Side-by-side comparison of the two widely studied GRPR radiotracers, radiolabeled NeoB and RM2, in a preclinical setting.

INTRODUCTION: NeoB and RM2 are the most investigated gastrin-releasing peptide receptor (GRPR)-targeting radiotracers in preclinical and clinical studies. Therefore, an extensive side-by-side comparison of the two radiotracers is valuable to demonstrate whether one has advantages over the other. Accordingly, this study aims to compare the in vitro and in vivo characteristics of radiolabeled NeoB and RM2 to guide future clinical studies. METHOD: The stability of the radiolabeled GRPR analogs was determined in phosphate buffered saline (PBS), and commercially available mouse and human serum. Target affinity was determined by incubating human prostate cancer PC-3 cells with [177Lu]Lu-NeoB or [177Lu]Lu-RM2, + / - increasing concentrations of unlabeled NeoB, RM2, or Tyr4-bombesin (BBN). To determine uptake and specificity cells were incubated with [177Lu]Lu-NeoB or [177Lu]Lu-RM2 + / - Tyr4-BBN. Moreover, in vivo studies were performed to determine biodistribution and pharmacokinetics. Finally, radiotracer binding to various GRPR-expressing human cancer tissues was investigated. RESULTS: Both radiotracers demonstrated high stability in PBS and human serum, but stability in mouse serum decreased substantially over time. Moreover, both radiotracers demonstrated high GRPR affinity and specificity, but a higher uptake of [177Lu]Lu-NeoB was observed in in vitro studies. In vivo, no difference in tumor uptake was seen. The most prominent difference in uptake in physiological organs was observed in the GRPR-expressing pancreas; [177Lu]Lu-RM2 had less pancreatic uptake and a shorter pancreatic half-life than [177Lu]Lu-NeoB. Furthermore, [177Lu]Lu-RM2 presented with a lower tumor-to-kidney ratio, while the tumor-to-blood ratio was lower for [177Lu]Lu-NeoB. The autoradiography studies revealed higher binding of radiolabeled NeoB to all human tumor tissues. CONCLUSION: Based on these findings, we conclude that the in vivo tumor-targeting capability of radiolabeled NeoB and RM2 is similar. Additional studies are needed to determine whether the differences observed in physiological organ uptakes, i.e., the pancreas, kidneys, and blood, result in relevant differences in organ absorbed doses when the radiotracers are applied for therapeutic purposes.

Epigenetic regulation of somatostatin and somatostatin receptors in neuroendocrine tumors and other types of cancer.

Both somatostatin (SST) and somatostatin receptors (SSTRs) are proteins with important functions in both physiological tissue and in tumors, particularly in neuroendocrine tumors (NETs). NETs are frequently characterized by high SSTRs expression levels. SST analogues (SSAs) that bind and activate SSTR have anti-proliferative and anti-secretory activity, thereby reducing both the growth as well as the hormonal symptoms of NETs. Moreover, the high expression levels of SSTR type-2 (SSTR2) in NETs is a powerful target for therapy with radiolabeled SSAs. Due to the important role of both SST and SSTRs, it is of great importance to elucidate the mechanisms involved in regulating their expression in NETs, as well as in other types of tumors. The field of epigenetics recently gained interest in NET research, highlighting the importance of this process in regulating the expression of gene and protein expression. In this review we will discuss the role of the epigenetic machinery in controlling the expression of both SSTRs and the neuropeptide SST. Particular attention will be given to the epigenetic regulation of these proteins in NETs, whereas the involvement of the epigenetic machinery in other types of cancer will be discussed as well. In addition, we will discuss the possibility to target enzymes involved in the epigenetic machinery to modify the expression of the SST-system, thereby possibly improving therapeutic options.

Swim posture of mice does not affect performance in the water maze.

We quantified swim postures of mice in relation to their cognitive performance. After training in a water maze, young (5-6 months) and aged (14-16 months) female apolipoprotein E-knockout (apoE0/0) mice and wild type controls were video taped while swimming. Subsequently, angles of body points with the water surface were calculated. Mice with a more horizontal swim posture (young and aged apoE0/0, aged wild type mice) also showed an increased body weight. However, swim posture was not related to cognitive performance.

Differential effect of corticosterone on spatial learning abilities in apolipoprotein E knockout and C57BL/6J mice.

Previously, we found that repeated exposure to predator stress corrected the deficit in spatial learning of apolipoprotein E-knockout (apoE0/0) mice, but impaired cognitive performance of wild-type mice. Here we show that elevated corticosterone concentrations, accomplished by subcutaneously implanted pellets, results in similar genotype-related effects on water maze learning: while apoE0/0 mice improved their spatial learning abilities, wild-type mice (C57/Bl6J) became impaired. These results suggest that corticosterone mediates the lasting effects of environmental challenges on apoE-genotype related cognitive performance.

Reversal of cognitive deficit of apolipoprotein E knockout mice after repeated exposure to a common environmental experience.

