A nanobody-based tracer targeting DPP6 for non-invasive imaging of human pancreatic endocrine cells.

There are presently no reliable ways to quantify endocrine cell mass (ECM) in vivo, which prevents an accurate understanding of the progressive beta cell loss in diabetes or following islet transplantation. To address this unmet need, we coupled RNA sequencing of human pancreatic islets to a systems biology approach to identify new biomarkers of the endocrine pancreas. Dipeptidyl-Peptidase 6 (DPP6) was identified as a target whose mRNA expression is at least 25-fold higher in human pancreatic islets as compared to surrounding tissues and is not changed by proinflammatory cytokines. At the protein level, DPP6 localizes only in beta and alpha cells within the pancreas. We next generated a high-affinity camelid single-domain antibody (nanobody) targeting human DPP6. The nanobody was radiolabelled and in vivo SPECT/CT imaging and biodistribution studies were performed in immunodeficient mice that were either transplanted with DPP6-expressing Kelly neuroblastoma cells or insulin-producing human EndoC-ßH1 cells. The human DPP6-expressing cells were clearly visualized in both models. In conclusion, we have identified a novel beta and alpha cell biomarker and developed a tracer for in vivo imaging of human insulin secreting cells. This provides a useful tool to non-invasively follow up intramuscularly implanted insulin secreting cells.

Acinar phenotype is preserved in human exocrine pancreas cells cultured at low temperature: implications for lineage-tracing of ß-cell neogenesis.

The regenerative medicine field is expanding with great successes in laboratory and preclinical settings. Pancreatic acinar cells in diabetic mice were recently converted into ß-cells by treatment with ciliary neurotrophic factor (CNTF) and epidermal growth factor (EGF). This suggests that human acinar cells might become a cornerstone for diabetes cell therapy in the future, if they can also be converted into glucose-responsive insulin-producing cells. Presently, studying pancreatic acinar cell biology in vitro is limited by their high plasticity, as they rapidly lose their phenotype and spontaneously transdifferentiate to a duct-like phenotype in culture. We questioned whether human pancreatic acinar cell phenotype could be preserved in vitro by physico-chemical manipulations and whether this could be valuable in the study of ß-cell neogenesis. We found that culture at low temperature (4°C) resulted in the maintenance of morphological and molecular acinar cell characteristics. Specifically, chilled acinar cells did not form the spherical clusters observed in controls (culture at 37°C), and they maintained high levels of acinar-specific transcripts and proteins. Five-day chilled acinar cells still transdifferentiated into duct-like cells upon transfer to 37°C. Moreover, adenoviral-mediated gene transfer evidenced an active Amylase promoter in the 7-day chilled acinar cells, and transduction performed in chilled conditions improved acinar cell labelling. Together, our findings indicate the maintenance of human pancreatic acinar cell phenotype at low temperature and the possibility to efficiently label acinar cells, which opens new perspectives for the study of human acinar-to-ß-cell transdifferentiation.

A standardized method for in vivo mouse pancreas imaging and semiquantitative ß cell mass measurement by dual isotope SPECT.

PURPOSE: In order to evaluate future ß cell tracers in vivo, we aimed to develop a standardized in vivo method allowing semiquantitative measurement of a prospective ß cell tracer within the pancreas. PROCEDURES: 2-[(123)I]Iodo-L-phenylalanine ([(123)I]IPA) and [Lys(40)([(111)In]DTPA)]exendin-3 ([(111)In]Ex3) pancreatic uptake and biodistribution were evaluated using SPECT, autoradiography, and an ex vivo biodistribution study in a controlled unilaterally nephrectomized mouse ß cell depletion model. Semiquantitative measurement of the imaging results was performed using [(123)I]IPA to delineate the pancreas and [(111)In]Ex3 as a ß cell tracer. RESULTS: The uptake of [(123)I]IPA was highest in the pancreas. Aside from the kidneys, the uptake of [(111)In]Ex3 was highest in the pancreas and lungs. Autoradiography showed only uptake of [(111)In]Ex3 in insulin-expressing cells. Semiquantitative measurement of [(111)In]Ex3 in the SPECT images based on the delineation of the pancreas with [(123)I]IPA showed a high correlation with the [(111)In]Ex3 uptake data of the pancreas obtained by dissection. A strong positive correlation was observed between the relative insulin positive area and the pancreas-to-blood ratios of [(111)In]Ex3 uptake as determined by counting with a gamma counter and the semiquantitative analysis of the SPECT images. CONCLUSIONS: [(123)I]IPA is a promising tracer to delineate pancreatic tissue on SPECT images. It shows a high uptake in the pancreas as compared to other abdominal tissues. This study also demonstrates the feasibility and accuracy to measure the ß cell mass in vivo in an animal model of diabetes.

