CMKLR1-targeting peptide tracers for PET/MR imaging of breast cancer.

Background: Molecular targeting remains to be a promising approach in oncology. Overexpression of G protein-coupled receptors (GPCRs) in human cancer is offering a powerful opportunity for tumor-selective imaging and treatment employing nuclear medicine. We utilized novel chemerin-based peptide conjugates for chemokine-like receptor 1 (CMKLR1) targeting in a breast cancer xenograft model. Methods: By conjugation with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), we obtained a family of five highly specific, high-affinity tracers for hybrid positron emission tomography/magnetic resonance (PET/MR) imaging. A xenograft model with target-positive DU4475 and negative A549 tumors in immunodeficient nude mice enabled CMKLR1-specific imaging in vivo. We acquired small animal PET/MR images, assessed biodistribution by ex vivo measurements and investigated the tracer specificity by blocking experiments. Results: Five CMKLR1-targeting peptide tracers demonstrated high biological activity and affinity in vitro with EC50 and IC50 values below 2 nM. Our target-positive (DU4475) and target-negative (A549) xenograft model could be validated by ex vivo analysis of CMKLR1 expression and binding. After preliminary PET imaging, the three most promising tracers [68Ga]Ga-DOTA-AHX-CG34, [68Ga]Ga-DOTA-KCap-CG34 and [68Ga]Ga-DOTA-ADX-CG34 with best tumor uptake were further analyzed. Hybrid PET/MR imaging along with concomitant biodistribution studies revealed distinct CMKLR1-specific uptake (5.1% IA/g, 3.3% IA/g and 6.2% IA/g 1 h post-injection) of our targeted tracers in DU4475 tumor tissue. In addition, tumor uptake was blocked by excess of unlabeled peptide (6.4-fold, 5.5-fold and 3.4-fold 1 h post-injection), further confirming CMKLR1 specificity. Out of five tracers, we identified these three tracers with moderate, balanced hydrophilicity to be the most potent in receptor-mediated tumor targeting. Conclusion: We demonstrated the applicability of 68Ga-labeled peptide tracers by visualizing CMKLR1-positive breast cancer xenografts in PET/MR imaging, paving the way for developing them into theranostics for tumor treatment.

Increasing molar activity by HPLC purification improves 68Ga-DOTA-NAPamide tumor accumulation in a B16/F1 melanoma xenograft model.

PURPOSE: Melanocortin receptor 1 (MC1R) is overexpressed in melanoma and may be a molecular target for imaging and peptide receptor radionuclide therapy. 68Gallium (68Ga) labeling of DOTA-conjugated peptides is an established procedure in the clinic for use in positron emission tomography (PET) imaging. Aim of this study was to compare a standard labeling protocol against the 68Ga-DOTA peptide purified from the excess of unlabeled peptide. PROCEDURES: The MC1R ligand DOTA-NAPamide was labeled with 68Ga using a standard clinical protocol. Radioactive peptide was separated from the excess of unlabeled DOTA-NAPamide by HPLC. Immediately after the incubation of peptide and 68Ga (95°C, 15 min), the reaction was loaded on a C18 column and separated by a water/acetonitrile gradient, allowing fractionation in less than 20 minutes. Radiolabeled products were compared in biodistribution studies and PET imaging using nude mice bearing MC1R-expressing B16/F1 xenograft tumors. RESULTS: In biodistribution studies, non-purified 68Ga-DOTA-NAPamide did not show significant uptake in the tumor at 1 h post injection (0.78% IA/g). By the additional HPLC step, the molar activity was raised around 10,000-fold by completely removing unlabeled peptide. Application of this rapid purification strategy led to a more than 8-fold increase in tumor uptake (7.0% IA/g). The addition of various amounts of unlabeled DOTA-NAPamide to the purified product led to a blocking effect and decreased specific tumor uptake, similar to the result seen with non-purified radiopeptide. PET imaging was performed using the same tracer preparations. Purified 68Ga-DOTA-NAPamide, in comparison, showed superior tumor uptake. CONCLUSIONS: We demonstrated that chromatographic separation of radiolabeled from excess unlabeled peptide is technically feasible and beneficial, even for short-lived isotopes such as 68Ga. Unlabeled peptide molecules compete with receptor binding sites in the target tissue. Purification of the radiopeptide therefore improved tumor uptake.

Photoacoustic pump-probe tomography of fluorophores in vivo using interleaved image acquisition for motion suppression.

