Overview of Genetically Engineered Mouse Models of Papillary and Anaplastic Thyroid Cancers: Enabling Translational Biology for Patient Care Improvement.

The prognosis from thyroid cancer subtypes in humans covers a spectrum from "cured at almost 90%" to "100% lethal." Invasive and poorly differentiated forms of thyroid cancer are among the most aggressive human cancers, and there are few effective therapeutic options. Genetically engineered mice, based on mutations observed in patients, can accurately recapitulate the human disease and its progression, providing invaluable tools for the preclinical evaluation of novel therapeutic approaches. This overview details models developed to date as well as their uses for identifying novel anticancer agents. © 2015 by John Wiley & Sons, Inc.

Genetically engineered silk and genipin-enhanced fibrin hydrogel for annulus fibrosus repair

INTRODUCTION: Around 80% of people are affected by low back pain at least once in their life, often caused by trauma provoking intervertebral disc (IVD) herniation and/or IVD degeneration. Apart from some promising approaches for nucleus pulposus repair, so far no treatment or repair is available for the outer fibrous tissue, annulus fibrosus (AF). We aimed for sealing and repairing an AF injury in a bovine IVD organ culture model in vitro over 14 days under different loading conditions. For this purpose, a silk fleece composite from Bombyx mori silk was combined with genipin-enhanced fibrin hydrogel [1]. METHODS: Bovine IVDs of 12-17 months old animals were isolated by first removing all surrounding tissue, followed by cutting out the IVDs [2]. Culturing of discs occurred in high glucose Dulbecco's Modified Eagle Medium (HG-DMEM) supplemented with 5% serum as previously described. On the next day, injury was induced using a 2mm biopsy punch (Polymed, Switzerland). The formed cavity was filled with (0.4%) genipin-enhanced human based fibrin hydrogel (35- 55mg/mL human fibrinogen, Baxter, Austria) and sealed with a silk fleece-membrane composite (Spintec Engineering, Germany). Different culture conditions were applied: free swelling, static diurnal load of 0.2MPa for 8h/d and complex loading at 0.2MPa compression combined with ± 2° torsion at 0.2Hz for 8h/d. Complex loading was applied by a custom built 2 degree of freedom bioreactor [3]. After 14 days of culture cell activity was determined with resazurin assay. Additionally, glycosaminoglycan (dimethyl-methylene blue), DNA (Hoechst) and collagen content (hydroxy-proline) were determined. Finally, real-time qPCR of major IVD marker genes was performed. RESULTS: The silk seal closing the injury site could successfully withstand the forces of all three loading conditions with no misplacement over the two weeks’ culture. Nevertheless, disc height of the repaired discs did not significantly differ from the injured group. The disc phenotype could be maintained as demonstrated by biochemical analysis of gene expression, cell activity, DNA-, collagen- and GAG content. The silk itself was evaluated to be highly biocompatible for hMSC, as revealed by cytotoxicity assays. DISCUSSION & CONCLUSIONS: The silk can be considered a highly-elastic and biocompatible material for AF closure and the genipin-enhanced fibrin hydrogel has also good biomechanical properties. However, the cyto-compatibility of genipin seems rather poor and other hydrogels and/or cross-linkers should be looked into. REFERENCES: 1 C.C. Guterl et al. (2014) Characterization of Mechanics and Cytocompatibility of Fibrin Genipin Annulus Fibrosus Sealant with the Addition of Cell Adhesion Molecules, Tissue Eng Part A 2 S.C. Chan, B. Gantenbein-Ritter (2012) Preparation of intact bovine tail intervertebral discs for organ culture, J Vis Exp 3 B Gantenbein et al. (2015) Organ Culture Bioreactors - Platforms to Study Human Intervertebral Disc Degeneration and Regenerative Therapy, Curr Stem Cell Res Ther [epub ahead of print] ACKNOWLEDGEMENTS: This project is supported by the Gebert Rüf Stiftung project # GRS-028/13.

