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Neuroendocrine Phenotype and mutational status NT-3

Clinical options for systemic therapy of neuroendocrine tumors (NET) are limited. Development of new drugs requires suitable representative in vitro and in vivo model systems. So far, the unavailability of a human model with a well-differentiated phenotype and typical growth characteristics has impaired preclinical research in NET. Herein, we establish and characterize a lymph node–derived cell line (NT-3) from a male patient with well-differentiated pancreatic NET. Neuroendocrine differentiation and tumor biology was compared with existing NET cell lines BON and QGP-1. In vivo growth was assessed in a xenograft mouse model. The neuroendocrine identity of NT-3 was verified by expression of multiple NET-specific markers, which were highly expressed in NT-3 compared with BON and QGP-1. In addition, NT-3 expressed and secreted insulin. Until now, this well-differentiated phenotype is stable since 58 passages. The proliferative labeling index, measured by Ki-67, of 14.6% ± 1.0% in NT-3 is akin to the original tumor (15%–20%), and was lower than in BON (80.6% ± 3.3%) and QGP-1 (82.6% ± 1.0%). NT-3 highly expressed somatostatin receptors (SSTRs: 1, 2, 3, and 5). Upon subcutaneous transplantation of NT-3 cells, recipient mice developed tumors with an efficient tumor take rate (94%) and growth rate (139% ± 13%) by 4 weeks. Importantly, morphology and neuroendocrine marker expression of xenograft tumors resembled the original human tumor.Implications: High expression of somatostatin receptors and a well-differentiated phenotype as well as a slow growth rate qualify the new cell line as a relevant model to study neuroendocrine tumor biology and to develop new tumor treatments. Mol Cancer Res; 16(3); 496–507. ©2018 AACR.

Supplementary Methods

Myeloid cells play an essential role in the maintenance of liver homeostasis, as well as the initiation and termination of innate and adaptive immune responses. In chronic hepatic inflammation, the production of transforming growth factor beta (TGF-β) is pivotal for scarring and fibrosis induction and progression. TGF-β signalling is tightly regulated via the Smad protein family. Smad7 acts as an inhibitor of the TGF-β-signalling pathway, rendering cells that express high levels of it resistant to TGF-β-dependent signal transduction. In hepatocytes, the absence of Smad7 promotes liver fibrosis. Here, we examine whether Smad7 expression in myeloid cells affects the extent of liver inflammation, injury and fibrosis induction during chronic liver inflammation. Using the well-established model of chronic carbon tetrachloride (CCl4)-mediated liver injury, we investigated the role of Smad7 in myeloid cells in LysM-Cre Smadfl/fl mice that harbour a myeloid-specific knock-down of Smad7. We found that the chronic application of CCl4 induces severe liver injury, with elevated serum alanine transaminase (ALT)/aspartate transaminase (AST) levels, centrilobular and periportal necrosis and immune-cell infiltration. However, the myeloid-specific knock-down of Smad7 did not influence these and other parameters in the CCl4-treated animals. In summary, our results suggest that, during long-term application of CCl4, Smad7 expression in myeloid cells and its potential effects on the TGF-β-signalling pathway are dispensable for regulating the extent of chronic liver injury and inflammation.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR&gamma, ) regulates target gene expression upon ligand binding. Apart from its effects on metabolism, PPAR&gamma, activity can inhibit the production of pro-inflammatory cytokines by several immune cells, including dendritic cells and macrophages. In chronic inflammatory disease models, PPAR&gamma, activation delays the onset and ameliorates disease severity. Here, we investigated the effect of PPAR&gamma, activation by the agonist Pioglitazone on the function of hepatic immune cells and its effect in a murine model of immune-mediated hepatitis. Cytokine production by both liver sinusoidal endothelial cells (IL-6) and in T cells ex vivo (IFN&gamma, ) was decreased in cells from Pioglitazone-treated mice. However, PPAR&gamma, activation did not decrease pro-inflammatory tumor necrosis factor alpha TNF&alpha, production by Kupffer cells after Toll-like receptor (TLR) stimulation ex vivo. Most interestingly, although PPAR&gamma, activation was shown to ameliorate chronic inflammatory diseases, it did not improve hepatic injury in a model of immune-mediated hepatitis. In contrast, Pioglitazone-induced PPAR&gamma, activation exacerbated D-galactosamine (GalN)/lipopolysaccharide (LPS) hepatitis associated with an increased production of TNF&alpha, by Kupffer cells and increased sensitivity of hepatocytes towards TNF&alpha, after in vivo Pioglitazone administration. These results unravel liver-specific effects of Pioglitazone that fail to attenuate liver inflammation but rather exacerbate liver injury in an experimental hepatitis model.

