Supplementary Figure S1 from Poly(ADP-Ribose) Polymerase 1 Promotes Tumor Cell Survival by Coactivating Hypoxia-Inducible Factor-1–Dependent Gene Expression

Inflammation is one of the factors that may increase the sensitivity of hepatic cells to acetaminophen (APAP) induced toxicity. To investigate the mechanisms, we exposed 3-dimensional (3D) Human Liver Microtissues, a co-culture of primary human hepatocytes (PHH) and Kupffer cells (KCs), to 0, 0.5 (low), 5 (median) and 10 mM (high dose) APAP for 24 h, with/without lipopolysaccharide (LPS). Microarray-technology was used to evaluate the transcriptome changes. In the presence of LPS, the median-dose of APAP is sufficient to inhibit the expression of respiratory chain-and antioxidant-related genes, suggesting the involvement of reactive oxygen species (ROS) and oxidative stress. Furthermore, the median- and high-dose of APAP inhibited the expression of Fc fragment receptor (Fc gamma R)-coding genes, regardless of the presence of LPS. The toll-like receptor 4 (TLR4) expression, however, was continuously elevated after the LPS/APAP co-exposures, which may result in reduced KC-phagocytosis and unbalanced cytokine patterns. Compared to the treatment with LPS only, LPS/APAP co-exposures induced the production of interleukin (IL)-8, a pro-inflammatory cytokine, but suppressed the secretion of IL-6, a cytokine regulating hepatic regeneration, along with the increase in APAP dosages. In addition to the disrupted mitochondrial functions, the presence of LPS exacerbated APAP toxicity. These findings suggest that 3D Microtissues are a suitable model for the mechanistic exploration of inflammation-associated drug toxicity.

Abstract Background A major issue for improving the overall success rate in drug development is the lack of accurate experimental human in-vitro models. While there is a significant and increasing need for such better ex-vivo cell-based models, the question on how closely these systems resemble and recapitulate the original tumors is of great importance. Patient-derived xenograft (PDX) models act as vehicle systems to propagate human tumor specimens, faithfully preserving the biological features and the genetic expression profile. The retainment of such criteria in in vitro 3D InSightTM Tumor Microtissues derived from PDX lines is crucial to provide a relevant physiological environment and strategy to assess candidate drugs for novel therapeutic approaches. Aim Development and characterization of in vitro 3D InSightTM Tumor Microtissues from patient-derived xenograft models. Material & Methods and Results PDX cell suspensions of Lung, Breast and Melanoma origin were successfully used to assess 3D aggregation in 96 well format and characterized over 10 days in culture. After careful removal of mouse cell contaminants in each in vitro 3D PDX sample, PDX cell cultures were supplied with exogenous normal human dermal fibroblasts (nHDF). The morphology, biomarker phenotype (IHC) as well as cell proliferation were assessed by histological analysis. In addition to screening for standard diagnostic marker such as proliferating vs. dead cells (e.g. Ki67, ClCasp3) and stromal vs. epithelial-tumor cells (e.g. FAP, pan-CK, E-Cadherin), we also assessed the expression of cancer type-specific biomarkers. Moreover, to monitor the dynamics of cancer phenotypic alterations, epithelial-to-mesenchymal transition (EMT) marker were evaluated and scored. Immunohistochemistry assessment of 3D microtumors validated the resemblance with their respective PDX tumor models. The viability and growth rate of PDX-derived microtumors were assessed by size analysis (cell scanner) and ATP assay. 3D tumor growth rate and cell behavior observations reflected the diversity of disease progression in vivo. Further efforts will focus on employing this platform to investigate the efficacy of specific targeted therapies based on the distinct molecular signatures of PDX tumor models. Conclusion Development and characterization of In vitro 3D InSightTM Tumor Microtissues from patient-derived xenograft (PDX) lines demonstrated that the morphological and molecular features of the parental tumors are well retained. We suggest that in vitro 3D PDX models offer a more suitable and robust approach to expedite faithful efficacy assessment and approval of optimal drug candidates. Citation Format: Francesca Chiovaro, Irina Agarkova, Armin Maier, Simon Messner, Julia Schueler, Patrick Guye. Development and characterization of PDX-derived 3D tumor microtissues as platform for screening targeted molecular therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-034.