The role of the interplay between polymer architecture and bacterial surface properties on the microbial adhesion to polyoxazoline-based ultrathin films

Surface platforms were engineered from poly(L-lysine)-graft-poly(2-methyl-2-oxazoline) (PLL-g-PMOXA) copolymers to study the mechanisms involved in the non-specific adhesion of Escherichia coli (E. coli) bacteria. Copolymers with three different grafting densities (PMOXA chains/Lysine residue of 0.09, 0.33 and 0.56) were synthesized and assembled on niobia (Nb O ) surfaces. PLL-modified and bare niobia surfaces served as controls. To evaluate the impact of fimbriae expression on the bacterial adhesion, the surfaces were exposed to genetically engineered E. coli strains either lacking, or constitutively expressing type 1 fimbriae. The bacterial adhesion was strongly influenced by the presence of bacterial fimbriae. Non-fimbriated bacteria behaved like hard, charged particles whose adhesion was dependent on surface charge and ionic strength of the media. In contrast, bacteria expressing type 1 fimbriae adhered to the substrates independent of surface charge and ionic strength, and adhesion was mediated by non-specific van der Waals and hydrophobic interactions of the proteins at the fimbrial tip. Adsorbed polymer mass, average surface density of the PMOXA chains, and thickness of the copolymer films were quantified by optical waveguide lightmode spectroscopy (OWLS) and variable-angle spectroscopic ellipsometry (VASE), whereas the lateral homogeneity was probed by time-of-flight secondary ion mass spectrometry (ToF-SIMS). Streaming current measurements provided information on the charge formation of the polymer-coated and the bare niobia surfaces. The adhesion of both bacterial strains could be efficiently inhibited by the copolymer film only with a grafting density of 0.33 characterized by the highest PMOXA chain surface density and a surface potential close to zero.

Inflammation induces hemorrhage in thrombocytopenia

The role of platelets in hemostasis is to produce a plug to arrest bleeding. During thrombocytopenia, spontaneous bleeding is seen in some patients but not in others; the reason for this is unknown. Here, we subjected thrombocytopenic mice to models of dermatitis, stroke, and lung inflammation. The mice showed massive hemorrhage that was limited to the area of inflammation and was not observed in uninflamed thrombocytopenic mice. Endotoxin-induced lung inflammation during thrombocytopenia triggered substantial intra-alveolar hemorrhage leading to profound anemia and respiratory distress. By imaging the cutaneous Arthus reaction through a skin window, we observed in real time the loss of vascular integrity and the kinetics of skin hemorrhage in thrombocytopenic mice. Bleeding-observed mostly from venules-occurred as early as 20 minutes after challenge, pointing to a continuous need for platelets to maintain vascular integrity in inflamed microcirculation. Inflammatory hemorrhage was not seen in genetically engineered mice lacking major platelet adhesion receptors or their activators (alphaIIbbeta3, glycoprotein Ibalpha [GPIbalpha], GPVI, and calcium and diacylglycerol-regulated guanine nucleotide exchange factor I [CalDAG-GEFI]), thus indicating that firm platelet adhesion was not necessary for their supporting role. While platelets were previously shown to promote endothelial activation and recruitment of inflammatory cells, they also appear indispensable to maintain vascular integrity in inflamed tissue. Based on our observations, we propose that inflammation may cause life-threatening hemorrhage during thrombocytopenia.

BRCA2-Deficient Sarcomatoid Mammary Tumors Exhibit Multidrug Resistance.

Pan- or multidrug resistance is a central problem in clinical oncology. Here, we use a genetically engineered mouse model of BRCA2-associated hereditary breast cancer to study drug resistance to several types of chemotherapy and PARP inhibition. We found that multidrug resistance was strongly associated with an EMT-like sarcomatoid phenotype and high expression of the Abcb1b gene, which encodes the drug efflux transporter P-glycoprotein. Inhibition of P-glycoprotein could partly resensitize sarcomatoid tumors to the PARP inhibitor olaparib, docetaxel, and doxorubicin. We propose that multidrug resistance is a multifactorial process and that mouse models are useful to unravel this. Cancer Res; 75(4); 732-41. ©2014 AACR.