In fluorophores, the excited state lifetime can be modulated using pump-probe excitation. By generating photoacoustic (PA) signals using simultaneous and time-delayed pump and probe excitation pulses at fluences below the maximum permissible exposure, a modulation of the signal amplitude is observed in fluorophores but not in endogenous chromophores. This provides a highly specific contrast mechanism that can be used to recover the location of the fluorophore using difference imaging. The practical challenges in applying this method to in vivo PA tomography include the typically low concentrations of fluorescent contrast agents, and tissue motion. The former results in smaller PA signal amplitudes compared to those measured in blood, while the latter gives rise to difference image artefacts that compromise the unambiguous and potentially noise-limited detection of fluorescent contrast agents. To address this limitation, a method based on interleaved pump-probe image acquisition was developed. It relies on fast switching between simultaneous and time-delayed pump-probe excitation to acquire PA difference signals in quick succession, and to minimise the effects of tissue motion. The feasibility of this method is demonstrated in tissue phantoms and in initial experiments in vivo.

Changes in metabolite profiles caused by genetically determined obesity in mice.

The Berlin Fat Mouse Inbred (BFMI) line harbors a major recessive gene defect on chromosome 3 (jobes1) leading to juvenile obesity and metabolic syndrome. The present study aimed at the identification of metabolites that might be linked to recessively acting genes in the obesity locus. Firstly, serum metabolites were analyzed between obese BFMI and lean B6 and BFMI × B6 F1 mice to identify metabolites that are different. In a second step, a metabolite-protein network analysis was performed linking metabolites typical for BFMI mice with genes of the jobes1 region. The levels of 22 diacyl-phosphatidylcholines (PC aa), two lyso-PC and three carnitines were found to be significantly lower in obese mice compared with lean mice, while serine, glycine, arginine and hydroxysphingomyelin were higher for the same comparison. The network analysis identified PC aa C42:1 as functionally linked with the genes Ccna2 and Trpc3 via the enzymes choline kinase alpha and phospholipase A2 group 1B (PLA2G1B), respectively. Gene expression analysis revealed elevated Ccna2 expression in adipose tissue of BFMI mice. Furthermore, unique mutations were found in the Ccna2 promoter of BFMI mice which are located in binding sites for transcription factors or micro RNAs and could cause differential Ccna2 mRNA levels between BFMI and B6 mice. Increased expression of Ccna2 was consistent with higher mitotic activity of adipose tissue in BFMI mice. Therefore, we suggest a higher demand for PC necessary for adipose tissue growth and remodeling. This study highlights the relationship between metabolite profiles and the underlying genetics of obesity in the BFMI line.

Relationship between obesity phenotypes and genetic determinants in a mouse model for juvenile obesity.

Obesity, a state of imbalance between lean mass and fat mass, is important for the etiology of diseases affected by the interplay of multiple genetic and environmental factors. Although genome-wide association studies have repeatedly associated genes with obesity and body weight, the mechanisms underlying the interaction between the muscle and adipose tissues remain unknown. Using 351 mice (at 10 wk of age) of an intercross population between Berlin Fat Mouse Inbred (BFMI) and C57BL/6NCrl (B6N) mice, we examined the causal relationships between genetic variations and multiple traits: body lean mass and fat mass, adipokines, and bone mineral density. Furthermore, evidence from structural equation modeling suggests causality among these traits. In the BFMI model, juvenile obesity affects lean mass and impairs bone mineral density via adipokines secreted from the white adipose tissues. While previous studies have indicated that lean mass has a causative effect on adiposity, in the Berlin Fat Mouse model that has been selected for juvenile obesity (at 9 wk of age) for >90 generations, however, the causality is switched from fat mass to lean mass. In addition, linkage studies and statistical modeling have indicated that quantitative trait loci on chromosomes 5 and 6 affect both lean mass and fat mass. These lines of evidence indicate that the muscle and adipose tissues interact with one another and the interaction is modulated by genetic variations that are shaped by selections. Experimental examinations are necessary to verify the biological role of the inferred causalities.

IGF-I contributes to glucose homeostasis in the Berlin Fat Mouse Inbred line.

This study aimed to investigate the tissue-specific role of the insulin-like growth factor 1 (IGF-I) on glucose homeostasis in the high-fatness selected Berlin Fat Mouse Inbred (BFMI) line. Therefore, the expression of different IGF-I transcripts and IGF-I protein, IGF-binding proteins, insulin as well as glucose tolerance was analyzed in BFMI in comparison with that in lean mice. In addition, dietary effects were investigated. The BFMI line showed normal blood glucose clearance on standard diet, but on high-fat diet the clearance was impaired, indicating the beginning of insulin resistance. Circulating IGF-I and insulin levels were elevated in BFMI than in lean mice on both diets along with a down-regulation of three IGF-I binding proteins in BFMI mice. Serum IGF-I levels corresponded with the expression pattern for both hepatic and one class II splice variants in reproductive adipose tissue, but not in muscle. High insulin and high IGF-I levels likely prevent BFMI mice from diabetes.