Introduction In vitro cell based drug testing tools have been widely used in drug discovery and early development to evaluate novel drug entities for further evaluation in preclinical in vivo models. However, the question on how closely these systems resemble and recapitulate the original tumours has prompted researchers and clinical oncologists to seek complementary in vitro strategies. Patient-derived xenograft (PDX) mouse models are a widely accepted tool for propagation of primary human tumour specimens, faithfully preserving the biological features and the genetic expression profiles. In order to increase accessibility of these physiological relevant tumour surrogates in drug development, we investigated whether ex-vivo culturing of PDX-derived tumours as 3D Microtissues retain the in-vivotumour characteristics. The resulting 3D PDX Tumour Microtissues will increase throughput and compatibility with existing platforms while reducing costs and animal testing. Material and methods To this end, PDX cell suspensions of Lung, Breast and Melanoma origin were used to aggregated in Akura 96 plates and characterised over 10 days in culture. The 3D tumour microtissues were monitored for growth characteristics, phenotype, cancer biomarkers and drug efficacy. In addition to screening for standard diagnostic marker for proliferating (Ki67), apoptotic cells (cleaved Caspase 3), stromal (FAP) and epithelial-tumour cells (pan-CK, E-Cadherin), we also assessed the expression of cancer type specific biomarkers. Immunohistochemistry assessment of 3D tumour microtissues demonstrated the resemblance with the respective in-vivo PDX tumour model. The viability and growth rate of PDX-derived tumour microtissues was assessed by size analysis and ATP assay. 3D tumour growth rate reflected the diversity of tumour growth progression in vivo. Subsequently, these tumour microtissues were assessed using either non-specific cytotoxic drugs or molecular-targeted drug therapy in a single agent treatment regimen. The efficacy of specific targeted therapies was based on the distinct molecular signatures of PDX tumour models. Results and discussions In conclusion, in vitro 3D InSight Tumour Microtissues from patient-derived xenograft (PDX) lines demonstrated retention of morphological and molecular features of the parental tumours. Conclusion We suggest that 3D PDX Tumour Microtissues are physiological relevant and higher throughput compatible in vitro models for efficacy assessment and drug candidate screenings.

Environmental exposure to the highly persistent chlorinated pesticides including dieldrin and lindane is postulated to be a risk factor to the development of Parkinson’s disease, a devastating movement disorder. We have previously reported that the combined treatment with dieldrin and lindane induces a cooperative toxicity in the rat N27 dopaminergic neuronal cells through increased oxidative stress and mitochondrial dysfunction. In this study, we investigated the involvement of NADPH oxidase (NOX) proteins in the combined treatment with dieldrin and lindane-induced dopaminergic neurotoxicity. Immunoblot analysis demonstrated the presence of NADPH Oxidase 1 (Nox1) isoform and p67phox in N27 neurons. Furthermore, treatment with dieldrin and lindane upregulated the cellular expression of Nox1 but not p67phox protein. Functionally, dieldrin and lindane-induced ROS production was attenuated, in a dose-dependent manner, by Nox inhibitors diphenylene iodonium and apocynin. Subcellular localization analysis of Nox1 and p67phox proteins indicated colocalization of both subunits with mitochondria in untreated cells. Treatment with dieldrin and lindane further increased mitochondrial colocalization of Nox1 protein, suggesting a potentially prominent role for mitochondrial Nox1 protein in dieldrin and lindane-induced ROS generation in dopaminergic neurons and its contribution to the combined organochlorinated pesticide-induced neurotoxicity.