Minimal residual disease in cancer therapy - Small things make all the difference

Minimal residual disease (MRD) is a major hurdle in the eradication of malignant tumors. Despite the high sensitivity of various cancers to treatment, some residual cancer cells persist and lead to tumor recurrence and treatment failure. Obvious reasons for residual disease include mechanisms of secondary therapy resistance, such as the presence of mutant cells that are insensitive to the drugs, or the presence of cells that become drug resistant due to activation of survival pathways. In addition to such unambiguous resistance modalities, several patients with relapsing tumors do not show refractory disease and respond again when the initial therapy is repeated. These cases cannot be explained by the selection of mutant tumor cells, and the precise mechanisms underlying this clinical drug resistance are ill-defined. In the current review, we put special emphasis on cell-intrinsic and -extrinsic mechanisms that may explain mechanisms of MRD that are independent of secondary therapy resistance. In particular, we show that studying genetically engineered mouse models (GEMMs), which highly resemble the disease in humans, provides a complementary approach to understand MRD. In these animal models, specific mechanisms of secondary resistance can be excluded by targeted genetic modifications. This allows a clear distinction between the selection of cells with stable secondary resistance and mechanisms that result in the survival of residual cells but do not provoke secondary drug resistance. Mechanisms that may explain the latter feature include special biochemical defense properties of cancer stem cells, metabolic peculiarities such as the dependence on autophagy, drug-tolerant persisting cells, intratumoral heterogeneity, secreted factors from the microenvironment, tumor vascularization patterns and immunosurveillance-related factors. We propose in the current review that a common feature of these various mechanisms is cancer cell dormancy. Therefore, dormant cancer cells appear to be an important target in the attempt to eradicate residual cancer cells, and eventually cure patients who repeatedly respond to anticancer therapy but lack complete tumor eradication.

Selective resistance to the PARP inhibitor olaparib in a mouse model for BRCA1-deficient metaplastic breast cancer

Metaplastic breast carcinoma (MBC) is a rare histological breast cancer subtype characterized by mesenchymal elements and poor clinical outcome. A large fraction of MBCs harbor defects in breast cancer 1 (BRCA1). As BRCA1 deficiency sensitizes tumors to DNA cross-linking agents and poly(ADP-ribose) polymerase (PARP) inhibitors, we sought to investigate the response of BRCA1-deficient MBCs to the PARP inhibitor olaparib. To this end, we established a genetically engineered mouse model (GEMM) for BRCA1-deficient MBC by introducing the MET proto-oncogene into a BRCA1-associated breast cancer model, using our novel female GEMM ES cell (ESC) pipeline. In contrast to carcinomas, BRCA1-deficient mouse carcinosarcomas resembling MBC show intrinsic resistance to olaparib caused by increased P-glycoprotein (Pgp) drug efflux transporter expression. Indeed, resistance could be circumvented by using another PARP inhibitor, AZD2461, which is a poor Pgp substrate. These preclinical findings suggest that patients with BRCA1-associated MBC may show poor response to olaparib and illustrate the value of GEMM-ESC models of human cancer for evaluation of novel therapeutics.

