Biology subjects

Groetzinger, C.

Publications and source records attributed to Groetzinger, C..

8 recordsLinked to original sources

bioRxiv2023

Is the neuropeptide PEN a ligand of GPR83?

G protein-coupled receptor 83 (GPR83) is a class A G protein-coupled receptor with predominant expression in the cerebellum and proposed function in the regulation of food intake and in anxiety-like behavior. The neuropeptide PEN has been suggested as a specific GPR83 ligand. However, conflicting reports exist about whether PEN is indeed able to bind and activate GPR83. This study was initiated to evaluate PEN as a potential ligand of GPR83. Employing several second-messenger and other GPCR activation assays as well as a radioligand binding assay, and using multiple GPR83 plasmids and PEN peptides from different sources, no experimental evidence was found to support a role of PEN as a GPR83 ligand.

pharmacology and toxicology↗

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↗

bioRxiv2021

A chemerin peptide analog stimulates tumor growth in two xenograft mouse models of human colorectal carcinoma

BackgroundChemerin plasma concentration has been reported to be positively correlated with the risk of colorectal cancer. However, the potential regulation of CRC tumorigenesis and progression has not yet been investigated in an experimental setting. This study addresses this hypothesis by investigating proliferation, colony formation and migration of CRC cell lines in vitro as well as in animal models. MethodsIn vitro, microscopic assays to study proliferation as well as a scratch-wound assay for migration monitoring were applied using the human CRC cell lines HCT116, HT29 and SW620 under the influence of the chemerin analog CG34. The animal study investigated HCT116-luc and HT29-luc subcutaneous tumor size and bioluminescence during treatment with CG34 versus control, followed by ex-vivo analysis of vessel density and mitotic activity. ResultsWhile proliferation of the three CRC cell lines in monolayers was not clearly stimulated by CG34, the chemerin analog promoted colony formation in three-dimensional aggregates. An effect on cell migration was not observed. In the treatment study, CG34 significantly stimulated both growth and bioluminescence signal of HCT116-luc and HT29-luc xenografts. ConclusionsThe results of this study represent the first indication of a tumor growth-stimulating effect of chemerin signaling in CRC.

cancer biology↗

bioRxiv2021

SPECT/CT imaging, biodistribution and radiation dosimetry of a 177Lu-DOTA-integrin αvβ6 cystine knot peptide in a pancreatic cancer xenograft model

IntroductionPancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignant neoplasms, as many cases go undetected until they reach an advanced stage. Integrin v{beta}6 is a cell surface receptor overexpressed in PDAC. Consequently, it may serve as a target for the development of probes for imaging diagnosis and radioligand therapy. Engineered cystine knottin peptides specific for integrin v{beta}6 have recently been developed showing high affinity and stability. This study aimed to evaluate an integrin v{beta}6-specific knottin molecular probe containing the therapeutic radionuclide 177Lu for targeting of PDAC. MethodsThe expression of integrin v{beta}6 in PDAC cell lines BxPC3 and Capan2 was analyzed using RT-qPCR and immunofluorescence. In vitro competition and saturation radioligand binding assays were performed to calculate the binding affinity of the DOTA-coupled tracer loaded with and without lutetium to BxPC3 and Capan2 cell lines. To evaluate tracer accumulation in the tumor and organs, SPECT/CT, biodistribution and dosimetry projections were carried out using a Capan2 xenograft tumor mouse model. ResultsRT-qPCR and immunofluorescence results showed high expression of integrin v{beta}6 in BxPC3 and Capan2 cells. A competition binding assay revealed high affinity of the tracer with IC50 values of 1.69 nM and 9.46 nM for BxPC3 and Capan2, respectively. SPECT/CT and biodistribution analysis of the conjugate 177Lu-DOTA-integrin v{beta}6 knottin demonstrated accumulation in Capan2 xenograft tumors (3.13 {+/-} 0.63 %IA/g at day 1 post injection) with kidney uptake at 19.2 {+/-} 2.5 %IA/g, declining much more rapidly than in tumors. Conclusion177Lu-DOTA-integrin v{beta}6 knottin was found to be a high-affinity tracer for PDAC tumors with considerable tumor accumulation and moderate, rapidly declining kidney uptake. These promising results warrant a preclinical treatment study to establish therapeutic efficacy.