The available tools and biological models to study drug-induced mitochondrial toxicity have evolved over time. Two key organs were selected to exemplify analysis of mitochondrial impairment using advanced 3D models: primary human liver microtissues and human cardiac microtissues. The significantly higher spare respiratory capacity (SRC) measured in the microtissue format makes it a valuable parameter to evaluate the impact of drugs on tissues. The use of microtissues to evaluate the impact of drugs on mitochondrial function represents a next level of an in vitro tissue model. Especially for the liver, a metabolically relevant environment allows to test not only short-term effects but also effects of mitochondrial active metabolites and long-term effects. Overall, the integration of 3D microtissues within the Seahorse technology generates a highly versatile platform for investigating mitochondrial response upon exposure of compounds and compound combinations.

Elevations of liver enzymes have been observed in clinical trials with BAL30072, a novel antibiotic. In vitro assays have identified potential mechanisms for the observed hepatotoxicity, including electron transport chain (ETC) inhibition and reactive oxygen species (ROS) generation. DILIsym, a quantitative systems pharmacology (QSP) model of drug-induced liver injury, has been used to predict the likelihood that each mechanism explains the observed toxicity. DILIsym was also used to predict the safety margin for a novel BAL30072 dosing scheme; it was predicted to be low. DILIsym was then used to recommend potential modifications to this dosing scheme; weight-adjusted dosing and a requirement to assay plasma alanine aminotransferase (ALT) daily and stop dosing as soon as ALT increases were observed improved the predicted safety margin of BAL30072 and decreased the predicted likelihood of severe injury. This research demonstrates a potential application for QSP modeling in improving the safety profile of candidate drugs.

We sought to evaluate a new regional segmentation method for use with three-dimensional (3D) non-contrast abdominal CT images and to report the preliminary results.The proposed method was evaluated in ten cases. Manually segmented areas were used as the gold standard for evaluation. To compare the standard and the extracted liver regions, the degree of coincidence R% was redefined by transforming a volumetric overlap error. We also evaluated the influence of varying the density window size in terms of setting the starting points.We confirmed in ten cases that our method could segment the liver region more precisely than the conventional method. A size of window 15 voxels was optimal as the starting point in all cases.We demonstrated the accuracy of a 3D semiautomatic liver segmentation method for non-contrast CT. This method promises to offer radiologists a time-efficient segmentation aid.

Drug-induced liver injury (DILI) continues to be a major source of clinical attrition, precautionary warnings, and post-market withdrawal of drugs. Accordingly, there is a need for more predictive tools to assess hepatotoxicity risk in drug discovery. Three-dimensional (3D) spheroid hepatic cultures have emerged as promising tools to assess mechanisms of hepatotoxicity, as they demonstrate enhanced liver phenotype, metabolic activity, and stability in culture not attainable with conventional two-dimensional hepatic models. Increased sensitivity of these models to drug-induced cytotoxicity has been demonstrated with relatively small panels of hepatotoxicants. However, a comprehensive evaluation of these models is lacking. Here, the predictive value of 3D human liver microtissues (hLiMT) to identify known hepatotoxicants using a panel of 110 drugs with and without clinical DILI has been assessed in comparison to plated two-dimensional primary human hepatocytes (PHH). Compounds were treated long-term (14 days) in hLiMT and acutely (2 days) in PHH to assess drug-induced cytotoxicity over an 8-point concentration range to generate IC50 values. Regardless of comparing IC50 values or exposure-corrected margin of safety values, hLiMT demonstrated increased sensitivity in identifying known hepatotoxicants than PHH, while specificity was consistent across both assays. In addition, hLiMT out performed PHH in correctly classifying hepatotoxicants from different pharmacological classes of molecules. The hLiMT demonstrated sufficient capability to warrant exploratory liver injury biomarker investigation (miR-122, HMGB1, α-GST) in the cell-culture media. Taken together, this study represents the most comprehensive evaluation of 3D spheroid hepatic cultures up to now and supports their utility for hepatotoxicity risk assessment in drug discovery.