A synthetic biology-based device prevents liver injury in mice

BACKGROUND & AIMS The liver performs a panoply of complex activities coordinating metabolic, immunologic and detoxification processes. Despite the liver's robustness and unique self-regeneration capacity, viral infection, autoimmune disorders, fatty liver disease, alcohol abuse and drug-induced hepatotoxicity contribute to the increasing prevalence of liver failure. Liver injuries impair the clearance of bile acids from the hepatic portal vein which leads to their spill over into the peripheral circulation where they activate the G-protein-coupled bile acid receptor TGR5 to initiate a variety of hepatoprotective processes. METHODS By functionally linking activation of ectopically expressed TGR5 to an artificial promoter controlling transcription of the hepatocyte growth factor (HGF), we created a closed-loop synthetic signalling network that coordinated liver injury-associated serum bile acid levels to expression of HGF in a self-sufficient, reversible and dose-dependent manner. RESULTS After implantation of genetically engineered human cells inside auto-vascularizing, immunoprotective and clinically validated alginate-poly-(L-lysine)-alginate beads into mice, the liver-protection device detected pathologic serum bile acid levels and produced therapeutic HGF levels that protected the animals from acute drug-induced liver failure. CONCLUSIONS Genetically engineered cells containing theranostic gene circuits that dynamically interface with host metabolism may provide novel opportunities for preventive, acute and chronic healthcare. LAY SUMMARY Liver diseases leading to organ failure may go unnoticed as they do not trigger any symptoms or significant discomfort. We have designed a synthetic gene circuit that senses excessive bile acid levels associated with liver injuries and automatically produces a therapeutic protein in response. When integrated into mammalian cells and implanted into mice, the circuit detects the onset of liver injuries and coordinates the production of a protein pharmaceutical which prevents liver damage.

Biomechanical effects of environmental and engineered particles on human airway smooth muscle cells

The past decade has seen significant increases in combustion-generated ambient particles, which contain a nanosized fraction (less than 100 nm), and even greater increases have occurred in engineered nanoparticles (NPs) propelled by the booming nanotechnology industry. Although inhalation of these particulates has become a public health concern, human health effects and mechanisms of action for NPs are not well understood. Focusing on the human airway smooth muscle cell, here we show that the cellular mechanical function is altered by particulate exposure in a manner that is dependent upon particle material, size and dose. We used Alamar Blue assay to measure cell viability and optical magnetic twisting cytometry to measure cell stiffness and agonist-induced contractility. The eight particle species fell into four categories, based on their respective effect on cell viability and on mechanical function. Cell viability was impaired and cell contractility was decreased by (i) zinc oxide (40-100 nm and less than 44 microm) and copper(II) oxide (less than 50 nm); cell contractility was decreased by (ii) fluorescent polystyrene spheres (40 nm), increased by (iii) welding fumes and unchanged by (iv) diesel exhaust particles, titanium dioxide (25 nm) and copper(II) oxide (less than 5 microm), although in none of these cases was cell viability impaired. Treatment with hydrogen peroxide up to 500 microM did not alter viability or cell mechanics, suggesting that the particle effects are unlikely to be mediated by particle-generated reactive oxygen species. Our results highlight the susceptibility of cellular mechanical function to particulate exposures and suggest that direct exposure of the airway smooth muscle cells to particulates may initiate or aggravate respiratory diseases.

Effects of long-term feeding of genetically modified corn (event MON810) on the performance of lactating dairy cows

A long-term study over 25 months was conducted to evaluate the effects of genetically modified corn on performance of lactating dairy cows. Thirty-six dairy cows were assigned to two feeding groups and fed with diets based on whole-crop silage, kernels and whole-crop cobs from Bt-corn (Bt-MON810) or its isogenic not genetically modified counterpart (CON) as main components. The study included two consecutive lactations. There were no differences in the chemical composition and estimated net energy content of Bt-MON810 and CON corn components and diets. CON feed samples were negative for the presence of Cry1Ab protein, while in Bt-MON810 feed samples the Cry1Ab protein was detected. Cows fed Bt-MON810 corn had a daily Cry1Ab protein intake of 6.0 mg in the first lactation and 6.1 mg in the second lactation of the trial. Dry matter intake (DMI) was 18.8 and 20.7 kg/cow per day in the first and the second lactation of the trial, with no treatment differences. Similarly, milk yield (23.8 and 29.0 kg/cow per day in the first and the second lactation of the trial) was not affected by dietary treatment. There were no consistent effects of feeding MON810 or its isogenic CON on milk composition or body condition. Thus, the present long-term study demonstrated the compositional and nutritional equivalence of Bt-MON810 and its isogenic CON.