Features of the metabolic syndrome in the Berlin Fat Mouse as a model for human obesity.

BACKGROUND: The Berlin Fat Mouse BFMI860 is a polygenic obesity mouse model which harbors a natural major gene defect resulting in early onset of obesity. To elucidate adult bodily responses in BFMI860 mice that develop juvenile obesity, we studied features of the metabolic syndrome at 20 weeks. METHODS: We examined fat deposition patterns, adipokines, lipid profiles in serum, glucose homeostasis, and insulin sensitivity in mice that were fed either a standard maintenance (SMD) or a high-fat diet (HFD). RESULTS: Like many obese humans, BFMI860 mice showed hyperleptinemia accompanied by hypoadiponectinemia already at SMD that was further unbalanced as a result of HFD. Furthermore, BFMI860 mice had high triglyceride concentrations. However, triglyceride clearance after an oral oil gavage was impaired on SMD but improved on HFD. The oral and intraperitoneal glucose as well as the insulin tolerance tests provided evidence for reduced insulin sensitivity under SMD and insulin resistance on HFD. BFMI860 mice can maintain normal glucose clearance over a wide range of feeding conditions according to an adaptation via increasing the insulin concentrations. CONCLUSIONS: BFMI860 mice show obesity, dyslipidemia, and insulin resistance as three major components of the metabolic syndrome. As these mice develop the described phenotype as a result of a major gene defect, they are a unique model for the investigation of genetic and pathophysiological mechanisms underlying the observed features of the metabolic syndrome and to search for potential strategies to revert the adverse effects under controlled conditions.

Genetic and diet effects on Ppar-α and Ppar-γ signaling pathways in the Berlin Fat Mouse Inbred line with genetic predisposition for obesity.

BACKGROUND: The Berlin Fat Mouse Inbred (BFMI) line is a new mouse model for obesity, which was long-term selected for high fatness. Peroxisome proliferator-activated receptors (PPARs) are involved in the control of energy homeostasis, nutrient metabolism and cell proliferation. Here, we studied the expression patterns of the different Ppar genes and the genes in the PPAR pathway in the BFMI line in comparison to physiological changes. RESULTS: At the age of 10 weeks, the BFMI mice exhibited marked obesity with enlarged adipocytes and high serum triglycerides concentrations in comparison to the often used mouse line C57BL/6 (B6). Between these two lines, gene expression analyses revealed differentially expressed genes belonging to the PPAR pathway, in particular genes of the lipogenesis and the fatty acid transport. CONCLUSION: Surprisingly, the Ppar-α gene expression was up-regulated in liver and Ppar-γ gene expression was down-regulated in the white adipose tissue, indicating the activation of a mechanism that counteracts the rise of obesity.

High-fat diet leads to tissue-specific changes reflecting risk factors for diseases in DBA/2J mice.

The aim of this study was to characterize the responses of individual tissues to high-fat feeding as a function of mass, fat composition, and transcript abundance. We examined a panel of eight tissues [5 white adipose tissues (WAT), brown adipose tissue (BAT), liver, muscle] obtained from DBA/2J mice on either a standard breeding diet (SBD) or a high-fat diet (HFD). HFD led to weight gain, decreased insulin sensitivity, and tissue-specific responses, including inflammation, in these mice. The dietary fatty acids were partially metabolized and converted in both liver and fat tissues. Saturated fatty acids (SFA) were converted in the liver to monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA), and oleic acid (C18:1) was the preferred MUFA for storage of excess energy in all tissues of HFD-fed mice. Transcriptional changes largely reflected the tissue-specific fat deposition. SFA were negatively correlated with genes in the collagen family and processes involving the extracellular matrix. We propose a novel role of the tryptophan hydroxylase 2 (Tph2) gene in adipose tissues of diet-induced obesity. Tissue-specific responses to HFD were identified. Liver steatosis was evident in HFD-fed mice. Gonadal, retroperitoneal and subcutaneous adipose tissue and BAT exhibited severe inflammatory and immune responses. Mesenteric adipose tissue was the most metabolically active adipose tissue. Gluteal adipose tissue had the highest mass gain but was sluggish in its metabolism. In HFD conditions, BAT functioned largely like WAT in its role as a depot for excess energy, whereas WAT played a role in thermogenesis.

