Carsten Grötzinger

Receptor-targeted optical imaging of tumors with near-infrared fluorescent ligands.

We report here the in vivo diagnostic use of a peptide–dye conjugate consisting of a cyanine dye and the somatostatin analog octreotate as a contrast agent for optical tumor imaging. When used in whole-body in vivo imaging of mouse xenografts, indotricarbocyanine-octreotate accumulated in tumor tissue. Tumor fluorescence rapidly increased and was more than threefold higher than that of normal tissue from 3 to 24 h after application. The targeting conjugate was also specifically internalized by primary human neuroendocrine tumor cells. This imaging approach, combining the specificity of ligand/receptor interaction with near-infrared fluorescence detection, may be applied in various other fields of cancer diagnosis.

Functional peptide microarrays for specific and sensitive antibody diagnostics

Peptide microarrays displaying biologically active small synthetic peptides in a high‐density format provide an attractive technology to probe complex samples for the presence and/or function of protein analytes. We present a new approach for manufacturing functional peptide microarrays for molecular immune diagnostics. Our method relies on the efficiency of site‐specific solution‐phase coupling of biotinylated synthetic peptides to NeutrAvidin (NA) and localized microdispensing of peptide‐NA‐complexes onto activated glass surfaces. Antibodies are captured in a sandwich manner between surface immobilized peptide probes and fluorescence‐labeled secondary antibodies. Our work includes a total of 54 peptides derived from immunodominant linear epitopes of the T7 phage capsid protein, Herpes simplex virus glycoprotein D, c‐myc protein, and three domains of the Human coronavirus polymerase polyprotein and their cognate mAbs. By using spacer molecules of different type and length for NA‐mediated peptide presentation, we show that the incorporation of a minimum spacer length is imperative for antibody binding, whereas the peptide immobilization direction has only secondary importance for antibody affinity and binding. We further demonstrate that the peptide array is capable of detecting low‐picomolar concentrations of mAbs in buffered solutions and diluted human serum with high specificity.

A cross-species analysis in pancreatic neuroendocrine tumors reveals molecular subtypes with distinctive clinical, metastatic, developmental, and metabolic characteristics

UNLABELLED Seeking to assess the representative and instructive value of an engineered mouse model of pancreatic neuroendocrine tumors (PanNET) for its cognate human cancer, we profiled and compared mRNA and miRNA transcriptomes of tumors from both. Mouse PanNET tumors could be classified into two distinctive subtypes, well-differentiated islet/insulinoma tumors (IT) and poorly differentiated tumors associated with liver metastases, dubbed metastasis-like primary (MLP). Human PanNETs were independently classified into these same two subtypes, along with a third, specific gene mutation-enriched subtype. The MLP subtypes in human and mouse were similar to liver metastases in terms of miRNA and mRNA transcriptome profiles and signature genes. The human/mouse MLP subtypes also similarly expressed genes known to regulate early pancreas development, whereas the IT subtypes expressed genes characteristic of mature islet cells, suggesting different tumorigenesis pathways. In addition, these subtypes exhibit distinct metabolic profiles marked by differential pyruvate metabolism, substantiating the significance of their separate identities. SIGNIFICANCE This study involves a comprehensive cross-species integrated analysis of multi-omics profiles and histology to stratify PanNETs into subtypes with distinctive characteristics. We provide support for the RIP1-TAG2 mouse model as representative of its cognate human cancer with prospects to better understand PanNET heterogeneity and consider future applications of personalized cancer therapy.

Cyanine Dye Labeled Vasoactive Intestinal Peptide and Somatostatin Analog for Optical Detection of Gastroenteropancreatic Tumors

Many gastroenteropancreatic (GEP) tumors express vasoactive intestinal peptide (VIP) and/or somatostatin (SST) receptors in high densities. 1 These receptors can, therefore, be used as targets for the tumor directed delivery of contrast agents for detection of primary tumors as well as metastases. Radiolabeled SST analogues are used in daily clinical routine for receptor scintigraphy of neuroendocrine GEP tumors, whereas radiolabeled VIP has recently been introduced for the detection of GEP adenocarcinomas (for a review see Ref. 2). In the last few years optical (nearinfrared) imaging, a method that does not expose patients to ionizing radiation, has emerged as a new diagnostic modality for oncological applications. 3 The method is based on the detection of differences in the absorption and/or fluorescence of normal and tumor tissue. Due to the limited penetration depth of the excitation light, optical imaging is mainly applied to the detection of superficial lesions. A potential application is the detection of gastrointestinal tumors by fluorescence guided endoscopy after administration of a tumor specific, fluorescent contrast agent. We previously reported the synthesis and characterization of cyanine dyes as fluorescent contrast agents for optical imaging. 4 Conjugates consisting of a cyanine dye and a peptide that binds with high affinity to receptors on tumor cells may provide new tumor specific contrast agents for the optical detection of superficial GEP tumors.

