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ICCVAM Biennial Report 2018-2019

Biennial Progress Report 2018-2019 Interagency Coordinating Committee on the Validation of Alternative Methods

Assay Development

Many ICCVAM member agencies are developing new in vitro technologies and resources intended to reduce or replace animal use for chemical safety testing. They include technologies such as MPS and image analysis, and address important endpoints such as cardiotoxicity, inhalation toxicity, and neurotoxicity.

Prediction of Differential Responses to Toxicity from Genetically Diverse Cell Lines

To gain a deeper understanding of the biological effects of chemical exposures, AFRL has developed a cell-based toxicity analysis system based on the Clarity Bioanalytics software platform. In the lab, cells are exposed to chemical or biological agents and imaged using high-content, high-resolution microscopy. These images are processed through an analytics pipeline using DoD supercomputing resources. The Clarity Bioanalytics system studies the changes in cells after exposure, identifies the level of toxicity of different compounds, and discovers genetic elements (e.g., SNPs, genes, pathways) that could affect the cellular risk to certain exposures. This software platform represents a central analytics tool for exposure toxicology research within AFRL. It allows unbiased phenotyping of the toxic effects of a variety of chemical and biological agents, and genotype-phenotype analyses for personalized response assessment and prediction. Initial experiments on this system were performed using B-lymphocytes in suspension, but ideally the system must be able to analyze images from a wide variety of cell types with the best possible accuracy. Current efforts have established machine learning methods to allow identification of any cell type and extract feature/phenotype information from cells, allowing expanded capabilities for comparative, unbiased phenotyping.

Gut-endothelial Barrier Models Incorporating Microbial Colonies

AFRL has developed gut-endothelial barrier models using microfluidic technology. These gut-on-a-chip platforms emulate many of the microstructures (gut epithelial-blood endothelial interactions), morphologies (macro- and micro-villus structures), and functions of the human gut. The dynamic nature of the models provides a robust opportunity for assessing gut-blood barrier integrity, nutrient transport, and host-microbiome interactions. Moreover, a tailored oxygen environment, simulating the low-oxygen conditions seen in the human gut, promotes a more robust microbial colony inclusion. Current investigations are evaluating probiotics, cooperative microbial interactions, and complex microbial community dynamics in the gut-on-a-chip platform. The gut-on-a-chip models are also being combined with brain-on-a-chip models to allow analyses of complex molecular gut-brain axis interactions. These technologies in combination have the potential to enable assessment of operational stress and exposure outcomes due to thermal burden, nutrition, toxin ingestion and digestion, and their corresponding impact on the blood-brain barrier and neuronal activity. Furthermore, identification of molecular targets or pathways for countermeasure development can be developed, matured, and optimized in a human model prior to in vivo testing, thereby limiting animal usage and reducing the cost and time for deployment. Further validation and assessments of these promising capabilities will have to be completed before complete implementation.

Cell-based Assays to Identify Chemical Targets

CCDC CBC has been assessing and validating a number of cell-based in vitro platforms, which are currently being optimized as predictive toxicological tools. As a result of these efforts, the center has a fully functional, high-throughput G protein-coupled receptor screening assay, as well as single receptor tests using engineered cell lines to assay ion channel effects. These cell-based platforms should allow rapid assessment of the effects of different chemical threats.

Thus far, the center has used the PRESTO-TANGO assay system to test the activity of alpha adrenergic receptor agonists against an entire library of G protein-coupled receptors. Data currently collected align with results obtained using in vivo systems. Current and future efforts aim to test G protein-coupled receptor agonism and antagonism of a number of different compounds of interest.

Phenotypic Screening Platforms for Cardiotoxicity, Hepatotoxicity, and Neurotoxicity

CCDC CBC is developing, assessing, and implementing approaches that use MPS to assess complex phenotypes such as organ function. In this manner, three-dimensional, human cell-based platforms are being developed to better represent organ function and, more importantly, potential perturbation of normal phenotypes critical to health. These target organ effects are measurable on a phenotypic level, which serves as a mechanism to observe potential toxic effects without screening for a specific target of interest. Some examples of implementation efforts include:

  • Cardiotoxicity can be measured in iPSC-derived cardiomyocytes that synchronously beat in culture, with toxicity measured in real-time using impedance-based technology.
  • Hepatoxicity can be measured using the Emulate Biotech liver chip system and the CN Bio PhysioMimix system, which have allowed success in the prolonged maintenance of a fully functional and viable liver environment in vitro. As a result, CCDC CBC can confidently assess both acute and chronic physiologically relevant, molecular-level hepatic effects of chemical threats without having to use an animal model.
  • Neurotoxicity can be measured in two- or three-dimensional cultures of primary or iPSC-derived neurons that can be functionally assessed using microelectrode-based measurements of synaptic firing or cytotoxic endpoints measured via high-content fluorescent imaging.
Lung-on-a-chip Models to Assess Toxicity of Aerosols

AFRL has developed in vitro lung modeling tools, including lung-on-a-chip models, to enable robust toxicological assessments of aerosolized particulates. These models include technologies to aerosolize, characterize, and monitor dosimetry in real time. They also provide the control necessary to reproduce operational exposure conditions. The lung-on-a-chip models have cyclical stretch capability, simulating the mechanics of breathing, which has been shown to be important in the exposure mechanism. Together, these models and tools provide a platform to assess operationally relevant environmental or engineer toxicant exposures.

Human Stem Cell Neuronal Platform with Phenotypic and Physiological Output

The Biological Modeling Group of the AFRL 711th Human Performance Wing in collaboration with the Sanford Burnham Prebys Medical Discovery Institute is developing a human in vitro stem cell assay system to determine the mechanism of neurotoxins that potentially affect human brain function. This research includes benchmark testing of over 200 neurotoxins with known mechanisms. Effects on treated cells are assessed by neurophysiology (multi-electrode array), mitochondrial membrane potential changes, and reactive oxygen species generated in the cell culture system. The results of these ongoing studies are being analyzed to develop predictive toxicology capabilities using QSAR analysis and read-across computational methods. These approaches will enable predictions of toxicity of unknown chemicals with varying degrees of confidence, depending on how similar the unknown chemicals are to chemicals in the known database. The next phase of human neuron stem cell readout will involve a mechanistic large-scale neural network modeling of chemically induced changes in the in vitro firing activity of the networking neurons. By measuring these changes in networking firing rates in combination with behavior phenotypes in the case of known chemical entities, the goal is to be able to predict the response for unknown chemicals or chemicals with limited experimental data.

Human Cancer Models Initiative

The Human Cancer Models Initiative is an international consortium that is generating novel, next-generation, tumor-derived culture models annotated with genomic and clinical data. Models and related data developed through the initiative are available as a community resource. NCI is contributing to the initiative by supporting four Cancer Model Development Centers. These centers are tasked with producing next-generation cancer models, such as organoids and conditionally reprogrammed cells, from clinical samples. The cancer models include tumor types that are rare, originate from patients from underrepresented populations, lack precision therapy, or lack cancer model tools. In 2018, a funding opportunity was offered to increase the racial and ethnic diversity of the samples used to develop new culture models.

Cancer Tissue Engineering Collaborative

The NCI Cancer Tissue Engineering Collaborative Research Program supports the development and characterization of state-of-the-art biomimetic tissue-engineered technologies for cancer research. Collaborative, multidisciplinary projects that engage the fields of regenerative medicine, tissue engineering, biomaterials, and bioengineering with cancer biology will be essential for generating novel experimental models that mimic cancer pathophysiology to elucidate specific cancer phenomena that are otherwise difficult to examine in vivo. Projects are funded through grants offered by NCI; the current round of funding opened in 2019 and will continue through January 2022. Endpoints under investigation by current projects include brain, breast, ovarian, and colon cancer.

Biomimetic In Vitro Models for Assessment of Occupational Respirable Aerosol Hazards

Inhalation of respirable particles in the workplace can cause lung disease. Risk assessment of respirable particles exposure has been challenging because of the large number of new particulate chemicals used in the workplace and the limited availability of appropriate in vitro models for toxicity assessment of respirable particles. To address this challenge, scientists at NIOSH developed in vitro cell culture models that allow simple, rapid, and specific testing of respirable particle toxicity as well as detailed mechanistic investigations of exposure effects. The models employ human lung cells for potential use in the screening of occupational respiratory hazards and for potential use in risk assessment. Considerations include determining the relevant in vitro doses to reflect potential occupational exposures, specific lung cell types, and disease endpoints. In vitro models are being used to assess both acute toxicity hazard and cancer hazard from long term exposure to low doses of particles (Kornberg et al. 2019). Further improvements to these in vitro models are being made by integrating the 3D liquid-air interface platform to simulate the complexity of real respirable particle airway exposure conditions. Successful development of this integrated system will facilitate rapid and realistic assessment of respirable particle toxicities.