BACKGROUND & AIMS:Currently most liver fibrosis research is performed in vivo, since suitable alternative in vitro systems which are able to recapitulate the cellular events leading to liver fibrosis are lacking. Here we aimed at generating a system containing cells representing the three key players of liver fibrosis (hepatocyte, Kupffer cells and stellate cells) and assess their response to pro-fibrotic compounds such as TGF-β1, methotrexate (MTX) and thioacetamide (TAA).METHODS:Human cell lines representing hepatocytes (HepaRG), Kupffer cell (THP-1 macrophages) and stellate cells (hTERT-HSC) were co-cultured using the InSphero hanging drop technology to generate scaffold-free 3D microtissues, that were treated with pro-fibrotic compounds (TGF-β1, MTX, TAA) for up to 14 days. The response of the microtissues was evaluated by determining the expression of cytokines (TNF-α, TGF-β1 and IL6), the deposition and secretion of ECM proteins and induction of gene expression of fibrosis biomarkers (e.g. αSMA). Induction of Nrf2 and Keap1, as key player of defence mechanism, was also evaluated.RESULTS:We could demonstrate that the multicellular 3D microtissue cultures could be maintained in a non-activated status, based on the low expression levels of activation markers. Macrophages were activated by stimulation with LPS and hTERT-HSC showed activation by TGF-β1. In addition, MTX and TAA elicited a fibrotic phenotype, as assessed by gene-expression and protein-deposition of ECM proteins such as collagens and fibronectin. An involvement of the antioxidant pathway upon stimulation with pro-fibrotic compounds was also observed.CONCLUSION:Here, for the first time, we demonstrate the in vitro recapitulation of key molecular and cellular events leading to liver fibrosis: hepatocellular injury, antioxidant defence response, activation of Kupffer cells and activation of HSC leading to deposition of ECM.

Three-Dimensional (3D) liver microtissues, specifically prepared from primary human hepatocytes (PHH) in coculture with nonparenchymal cells (NPCs), have been shown to be a valuable tool for in vitro toxicology. However, a lack of thorough characterization on a functional, transcriptomic, and proteomic level of such models during long-term cultivation is evident. By integrating multiple omics technologies, we provide in this study an in-depth long-term characterization of 3D microtissues composed of PHH from three different donors cocultured with primary NPCs. The 3D human liver microtissues (hLiMTs) exhibited stable adenosine triphosphate (ATP) content and albumin secretion over 5 weeks. Histological analysis indicated a healthy liver tissue with polarized expression of bile salt export pump (BSEP) and multidrug resistance protein 2 (MRP2) in a structure reminiscent of bile canaliculi. The 3D microtissues exhibited stable basal and inducible cytochrome P450 activities up to 5 weeks in culture. Analysis of 40,716 transcripts using RNA arrays revealed distinct similarities to native human liver gene expression. Long-term culture showed a stable phenotype up to 5 weeks, with differences in liver gene expression primarily attributed to individual donors. Proteomic profiling of 2200 unique proteins by label-free LC-MS/MS revealed a relatively stable protein expression where only 7.3% were up- or downregulated more than twofold from day 7 to 35 in culture. Taken together, these results suggest that hLiMTs represent a responsive and physiologically relevant in vitro liver model that maintains stable function over 5 weeks and is therefore well suited for repeated-dose toxicity testing.