Transient Receptor Potential Channel TRPM8 Agonists Stimulate Calcium Influx and Neurotensin Secretion in Neuroendocrine Tumor Cells

TRPM8 is a member of the melastatin-type transient receptor potential ion channel family. Activation by cold or by agonists (menthol, icilin) induces a transient rise in intracellular free calcium concentration ([Ca2+]i). Our previous study demonstrated that Ca2+-permeable cation channels play a role in IGF-1-induced secretion of chromogranin A in human neuroendocrine tumor (NET) cell line BON [Mergler et al.: Neuroendocrinology 2006;82:87–102]. Here, we extend our earlier study by investigating the expression of TRPM8 and characterizing its impact on [Ca2+]i and the secretion of neurotensin (NT). We identified TRPM8 expression in NET BON cells by RT-PCR, Western blotting and immunofluorescence staining. Icilin increased [Ca2+]i in TRPM8-transfected human embryonic kidney cells (HEK293) but not in mock-transfected cells. Icilin and menthol induced Ca2+ transients in BON cells as well as in primary NET cell cultures of two different pancreatic NETs as detected by single cell fluorescence imaging. Icilin increased non-selective cation channel currents in BON cells as detected by patch-clamp recordings. This activation was associated with increased NT secretion. Taken together, this study demonstrates for the first time the expression TRPM8 in NET cells and its role in regulating [Ca2+]i and NT secretion. The regulation of NT secretion in NETs by TRPM8 may have a potential clinical implication in diagnosis or therapy.

Capsaicin induces cytotoxicity in pancreatic neuroendocrine tumor cells via mitochondrial action.

Capsaicin (CAP), the pungent ingredient of chili peppers, inhibits growth of various solid cancers via TRPV1 as well as TRPV1-independent mechanisms. Recently, we showed that TRPV1 regulates intracellular calcium level and chromogranin A secretion in pancreatic neuroendocrine tumor (NET) cells. In the present study, we characterize the role of the TRPV1 agonist - CAP - in controlling proliferation and apoptosis of pancreatic BON and QGP-1 NET cells. We demonstrate that CAP reduces viability and proliferation, and stimulates apoptotic death of NET cells. CAP causes mitochondrial membrane potential loss, inhibits ATP synthesis and reduces mitochondrial Bcl-2 protein production. In addition, CAP increases cytochrome c and cleaved caspase 3 levels in cytoplasm. CAP reduces reactive oxygen species (ROS) generation. The antioxidant N-acetyl-l-cysteine (NAC) acts synergistically with CAP to reduce ROS generation, without affecting CAP-induced toxicity. TRPV1 protein reduction by 75% reduction fails to attenuate CAP-induced cytotoxicity. In summary, these results suggest that CAP induces cytotoxicity by disturbing mitochondrial potential, and inhibits ATP synthesis in NET cells. Stimulation of ROS generation by CAP appears to be a secondary effect, not related to CAP-induced cytotoxicity. These results justify further evaluation of CAP in modulating pancreatic NETs in vivo.

Deciphering the Antibodyome - Peptide Arrays for Serum Antibody Biomarker Diagnostics

The analysis of antibodies in human serum is an established technique in the laboratory diagnosis of infectious as well as autoimmune diseases. The multitude of antibody reactions towards pathogens and likewise the antibody profile in autoimmune diseases does contain a wealth of proteomic (antibody) data that may constitute valuable diagnostic infor- mation with relevance for the patients prognosis and response to therapy. Hence the use of antibodies as diagnostic bio- markers may be one of the most promising strategies to identify patient subgroups. The presence or absence of antibodies directed against specific epitopes could represent a serologic biomarker that is able to predict the severity of a disease and assist in medical decision making. In addition, parallel detection of many different antibodies in a serum sample would be of great value in many areas of basic immunological research. Peptide arrays displaying biologically active small syn- thetic peptides in either low, medium or high-density formats represent an attractive technology to probe complex serum samples for the presence of such antibody analytes. Holding the unique capacity to break down the heterogeneous immu- nologic response into monoclonal antibody specificities and to differentiate subtle changes in antibody abundance and specificity, the peptide array technology by far extends the diagnostic potential of any conventional serologic assay. To- gether with an unrivalled parallelity, peptide (micro)array analysis opens new perspectives for the novel use of antibodies as diagnostic biomarkers and provides unique access to a more differentiated serological diagnosis. This review recapitu- lates the development of the peptide array technology with a focus on recent advances and current state of the art plat- forms for antibody diagnostics. Latest applications of peptide arrays for the serologic diagnosis of infectious diseases, autoimmunity and allergy are discussed, and conclusions for future developments and implications are drawn.

