Biology subjects

Niederstadt, L.

Publications and source records attributed to Niederstadt, L..

3 recordsLinked to original sources

bioRxiv2022

Secretin receptor as a target in gastrointestinal cancer: expression analysis and ligand development

Secretin was originally discovered as a gastrointestinal peptide that stimulates fluid secretion from pancreas and liver and delays gastric emptying. In disease, secretin receptor (SCTR) was found to occur as a splice variant in gastrinoma and pancreatic adenocarcinoma. Overexpression of SCTR has been described for gastrinomas, carcinoid tumors of the lung and cholangiocarcinoma. SCTR therefore remains a candidate target for molecular tumor imaging as well as for peptide receptor radioligand therapy (PRRT) in a number of oncological indications. The aim of this study was to characterize SCTR expression in esophageal and pancreatic cancer. 65 of 70 PDAC tissues stained strongly positive for SCTR in immunohistochemistry, as were most of 151 esophageal cancer samples, with minor influence of grading in both entities. In addition, the aim of this study was to further delineate residues in human secretin that are critical for binding to and activation of the human SCTR. For a potential development of short and metabolically stable analogs for clinical use, it was intended to probe the peptide for its capacity to incorporate deletions and substitutions without losing its affinity to SCTR. A library of 146 secretin variants containing single amino acid substitutions as well as truncations on either end was tested using {beta}-arrestin2-GFP translocation and fluorescent ligand internalization high-content analysis and cAMP assays run in agonist and antagonist mode as well as a radioligand binding. The main structural determinants of SCTR binding and activation were localized to the N-terminus, with His1, Asp3 being among the most sensitive positions, followed by Phe6, Thr7 and Leu10. Aminoterminal truncation caused a rapid decline in receptor activity and most of these variants proved to be partial agonists showing antagonistic properties. In this study, the most potent antagonist showed an IC50 of 309 {+/-} 74 nM in the {beta}-arrestin2-GFP translocation assay on human SCTR while remaining a weak partial agonist. Future studies will have to demonstrate the utility of further enhanced secretin analogues as tracers for in vivo imaging and therapy.

cancer biology↗

bioRxiv2019

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

Molecular targeting remains to be a promising approach in cancer medicine. Knowledge about molecular properties such as overexpression of G protein-coupled receptors (GPCRs) is thereby offering a powerful tool for tumor-selective imaging and treatment of cancer cells. We utilized chemerin-based peptides for CMKLR1 receptor targeting in a breast cancer xenograft model. By conjugation with radiolabeled chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), we obtained a family of highly specific and affine tracers for hybrid in vivo imaging with positron emission tomography (PET)/ magnetic resonance (MR) and concomitant biodistribution studies.\n\nMethodsWe developed five highly specific and affine peptide tracers targeting CMKLR1 by linker-based conjugation of chemerin peptide analogs (CG34 and CG36) with radiolabeled (68Ga) chelator DOTA. Our established xenograft model with target-positive DU4475 and negative A549 tumors in immunodeficient nude mice enabled CMKLR1-specific imaging in vivo. Therefore, we acquired small animal PET/MR images, assessed biodistribution by ex vivo measurements and investigated the tracer specificity by blocking experiments.\n\nResultsThe family of five CMKLR1-targeting peptide tracers demonstrated high biological activity and affinity in vitro with EC50 and IC50 values being below 2 nM. Our target-positive (DU4475) and target-negative (A549) xenograft model could be confirmed by ex vivo analysis of CMKLR1 expression and binding. After preliminary PET imaging, the three most promising tracers 68Ga-DOTA-AHX-CG34, 68Ga-DOTA-KCap-CG34 and 68Ga-DOTA-ADX-CG34 with apparent DU4475 tumor uptake were further analyzed. Hybrid PET/MR imaging along with concomitant biodistribution studies revealed distinct CMKLR1-specific uptake (5.1% IA/g, 4.5% IA/g and 6.2% IA/g 1 h post-injection) of our targeted tracers in DU4475 tumor tissue. More strikingly, the tumor uptake could be blocked by excess of unlabeled peptide (6.4-fold, 7.2-fold and 3.4-fold 1 h post-injection) and further confirmed the CMKLR1 specificity. As our five tracers, each with particular degree of hydrophobicity, showed different results regarding tumor uptake and organ distribution, we identified these three tracers with moderate, balanced properties to be the most potent in receptor-mediated tumor targeting.\n\nConclusionWith the breast cancer cell line DU4475, we established a model endogenously expressing our target CMKLR1 to evaluate our chemerin-based peptide tracers as highly affine and specific targeting agents. Eventually, we demonstrated the applicability of our 68Ga-labeled tracers by visualizing CMKLR1-positive breast cancer xenografts in PET/MR imaging and thus developed promising theranostics for tumor treatment.

cancer biology↗

bioRxiv2019

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

PurposeMelanocortin receptor 1 is overexpressed in melanoma and may be a molecular target for imaging and peptide receptor radionuclide therapy. 68Gallium labeling of DOTA-conjugated peptides is an established procedure in the clinic for use in positron emission tomography imaging. Aim of this study was to compare a standard labeling protocol against the 68Ga-DOTA peptide purified from the excess of unlabeled peptide. ProceduresThe 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 {degrees}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. ResultsIn biodistribution studies, the 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 a decreased specific tumor uptake, similar to the result seen with non-purified radiopeptide. PET imaging was performed using the same tracers for biodistribution. Purified 68Ga-DOTA-NAPamide, in comparison, showed superior tumor uptake. ConclusionsWe 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.

cancer biology↗