// Michael Thomaschewski 1 , Kristoffer Riecken 1 , Ludmilla Unrau 1 , Tassilo Volz 2 , Kerstin Cornils 1 , Harald Ittrich 3 , Denise Heim 2 , Henning Wege 2 , Ercan Akgun 1 , Marc Lutgehetmann 2 , Jan Dieckhoff 3 , Michael Kopke 2 , Maura Dandri 2 , Daniel Benten 2, 4, * and Boris Fehse 1, * 1 Research Department of Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Center (UMC) Hamburg-Eppendorf, Hamburg, Germany 2 Department of Medicine, Gastroenterology and Hepatology, UMC Hamburg-Eppendorf, Hamburg, Germany 3 Diagnostic and Interventional Radiology, UMC Hamburg-Eppendorf, Hamburg, Germany 4 Department of Gastroenterology, Helios Klinikum Duisburg, Duisburg, Germany * These authors have contributed equally to this work Correspondence to: Boris Fehse, email: fehse@uke.de Daniel Benten, email: d.benten@helios-gesundheit.de Keywords: HCC; insertional mutagenesis; RGB marking; LeGO vectors; hTERT Received: September 15, 2017 Accepted: December 04, 2017 Published: December 14, 2017 ABSTRACT We recently introduced red-green-blue (RGB) marking for clonal cell tracking based on individual color-coding. Here, we applied RGB marking to study clonal development of liver tumors. Immortalized, non-tumorigenic human fetal hepatocytes expressing the human telomerase reverse transcriptase (FH-hTERT) were RGB-marked by simultaneous transduction with lentiviral vectors encoding mCherry, Venus, and Cerulean. Multi-color fluorescence microscopy was used to analyze growth characteristics of RGB-marked FH-hTERT in vitro and in vivo after transplantation into livers of immunodeficient mice with endogenous liver damage (uPA/SCID). After initially polyclonal engraftment we observed oligoclonal regenerative nodules derived from transplanted RGB-marked FH-hTERT. Some mice developed monochromatic invasive liver tumors; their clonal origin was confirmed both on the molecular level, based on specific lentiviral-vector insertion sites, and by serial transplantation of one tumor. Vector insertions in proximity to the proto-oncogene MCF2 and the transcription factor MITF resulted in strong upregulation of mRNA expression in the respective tumors. Notably, upregulated MCF2 and MITF expression was also observed in 21% and 33% of 24 human hepatocellular carcinomas analyzed. In conclusion, liver repopulation with RGB-marked FH-hTERT is a useful tool to study clonal progression of liver tumors caused by insertional mutagenesis in vivo and will help identifying genes involved in liver cancer.

Clinical options for systemic therapy of neuroendocrine tumors (NET) are limited. Development of new drugs requires suitable representative in vitro and in vivo model systems. So far, the unavailability of a human model with a well-differentiated phenotype and typical growth characteristics has impaired preclinical research in NET. Herein, we establish and characterize a lymph node–derived cell line (NT-3) from a male patient with well-differentiated pancreatic NET. Neuroendocrine differentiation and tumor biology was compared with existing NET cell lines BON and QGP-1. In vivo growth was assessed in a xenograft mouse model. The neuroendocrine identity of NT-3 was verified by expression of multiple NET-specific markers, which were highly expressed in NT-3 compared with BON and QGP-1. In addition, NT-3 expressed and secreted insulin. Until now, this well-differentiated phenotype is stable since 58 passages. The proliferative labeling index, measured by Ki-67, of 14.6% ± 1.0% in NT-3 is akin to the original tumor (15%–20%), and was lower than in BON (80.6% ± 3.3%) and QGP-1 (82.6% ± 1.0%). NT-3 highly expressed somatostatin receptors (SSTRs: 1, 2, 3, and 5). Upon subcutaneous transplantation of NT-3 cells, recipient mice developed tumors with an efficient tumor take rate (94%) and growth rate (139% ± 13%) by 4 weeks. Importantly, morphology and neuroendocrine marker expression of xenograft tumors resembled the original human tumor. Implications: High expression of somatostatin receptors and a well-differentiated phenotype as well as a slow growth rate qualify the new cell line as a relevant model to study neuroendocrine tumor biology and to develop new tumor treatments. Mol Cancer Res; 16(3); 496–507. ©2018 AACR.

Myeloid cells are essential for the initiation and termination of innate and adaptive immunity that create homeostasis in the liver. Smad7 is an inhibitor of the transforming growth factor &beta, (TGF-&beta, ) signaling pathway, which regulates inflammatory cellular processes. Knockdown of Smad7 in hepatocytes has been shown to promote liver fibrosis, but little is known about the effects of Smad7 in myeloid cells during inflammatory responses in the liver. Using mice with a myeloid-specific knockdown of Smad7 (LysM-Cre Smad7fl/fl), we investigated the impact of Smad7 deficiency in myeloid cells on liver inflammation and regeneration using the well-established model of CCl4-mediated liver injury. Early (24/48 h) and late (7 d) time points were analyzed. We found that CCl4 induces severe liver injury, with elevated serum ALT levels, centrilobular and periportal necrosis, infiltrating myeloid cells and an increase of inflammatory cytokines in the liver. Furthermore, as expected, inflammation peaked at 24 h and subsided after 7 d. However, the knockdown of Smad7 in myeloid cells did not affect any of the investigated parameters in the CCl4-treated animals. In summary, our results suggest that the inhibition of TGF-&beta, signaling via Smad7 expression in myeloid cells is dispensable for the induction and control of acute CCl4-induced liver injury.

The sequential activation of distinct developmental gene networks governs the ultimate identity of a cell, but the mechanisms involved in initiating downstream programs are incompletely understood. The pre-B-cell receptor (pre-BCR) is an important checkpoint of B-cell development and is essential for a pre-B cell to traverse into an immature B cell. Here, we show that activation of myocyte enhancer factor 2 (Mef2) transcription factors (TFs) by the pre-BCR is necessary for initiating the subsequent genetic network. We demonstrate that B-cell development is blocked at the pre-B-cell stage in mice deficient for Mef2c and Mef2d TFs and that pre-BCR signaling enhances the transcriptional activity of Mef2c/d through phosphorylation by the Erk5 mitogen-activating kinase. This activation is instrumental in inducing Kruppel-like factor 2 and several immediate early genes of the AP1 and Egr family. Finally, we show that Mef2 proteins cooperate with the products of their target genes (Irf4 and Egr2) to induce secondary waves of transcriptional regulation. Our findings uncover a novel role for Mef2c/d in coordinating the transcriptional network that promotes early B-cell development.