cancer biology↗

bioRxiv2020

mTOR inhibitors as radiosensitizers in neuroendocrine neoplasms

Peptide receptor radioligand therapy (PRRT) has evolved as an important second-line treatment option in the management of inoperable and metastatic neuroendocrine neoplasms (NEN). Though high radiation doses can be delivered to the tumors, complete remission is still rare. Radiosensitization prior to PRRT is therefore considered to be a promising strategy to improve the treatment effect. In this study, effect and mechanism of mTOR inhibitors were investigated in a comprehensive panel of five NEN cell lines (BON, QGP-1, LCC-18, H727, UMC-11), employing assays for cellular proliferation, clonogenic survival, cell cycle modification and signaling. mTOR inhibition lead to growth arrest with a biphasic concentration-response pattern: a partial response at approximately 1 nM and full response at micromolar concentrations (8-48 μM). All cell lines demonstrated elevated p70S6K phosphorylation yet also increased phosphorylation of counterregulatory Akt. The pulmonary NEN cell line UMC-11 showed the lowest induction of phospho-Akt and strongest growth arrest by mTOR inhibitors. Radiation sensitivity of the cells (50% reduction versus control) was found to range between 4 and 8 Gy. Further, mTOR inhibition was employed together with irradiation to evaluate radiosensitizing effects of this combination treatment. mTOR inhibition was found to radiosensitize all five NEN cells in an additive manner with a moderate overall effect. The radiation-induced G2/M arrest was diminished under combination treatment, leading to an increased G1 arrest. Further investigation involving a suitable animal model as well as radioligand application such as 177Lu-DOTATATE or 177Lu-DOTATOC will have to demonstrate the full potential of this strategy for radiosensitization in NEN.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cancer biology↗

bioRxiv2019

AGTR1 is overexpressed in neuroendocrine neoplasms, regulates secretion and may serve as a target for molecular imaging and therapy

Peptide receptor targeting has proven to be a pivotal tool for diagnostic imaging and radioligand therapy of neuroendocrine neoplasms (NENs), which frequently express somatostatin receptors (SSTRs) on their cell surface. However, up to 30 % of NEN patients do not benefit from SSTR-based approaches, others develop a resistance. Consequently, alternative cell surface targets need to be identified. In this study, cell-based dynamic mass redistribution and calcium mobilization screening using a 998-compound library identified and confirmed angiotensin II (ATII) as a strong activator of cellular signaling in NEN cells. Expression analyses of the ATII receptor type 1 (AGTR1) revealed an upregulation of both mRNA levels (RT-qPCR) and radioligand binding (autoradiography) in pancreatic (n=42) and small-intestinal (n=71) NEN tissues compared to healthy controls (n=25). The two NEN cell lines BON (pancreas) and H727 (lung) with elevated AGTR1 expression exhibited concentration-dependent calcium mobilization and chromogranin A secretion upon stimulation with ATII, blocked by AGTR1 antagonism and Gq inhibition. To assess the applicability of AGTR1 for optical in vivo imaging, the receptor ligand saralasin was coupled to the near-infrared dye indotricarbocyanine and tested for its biodistribution in a NMRI Foxn1nu/Foxn1nu mouse model bearing AGTR1-positive BON and negative QGP-1 xenograft tumors. Near-infrared fluorescent imaging showed a significantly higher uptake in BON tumors 3-6 hours after injection. This successful targeting in an NEN model establishes AGTR1 as an interesting target in this tumor entity, paving the way for the development of translational chelator-based probes for diagnostic PET imaging and peptide receptor radioligand 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↗