Characterization of voltage operated R-type Ca2+ channels in modulating somatostatin receptor subtype 2-and 3-dependent inhibition of insulin secretion from INS-1 cells

Somatostatin (SST) inhibits Ca(2+) entry into pancreatic B-cells via voltage-operated Ca(2+) channels (VOCCs) of L-type, leading to the suppression of insulin secretion. Activation of R-type channels increases insulin secretion. However, the role of R-type Ca(2+) channels (Ca(V)2.3) in mediating the effects of SST on insulin secretion has not been so far investigated. Here, we identify the SST-receptor subtypes (SSTR) expressed on insulin-producing INS-1 cells by RT-PCR and by functional assays. The role of R-type channels in regulating [Ca(2+)](i) in response to SST-treatment was detected by cell fluorescence imaging and patch-clamp technique. INS-1 expressed SSTR2 and SSTR3 and agonists (ag.) selective for these receptors reduced 10 nM exendin-4/20 mM glucose-stimulated insulin secretion. Surprisingly, SST and SST2-ag. transiently increased [Ca(2+)](i). Subsequently, these agonists led to a decrease in [Ca(2+)](i) below the basal levels. In contrast, SST3-ag. failed to induce a transient peak of [Ca(2+)](i). Instead, a persistent minor suppression of [Ca(2+)](i) was detected from 25 min. R-type channel blocker SNX-482 altered [Ca(2+)](i) in SST- and SST2-ag.-treated cells. Notably, the inhibition of insulin secretion by SST and SST2-ag., but not SST3-ag. was attenuated by SNX-482. Taken together, SST and SSTR2 regulate [Ca(2+)](i) and insulin secretion in INS-1 cells via R-type channels. In contrast, the R-type calcium channel does not mediate the effects of SST3-ag. on insulin secretion. We conclude that R-type channels play a major role in the inhibition of insulin secretion by somatostatin in INS-1 cells.

Peptide microarrays with site-specifically immobilized synthetic peptides for antibody diagnostics

Abstract Peptide microarrays bear the potential to discover molecular recognition events on protein level, particularly in the field of molecular immunology, in a manner and with an efficiency comparable to the performance of DNA microarrays. We developed a novel peptide microarray platform for the detection of antibodies in liquid samples. The system comprises site-specific solution phase coupling of biotinylated peptides to NeutrAvidin, localized microdispensing of peptide–NeutrAvidin conjugates onto activated glass slides and a fluorescence immuno sandwich assay format for antibody capture and detection. Our work includes synthetic peptides deduced from amino acid sequences of immunodominant linear epitopes, such as the T7 phage capsid protein, Herpes simplex virus glycoprotein D, c-myc protein and three domains of the Human coronavirus 229E polymerase polyprotein. We demonstrate that our method produces peptide arrays with excellent spot morphology which are capable of specific and sensitive detection of monoclonal antibodies from fluid samples.

Tumour Biology of Gastroenteropancreatic Neuroendocrine Tumours

Neuroendocrine tumours of the gastroenteropancreatic tract (GEP NETs) represent a rare and heterogeneous group of tumours. Based on their ontogenetic origin, GEP NETs are classified into foregut, midgut and hindgut tumours. Although they have many features in common, their molecular backgrounds are obviously different. Elucidation of the key factors determining tumour biology has been hampered by the low incidence and high variability of these tumours in terms of origin, morphology and growth. However, recent years have shed some light on molecular genetics of these tumours, revealing important genetic factors as the RET proto-oncogene and the tumour suppressor menin as well as knowledge about the role of growth factors like IGF-1, TGF-β, VEGF and PDGF for the regulation of differentiation, growth and secretion. In the future, emerging molecular tools in rapid individual genome analysis and in proteomic and array technologies may help to delineate common patterns of NET disease.