BAL30072 is a new monocyclic β-lactam antibiotic under development which provides a therapeutic option for the treatment of severe infections caused by multi-drug-resistant Gram-negative bacteria. Despite the absence of liver toxicity in preclinical studies in rats and marmosets and in single dose clinical studies in humans, increased transaminase activities were observed in healthy subjects in multiple-dose clinical studies. We, therefore, initiated a comprehensive program to find out the mechanisms leading to hepatocellular injury using HepG2 cells (human hepatocellular carcinoma cell line), HepaRG cells (inducible hepatocytes derived from a human hepatic progenitor cell line), and human liver microtissue preparations. Our investigations demonstrated a concentration- and time-dependent reduction of the ATP content of BAL30072-treated HepG2 cells and liver microtissues. BAL30072 impaired oxygen consumption by HepG2 cells at clinically relevant concentrations, inhibited complexes II and III of the mitochondrial electron transport chain, increased the production of reactive oxygen species (ROS), and reduced the mitochondrial membrane potential. Furthermore, BAL 30072 impaired mitochondrial fatty acid metabolism, inhibited glycolysis, and was associated with hepatocyte apoptosis. Co-administration of N-acetyl-L-cysteine partially protected hepatocytes from BAL30072-mediated toxicity, underscoring the role of oxidative damage in the observed hepatocellular toxicity. In conclusion, BAL30072 is toxic for liver mitochondria and inhibits glycolysis at clinically relevant concentrations. Impaired hepatic mitochondrial function and inhibition of glycolysis can explain liver injury observed in human subjects receiving long-term treatment with this compound.

Genetic Engineering & Biotechnology NewsVol. 36, No. 16 OMICS TutorialMitotoxicity Testing in 3D Liver MicrotissuesNovel Two-Step Assay Integrates Toxicity Testing in 3D Liver Models and Assessment of OCRSimon Messner, Randy Strube, Katrin Roessger, and Jens M. KelmSimon MessnerSimon Messner is senior product manager (E-mail Address: simon.messner@insphero.com), Randy Strube is global marketing director, Katrin Roessger is application scientist, and Jens M. Kelm is chief technology officer at InSphero. Website: www.insphero.com.Search for more papers by this author, Randy StrubeSimon Messner is senior product manager (E-mail Address: simon.messner@insphero.com), Randy Strube is global marketing director, Katrin Roessger is application scientist, and Jens M. Kelm is chief technology officer at InSphero. Website: www.insphero.com.Search for more papers by this author, Katrin RoessgerSimon Messner is senior product manager (E-mail Address: simon.messner@insphero.com), Randy Strube is global marketing director, Katrin Roessger is application scientist, and Jens M. Kelm is chief technology officer at InSphero. Website: www.insphero.com.Search for more papers by this author, and Jens M. KelmSimon Messner is senior product manager (E-mail Address: simon.messner@insphero.com), Randy Strube is global marketing director, Katrin Roessger is application scientist, and Jens M. Kelm is chief technology officer at InSphero. Website: www.insphero.com.Search for more papers by this authorPublished Online:14 Sep 2016https://doi.org/10.1089/gen.36.16.12AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetails Volume 36Issue 16Sep 2016 InformationCopyright © by GEN PublishingTo cite this article:Simon Messner, Randy Strube, Katrin Roessger, and Jens M. Kelm.Mitotoxicity Testing in 3D Liver Microtissues.Genetic Engineering & Biotechnology News.Sep 2016.24-25.http://doi.org/10.1089/gen.36.16.12Published in Volume: 36 Issue 16: September 14, 2016PDF download

Synthesis of CoIII, CuII and PdII complexes containing 2-(2′-hydroxyphenyl)-2-thiazoline as a bidentate O,N-donor thiazoline ligand (tzol), [Co(tzol)3] (1), [Cu(tzol)2] (2), [Pd(tzol)2] (3) and characterization by various techniques such as IR, mass spectrometry, and elemental analysis are reported. The novel complex 1 was also characterized by X-ray crystallography. The structures of complexes 2 and 3 were analyzed by density functional theory. The representative synthetic procedure involves the reaction of a metal acetate with the thiazoline ligand in methanol. The catalytic activity of complexes 1–3 for azide–alkyne cycloaddition was tested. The reaction conditions for the azide–alkyne cycloaddition were optimized by varying the catalyst loading, temperature, and time of reaction.