Tumor cell integrin ß4 and tumor stroma E-/P-selectin cooperatively regulate tumor growth in vivo.

BACKGROUND: The immunological composition of the tumor microenvironment has a decisive influence on the biological course of cancer and is therefore of profound clinical relevance. In this study, we analyzed the cooperative effects of integrin ß4 (ITGB4) on tumor cells and E-/P-selectin on endothelial cells within the tumor stroma for regulating tumor growth by shaping the local and systemic immune environment. METHODS: We used several preclinical mouse models for different solid human cancer types (xenograft and syngeneic) to explore the role of ITGB4 (shRNA-mediated knockdown in tumor cells) and E-/P-selectins (knockout in mice) for tumor growth; effects on apoptosis, proliferation and intratumoral signaling pathways were determined by histological and biochemical methods and 3D in vitro experiments; changes in the intratumoral and systemic immune cell composition were determined by flow cytometry and immunohistochemistry; chemokine levels and their attracting potential were measured by ELISA and 3D invasion assays. RESULTS: We observed a very robust synergism between ITGB4 and E-/P-selectin for the regulation of tumor growth, accompanied by an increased recruitment of CD11b+ Gr-1Hi cells with low granularity (i.e., myeloid-derived suppressor cells, MDSCs) specifically into ITGB4-depleted tumors. ITGB4-depleted tumors undergo apoptosis and actively attract MDSCs, well-known to promote tumor growth in several cancers, via increased secretion of different chemokines. MDSC trafficking into tumors crucially depends on E-/P-selectin expression. Analyses of clinical samples confirmed an inverse relationship between ITGB4 expression in tumors and number of tumor-infiltrating leukocytes. CONCLUSIONS: These findings suggest a distinct vulnerability of ITGB4Lo tumors for MDSC-directed immunotherapies.

Pioglitazone-Mediated Peroxisome Proliferator-Activated Receptor γ Activation Aggravates Murine Immune-Mediated Hepatitis.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) regulates target gene expression upon ligand binding. Apart from its effects on metabolism, PPARγ activity can inhibit the production of pro-inflammatory cytokines by several immune cells, including dendritic cells and macrophages. In chronic inflammatory disease models, PPARγ activation delays the onset and ameliorates disease severity. Here, we investigated the effect of PPARγ 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 (IFNγ) was decreased in cells from Pioglitazone-treated mice. However, PPARγ activation did not decrease pro-inflammatory tumor necrosis factor alpha TNFα production by Kupffer cells after Toll-like receptor (TLR) stimulation ex vivo. Most interestingly, although PPARγ 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 PPARγ activation exacerbated D-galactosamine (GalN)/lipopolysaccharide (LPS) hepatitis associated with an increased production of TNFα by Kupffer cells and increased sensitivity of hepatocytes towards TNFα 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.

Establishment of the First Well-differentiated Human Pancreatic Neuroendocrine Tumor Model.

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.

Multi-color RGB marking enables clonality assessment of liver tumors in a murine xenograft model.

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.