Circannual changes in the expression of vascular endothelial growth factor in the testis of roe deer (Capreolus capreolus).

Adult roe deer males show seasonal cycles of testicular growth and involution. The exact timing of these cycles requires endocrine regulation and local testicular control by autocrine/paracrine factors. Recent findings suggest that the vascular endothelial growth factor (VEGF) might have effects on both vascular and germinative cells in testis. Thus, we studied the expression pattern of vascular endothelial growth factor (VEGF) in roe deer testis using quantitative RT-PCR. The strength of VEGF mRNA expression depended on season. It reached its highest level at the peak of spermatogenesis during the pre-rutting period and had its nadir at the end of the rut when involution already began. The results suggested that VEGF may directly affect the regulation of spermatogenesis but may not be involved predominantly in testicular microvasculature as initially expected.

High energy digestion efficiency and altered lipid metabolism contribute to obesity in BFMI mice.

To constitute a valuable resource to identify individual genes involved in the development of obesity, a novel mouse model, the Berlin Fat Mouse Inbred line 860 (BFMI860), was established. In order to characterize energy intake and energy expenditure in obese BFMI860 mice, we performed two independent sets of experiments in male BFMI860 and B6 control mice (10 per line). In experiment 1, we analyzed body fat content noninvasively by dual-energy X-ray absorptiometry and measured resting metabolic rate at thermoneutrality (RMRt) and respiratory quotient (RQ) in week 6, 10, and 18. In a second experiment, energy digested (energy intake minus fecal energy loss) was determined by bomb calorimetry from week 6 through week 12. BFMI860 mice were heavier and had higher fat mass (final body fat content was 24.7% compared with 14.6% in B6). They also showed fatty liver syndrome. High body fat accumulation in BFMI860 mice was restricted to weeks 6-10 and was accompanied by hyperphagia, higher energy digestion, higher RQs, and abnormally high blood triglyceride levels. Lean mass-adjusted RMRt was not altered between lines. These results indicate that in BFMI860 mice, the excessive accumulation of body fat is associated with altered lipid metabolism, high energy intake, and energy digestion. Assuming that BFMI860 mice and their obese phenotypes are of polygenic nature, this line is an excellent model for the study of obesity in humans, especially for juvenile obesity and hyperlipidemia.

Genetic, sex, and diet effects on body weight and obesity in the Berlin Fat Mouse Inbred lines.

Mouse lines long-term selected for high fatness offer the possibility to identify individual genes involved in the development of obesity. The Berlin Fat Mouse (BFM) line has been selected for low protein content and afterward for high fatness. Three Berlin Fat Mouse Inbred (BFMI) lines, which are derivates of the selection line BFM and an unselected control line (C57BL/6; B6) were systematically phenotyped between 3 and 20 wk. The body weights and body compositions were measured on a weekly basis. We demonstrated that the BFMI lines dispose of more body weight, body fat mass, and body lean mass than the control line B6 because of a better feed efficiency in these lines. In contrast to other growth-selected mouse lines, the BFMI lines exhibited a general increase in body fat mass but only a marginal increase in body lean mass. The three BFMI lines also showed line- and sex-specific patterns and varied in their response to high-fat diet. The phenotypic differences between the BFMI lines can be traced back to different sets of fixed alleles contributing to fat accumulation and diet-induced obesity. Our results demonstrate that the genetically related BFMI lines are novel models to study the genetic as well as the nutritional aspects of obesity.

Differential changes in expression of a and b FGF, IGF-1 and -2, and TGF-alpha during seasonal growth and involution of roe deer testis.

Growth factors are involved in the regulation of testicular growth and involution of seasonal breeders. Therefore, we studied the seasonal expression of several growth factors in roe deer: aFGF, bFGF, IGF-1, IGF-2, and TGF-alpha. Total RNA from testis tissue was extracted monthly and analyzed using quantitative RT-PCR. Localization of mRNAs was examined by in situ-hybridization. Levels of expression differed by more than three orders of magnitude. Expression also showed different seasonal patterns. IGF-1, IGF-2 and bFGF were maximally transcribed during testis recrudescence in spring. In contrast, the mRNA amount of aFGF reached its maximum between July (breeding season) and January. TGF-alpha mRNA-levels were very low and showed poor seasonal variation. Each growth factor showed its own typical expression localization in testicular tissues and cell types. The results suggest the specific role of different growth factors in the paracrine control of spermatogenesis and its seasonal regulation.