Efficacy Of Candidate Therapeutics Research Articles

Abstract B12: 3D perfusion bioreactor system as a model for studying cell biology of ovarian cancer

Abstract Introduction: Epithelial ovarian cancer (EOC) is the most common cause of death among gynecologic malignancies. This is a result of challenging clinical management due to the high rate of recurrence and the development of chemoresistance in EOC patients. Therefore, the development of new therapeutics is crucial. Unfortunately, the success rate of establishing new drugs is very low. A major factor contributing to this is the lack of preclinical models that accurately predict in vivo efficacy of candidate therapeutics. Preclinical models do not accurately mimic the biomechanical regulation of the tumor microenvironment (TME) and cannot predict in vivo efficacy of candidate therapeutics. We have developed an EOC 3D perfused bioreactor system that recapitulates EOC tumor biology; better predicts the clinical response of new drug candidates, thereby eliminating ineffective candidates prior to clinical trials; and incorporates tumor biomechanical regulation. Materials and Methods: EOC cell lines (luciferase-tagged SKOV-3 and OVCAR-8) were embedded in a relevant ECM (5.25 × 105 total cells/100 uL ECM) containing 90% bovine collagen I + 10% basement membrane (Matrigel) and injected into a polydimethylsiloxane bioreactor (PDMS). The volume was then perforated by five wires and the bioreactor was placed in the incubator (37C, 5%CO2) for polymerization. After that, the wires were removed to create microchannels so that the cell culture media (RPMI 10% FBS, 1% penicillin-streptomycin-gentamicin) can flow through the perfusion bioreactor system and provide nutrient delivery and gas exchange, which helps maintain viability and function of surrounding cells. Finally, the bioreactors were connected to a peristaltic pump that allows the cell culture media to run over 14 days, along the channels and through the matrix. We monitored cell growth over 14 days using bioluminescence (BLI) and these results were validated with histologic analysis of the cell density. In order to prove that the bioreactor keeps EOC biology, by immunohistochemical (IHC) analysis with p53 and PAX8, clinically used ovarian cancer markers. Results and Discussion: For our preliminary data utilizing the 3D perfusion bioreactor system in ovarian cancer, we first used the luciferase-tagged OVCAR-8 cell line to evaluate cell growth. The bioluminescence signal showed a linear increase over 14 days. These results were validated histologically with cell density by measuring the number of nucleated cells per micron2. Graphical representation of the region of interest (ROI) showed a high correlation between manual determination of cell number and bioluminescence score. In our second experiment, we used the luciferase-tagged SKOV-3 cell line (high-grade serous origin) and we also monitored cell growth over 14 days using BLI and histologic analysis of the cell density as described for OVCAR-8 using the same conditions in the bioreactor. IHC analysis with p53 and PAX8 was positive and proved that the perfusion bioreactor system keeps EOC biology. Citation Format: Alba Martinez, Molly Buckley, Joel Berry, Rebecca Arend, Michael Birrer. 3D perfusion bioreactor system as a model for studying cell biology of ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B12.

Metabolomic study of disease progression in scrapie prion infected mice; validation of a novel method for brain metabolite extraction.

Prion disease is a form of neurodegenerative disease caused by the misfolding and aggregation of cellular prion protein (PrPC). The neurotoxicity of the misfolded form of prion protein, PrPSc still remains understudied. Here we try to investigate this issue using a metabolomics approach. The intention was to identify and quantify the small-in-size and water-soluble metabolites extracted from mice brains infected with the Rocky Mountain Laboratory isolate of mouse-adapted scrapie prions (RML) and track changes in these metabolites during disease evolution. A total of 73 mice were inoculated with RML prions or normal brain homogenate control; brains were harvested at 30, 60, 90, 120 and 150days post-inoculation (dpi). We devised a high-efficiency metabolite extraction method and used nuclear magnetic resonance spectroscopy to identify and quantify 50 metabolites in the brain extracts. Data were analyzed using multivariate approach. Brain metabolome profiles of RML infected animals displayed continuous changes throughout the course of disease. Among the analyzed metabolites, the most noteworthy changes included increases in myo-inositol and glutamine as well as decreases in 4-aminobutyrate, acetate, aspartate and taurine. We report a novel metabolite extraction method for lipid-rich tissue. As all the major metabolites are identifiable and quantifiable by magnetic resonance spectroscopy, this study suggests that tracking of neurochemical profiles could be effective in monitoring the progression of neurodegenerative diseases and useful for assessing the efficacy of candidate therapeutics.