This study tests the hypothesis that a history of common stressful experiences further promotes the cognitive deficit of apolipoprotein E (apoE)-knockout mice, an animal model to study aspects of Alzheimer's disease. In experiment 1, apoE-knockout and wild-type mice were repeatedly subjected to an environmental challenge (i.e. exposure to rats) and the effect was monitored on Morris water maze performance. Naive apoE-knockout mice were impaired, but surprisingly after rat stress their water maze performance improved and switched to a goal-directed search strategy. Rat stress induced in wild-type mice spatial learning deficits and an inefficient search strategy. Swim ability was not affected by rat stress and under basal conditions measures for locomotion and anxiety were similar for both genotypes. In experiments 2 and 3, we found that the rat stress paradigm attenuated the elevation of basal and stress-induced corticosterone concentrations in the apoE-knockout mice towards concentrations observed in wild-type mice. The expression of hippocampal mineralocorticoid and glucocorticoid receptor mRNA was similar in both genotypes, but in response to rat stress, the level of glucocorticoid receptor mRNA increased selectively in the CA1 pyramidal field. In conclusion, repeated exposure to a common environmental experience did abolish and reverse the difference in cognitive performance and corticosterone concentrations of apoE-knockout and wild-type mice.

Stress alleviates reduced expression of cell adhesion molecules (NCAM, L1), and deficits in learning and corticosterone regulation of apolipoprotein E knockout mice.

Cell adhesion molecules (CAMs) involved in synaptic changes underlying learning and memory processes, are implicated in the effect of stress on behavioural performance. The present study was designed to test the hypothesis that (i) expression of CAMs is apolipoprotein E- (apoE) genotype dependent and (ii) repeated exposure to stress modulates the synthesis of CAMs in an apoE-genotype dependent manner. Using ELISA we tested this hypothesis and measured expression of NCAM and L1 in different brain regions of naïve and stressed apolipoprotein E-knockout (apoE0/0) and C57Bl6 (wild-type) mice. Naïve apoE0/0 mice had elevated basal morning corticosterone and ACTH concentrations and decreased expression of NCAM and L1 compared to wild-type mice. Repeated exposure of mice to rats, as the common stressor, alleviated the reduction in expression of CAMs in apoE0/0 mice; seven days after the last rat exposure, expression of NCAM was increased in frontal brain and hippocampus whereas expression of L1 was increased in hippocampus and cerebellum. Rat stress attenuated the elevation of basal morning corticosterone concentration in apoE0/0 mice towards concentrations detected in wild-type mice. Moreover, rat stress improved learning and memory of apoE0/0 mice in the water maze. In conclusion, repeated exposure to stress eliminated apoE-genotype-related differences in expression of CAMs. Under these same conditions the differences in cognitive performance and corticosterone concentrations were abolished between wild type and apoE0/0 mice.

Repeated exposure to rats has persistent genotype-dependent effects on learning and locomotor activity of apolipoprotein E knockout and C57Bl/6 mice.

Recently we have shown that an experimentally controlled encounter of mice with rats ("rat stress") some time before actual behavioural testing either abolished or induced behavioural deficits in the Morris water maze, depending on the genotype of the mice: apolipoprotein E knockout mice (apoE0/0) and wild type mice. Here we report that previous rat stress: (i) facilitated learning of a circular hole board task in apoE0/0 mice and impaired learning in wild type mice, thereby abolishing genotype-dependent differences; (ii) although both genotypes preferred the dark compartment when tested in a light/dark-preference task 3 months after rat stress, locomotor activity was reduced in apoE0/0 and increased in wild type mice, thus genotype differences were amplified; (iii) both genotypes responded with a differential regulation of bodyweight during exposure to rats, which persisted for 3 months: apoE0/0 mice decreased while wild type mice increased their body weight; (iv) the high emotional reactivity (defecation boli) measured during behavioural tasks was not affected in apoE0/0 mice, whereas a decrease was observed in wild type mice. Thus, pre-experimental confrontation of mice with rats shifts behaviour and physiological responses and eliminates some of the genotype-dependent differences.

A refined method for sequential blood sampling by tail incision in rats.

Levels of endogenous or administered substances can be estimated by blood sampling. This allows an evaluation of the relationship between clinical signs, physiological parameters, pharmacological treatments and behaviour of the animal. We show that blood samples can be taken occasionally as well as sequentially by means of a small incision at the end of the rats' tails. Up to 300 microl of blood can be collected within 90 s. The advantages of this method are: (i) anaesthesia and surgery or restraint of the animal are not necessary; (ii) the procedure can be considered stress-free as indicated by the low, basal levels of the stress hormone corticosterone, even with frequent sequential blood sampling over 3 h; and (iii) it can be used for longitudinal studies allowing intra-individual comparisons over months and even years. Blood samples collected via an intravenous catheter and, at the same time, by our tail incision method resulted in comparable amounts of corticosterone. Moreover, we consider the tail incision method for rats to be 'animal-friendly' and a real alternative to other conventionally used blood sampling techniques.

Quantification of swim patterns in the Morris water maze.

Spatial learning and memory in rodents is most often assessed in the Morris water maze. Neurobiologists have to distinguish behavioral patterns to unravel underlying neuronal systems. We analyzed swim patterns of mice videotaped before and after training with a multi-trial procedure in the water maze. In addition to traditional parameters, the animals' position in relation to trained and other possible platform locations was estimated five times per second by an image analysis system. This parameter, cumulative distance to platform, was correlated with time spent in the platform quadrant but not with latency to and crossings of the platform location. We detected a subgroup of animals with concentric patterns within the group of spatial/persistent patterns. Random patterns were classified as well. Swim patterns before training were not predictive for the one after training. In summary, image analysis systems have made it very convenient to quantify behavior. Using their capacity, we have further improved the analysis of swim patterns, revealing animals' different approaches to solve a problem.