Phenylpropenoic acid glucoside augments pancreatic beta cell mass in high-fat diet-fed mice and protects beta cells from ER stress-induced apoptosis.

SCOPE: A major goal of diabetes therapy is to identify novel drugs that preserve or expand pancreatic beta cell mass. Here, we examined the effect of a phenylpropenoic acid glucoside (PPAG) on the beta cell mass, and via which mechanism this effect is established. METHODS AND RESULTS: Mice were fed a high-fat and fructose-containing diet to induce obesity and hyperglycemia. PPAG treatment protected obese mice from diet-induced hyperglycemia and resulted in a tripling of beta cell mass. The effect of the phytochemical on beta cell mass was neither due to increased proliferation, as determined by Ki67 immunostaining, nor to neogenesis, which was assessed by genetic lineage tracing. TUNEL staining revealed suppressed apoptosis in PPAG-treated obese mice. In vitro, PPAG protected beta cells from palmitate-induced apoptosis. It protected beta cells against ER stress by increasing expression of antiapoptotic B-cell lymphoma 2 (BCL2) protein without affecting proapoptotic signals. CONCLUSIONS: We identified an antidiabetic phytochemical that protects pancreatic beta cells from ER stress and apoptosis induced by high-fat diet/lipotoxicity. At the tissue level, this led to a tripling of beta cell mass. At the molecular level, the protective effect of the phytochemical was mediated by increasing BCL2 expression in beta cells.

Specific targeting of atherosclerotic plaques in ApoE(-/-) mice using a new Camelid sdAb binding the vulnerable plaque marker LOX-1.

PURPOSE: Molecular imaging has the potential to provide quantitative information about specific biological aspects of developing atherosclerotic lesions. This requires the generation of reliable, highly specific plaque tracers. This study reports a new camelid single-domain antibody fragment (sdAb) targeting the Lectin-like oxidized low-density lipoprotein receptor (LOX-1), a biomarker for the detection and molecular phenotyping of vulnerable atherosclerotic plaques. PROCEDURES: A camelid sdAb was generated and selected for high affinity binding to LOX-1. Ex vivo biodistribution and in vivo single photon emission computed tomography (SPECT)/computed tomography (CT) imaging studies were performed in wild-type mice and in fat-fed atherosclerotic apolipoprotein E-deficient mice with (99m)Tc-labeled sdAbs. Gamma-counting and autoradiography analyses were performed on dissected aorta segments with different degrees of plaque burden. The specificity of the LOX-1-targeting sdAb was evaluated by blocking with unlabeled sdAb or by comparison with a nontargeting (99m)Tc-labeled control sdAb. RESULTS: We generated a sdAb binding LOX-1 with a KD of 280 pM ± 62 pM affinity. After (99m)Tc-labeling, the tracer had radiochemical purity higher then 99 % and retained specificity in in vitro binding studies. Tracer blood clearance was fast with concomitant high kidney retention. At 3 h after injection, uptake in tissues other than plaques was low and not different than background, suggesting a restricted expression pattern of LOX-1. Conversely, uptake in aortic segments increased with plaque content and was due to specific LOX-1 binding. In vivo SPECT/CT imaging 160 min after injection in atherosclerotic mice confirmed specific targeting of LOX-1-expressing aortic plaques. CONCLUSIONS: The LOX-sdAb specifically targets LOX-1-expressing atherosclerotic plaques within hours after injection. The possibility to image LOX-1 rapidly after administration combined with the favourable biodistribution of a sdAb are beneficial for molecular phenotyping of atherosclerotic plaques and the generation of a future prognostic tracer.

Lineage tracing of pancreatic stem cells and beta cell regeneration.

Restoring a functional ß cell mass in diabetes patients by ß cell transplantation or stimulation of ß cell regeneration are promising approaches. It requires knowledge on the mechanisms of ß cell neogenesis, an issue that is still quite controversial. Postnatal islet regeneration may or may not depend on an influx of new islet cells from adult progenitors. To solve this issue in animal models, genetic lineage tracing has become a crucial research method. This method allows to test the various hypotheses that have been proposed concerning ß cell neogenesis and regeneration.

Genetic lineage tracing of beta cell neogenesis.

Genetic lineage tracing is an invaluable tool to demonstrate and measure neogenesis of beta cells from putative precursor cells. Cre-Lox recombination technology can be used for indelible labeling of a cohort of cells and following the fate of these cells and their progeny in animal models. Here, the combination is described of beta-galactosidase enzymatic staining with immunohistochemical staining to demonstrate labeled cells. This technique is performed in tissue cryosections.