T Helper 17 Cells in Primary Sjögren’s Syndrome

Primary Sjögren’s syndrome is an autoimmune disease characterized by diffuse infiltration of lymphocytes into exocrine glands and other tissues. The infiltrating lymphocytes have been identified as subsets of B cells and T cells, including T helper 17 cells, T regulatory cells and follicular helper T cells. The role of these cells in the development of the syndrome is now known, as is their impact on the production of proinflammatory cytokines such as IL-6, IL-17, IL-22 and IL-23. In particular, experimental animal models and patients suggest that a shift in Th17/Treg balance toward the proinflammatory Th17 axis exacerbates primary Sjögren’s syndrome and other autoimmune disorders. Nevertheless, the pathogenesis of the disorder is not yet fully elucidated. This review summarizes the recent advances in therapeutic control of the Treg/Th17 balance, as well as the efficacy of candidate therapeutics against primary Sjögren’s syndrome.

Therapeutic Potential of Targeting the Th17/Treg Axis in Autoimmune Disorders.

A disruption of the crucial balance between regulatory T-cells (Tregs) and Th17-cells was recently implicated in various autoimmune disorders. Tregs are responsible for the maintenance of self-tolerance, thus inhibiting autoimmunity, whereas pro-inflammatory Th17-cells contribute to the induction and propagation of inflammation. Distortion of the Th17/Treg balance favoring the pro-inflammatory Th17 side is hence suspected to contribute to exacerbation of autoimmune disorders. This review aims to summarize recent data and advances in targeted therapeutic modification of the Th17/Treg-balance, as well as information on the efficacy of candidate therapeutics with respect to the treatment of autoimmune diseases.

The CounterACT Research Network: Basic Mechanisms and Practical Applications

The National Institutes of Health has developed a comprehensive research program that includes research centers of excellence, individual research projects, small business projects, contracts, and interagency agreements to conduct basic, translational, and clinical research aimed at the discovery and/or identification of better medical countermeasures against chemical threat agents. Chemical threats include chemical warfare agents, toxic industrial and agricultural chemicals, and toxins and other chemicals that could be used intentionally as an act of terror or by large-scale accidents or natural disasters. The overarching goal of this research program is to enhance our medical response capabilities during an emergency. The program is named Countermeasures Against Chemical Threats (CounterACT). It supports translational research, applying ideas, insights, and discoveries generated through basic scientific inquiry to the treatment or prevention of mortality and morbidity caused by chemical threat agents. The categories of research supported under this program include creation and development of screening assays and animal models for therapy development, identification of candidate therapeutics, obtaining preliminary proof-of-principle data on the efficacy of candidate therapeutics, advanced efficacy and preclinical safety studies with appropriate animal models using Good Laboratory Practices (GLP), and clinical studies, including clinical trials with new drugs. Special consideration is given to research relevant to people who are particularly vulnerable, including the young, the elderly, and individuals with pre-existing medical conditions.

Bioluminescent imaging: a critical tool in pre‐clinical oncology research

Bioluminescent imaging (BLI) is a non-invasive imaging modality widely used in the field of pre-clinical oncology research. Imaging of small animal tumour models using BLI involves the generation of light by luciferase-expressing cells in the animal following administration of substrate. This light may be imaged using an external detector. The technique allows a variety of tumour-associated properties to be visualized dynamically in living models. The increasing use of BLI as a small-animal imaging modality has led to advances in the development of xenogeneic, orthotopic, and genetically engineered animal models expressing luciferase genes. This review aims to provide insight into the principles of BLI and its applications in cancer research. Many studies to assess tumour growth and development, as well as efficacy of candidate therapeutics, have been performed using BLI. More recently, advances have also been made using bioluminescent imaging in studies of protein-protein interactions, genetic screening, cell-cycle regulators, and spontaneous cancer development. Such novel studies highlight the versatility and potential of bioluminescent imaging in future oncological research.