = 0.046; 95% confidence interval (CI): 0.1; 10.3] in fat pad tumors. Low- and moderate-intensity exercises did not. Following experiments investigating the duration of the effect-as well as experiments in mice with back tumors-failed to show any exercise-induced changes in hypoxia. Interestingly, prolonged daily training significantly reduced hypoxic fraction by 60% (P = 0.002; 95% CI: 2.5; 10.1) compared with CON. Despite diverging findings on the acute effect of exercise on hypoxia, our data indicate that if exercise has a diminishing effect, high-intensity exercise is needed. Prolonged training reduced tumor hypoxic fraction-cautiously suggesting a potential clinical potential.NEW & NOTEWORTHY This study provides novel information on the effects of acute and chronic exercise on tumor hypoxia in mice. In contrast to the few related existing studies, diverging findings on tumor hypoxia after acute exercise were observed, suggesting that tumor model and location should be considered in future studies. Highly significant reductions in tumor hypoxia following chronic high-intensity exercise propose a future clinical potential but this should be investigated in patients.

Nuclear Medicine Preclinical Research: The Role of Cell Cultures.

Cell lines are essential in biomedical research due to their adaptability and precise simulation of physiological and pathophysiological conditions. Cell culture techniques have greatly advanced our understanding of biology in various fields and are widely regarded as a reliable and durable tool. Their diverse applications make them indispensable in scientific research. Radiation-emitting compounds are commonly used in cell culture research to investigate biological processes. Radiolabeled compounds are utilized to study cell function, metabolism, molecular markers, receptor density, drug binding and kinetics, as well as to analyze the direct interaction of radiotracers with target organ cells. This allows for the examination of normal physiology and disease states. The In Vitro system simplifies the study and filters out nonspecific signals from the In Vivo environment, leading to more specific results. Moreover, cell cultures offer ethical advantages when evaluating new tracers and drugs in preclinical studies. While cell experiments cannot entirely replace animal experiments, they reduce the need for live animals in experimentation.

In Vitro Characterization of the Bacteria-derived Hypoxia-selective Cytotoxin BE-43547.

BACKGROUND/AIM: Hypoxia-activated pro-drugs, such as TH-302, may kill hypoxic treatment-resistant tumor cells, but have failed in clinical trials. This may be related to variable levels of drug-activating reductases. Compounds such as bacteria-derived BE-43547, which target hypoxic cells independently of reductases, may be beneficial. This study characterized the in vitro potency and hypoxia selectivity of BE-43547 and TH-302. MATERIALS AND METHODS: Tumor cells were exposed to different oxygenation levels in the presence/absence of drug, and survival was quantified using total cell number (BE-43547) or clonogenic survival (BE-43547 and TH-302) assays. Half-maximal inhibitory concentration (IC50) values and the hypoxia-cytotoxicity-ratio (HCR: normoxic IC50/hypoxic IC50) were determined from dose-response curves. Finally, both drugs were tested in spheroids exposed to 20% or 0% O2 for 24 h followed by assessment of clonogenic survival. RESULTS: BE-43547 was highly potent and displayed little inter-cell line variability. Strongly enhanced cytotoxicity was observed under oxygen-restricted conditions with HCR's of ~100 and ~20 after 24 h of treatment with 0 or 0.5% O2, respectively. Reducing treatment time somewhat reduced hypoxia selectivity. Hypoxia selectivity was observed regardless of whether the drug was added before or during the hypoxic challenge. TH-302 IC50 values varied 10-fold under oxic conditions, whereas those of the anoxic-to-normoxic HCR varied from 15 to 88. Both BE-43547 and TH-302 were unable to completely sterilize anoxic incubated spheroids. CONCLUSION: BE-43547 is highly hypoxia-selective, and unlike TH-302, displayed minimal variability between cell lines, suggesting that BE-43547 targets a fundamental feature/target that is only present, or of survival importance, during hypoxia. Spheroid experiments suggested inadequate tissue penetrability, which may be overcome by designing novel drug analogs.

Special Challenges in PET Imaging of Ectothermic Vertebrates.

The bulk of biomedical positron emission tomography (PET)-scanning experiments are performed on mammals (ie, rodents, pigs, and dogs), and the technique is only infrequently applied to answer research questions in ectothermic vertebrates such as fish, amphibians, and reptiles. Nevertheless, many unique and interesting physiological characteristics in these ectothermic vertebrates could be addressed in detail through PET. The low metabolic rate of ectothermic animals, however, may compromise the validity of physiological and biochemical parameters derived from the images created by PET and other scanning modalities. Here, we review some of the considerations that should be taken into account when PET scanning fish, amphibians, and reptiles. We present specific results from our own experiments, many of which remain previously unpublished, and we draw on examples from the literature. We conclude that knowledge on the natural history and physiology of the species studied and an understanding of the limitations of the PET scanning techniques are necessary to avoid the design of faulty experiments and erroneous conclusions.