Abstracts | SLAS Discovery 2017

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Corning Sponsored Articles and Research from SLAS Discovery 2017

Corning Sponsored Articles and Research from SLAS Discovery 2017

Corning Sponsored Articles from SLAS 2017


3D Models of the NCI60 Cell Lines for Screening Oncology Compounds

Mike Selby, Rene Delosh, Julie Laudeman, Chad Ogle, Russell Reinhart, Thomas Silvers, Scott Lawrence, Robert Kinders, Ralph Parchment, Beverly A. Teicher, David M. Evans

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A High-Throughput Screening Model of the Tumor Microenvironment for Ovarian Cancer Cell Growth

Madhu Lal-Nag, Lauren McGee, Rajarshi Guha, Ernst Lengyel, Hilary A. Kenny, Marc Ferrer

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A 1536-Well 3D Viability Assay to Assess the Cytotoxic Effect of Drugs on Spheroids

Franck Madoux, Allison Tanner, Michelle Vessels, Lynsey Willetts, Shurong Hou, Louis Scampavia, Timothy P. Spicer

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RNAi High-Throughput Screening of Single- and Multi-Cell-Type Tumor Spheroids: A Comprehensive Analysis in Two and Three Dimensions

Jiaqi Fu, Daniel Fernandez, Marc Ferrer, Steven A. Titus, Eugen Buehler, Madhu A. Lal-Nag

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Exploring Drug Dosing Regimens in Vitro using Real-Time 3D Spheroid Tumor Growth Assays

Madhu Lal-Nag, Lauren McGee, Steven A. Titus, Kyle Brimacombe, Sam Michael, Gurusingham Sittampalam, Marc Ferrer

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3D Models of the NCI60 Cell Lines for Screening Oncology Compounds
Abstract:

The NCI60 cell line panel screen includes 60 human tumor cell lines derived from nine tumor types that has been used over the past 20+ years to screen small molecules, biologics, and natural products for activity. Cells in monolayer culture in 96-well plates are exposed to compounds for 48 h, and Sulforhodamine B is used to determine cell viability. Data analysis tools such as COMPARE allow classification of compounds based on the pattern of cell line response. However, many compounds highly active in monolayer cell culture fail to show efficacy in vivo. Therefore, we explored 3D culture of the NCI60 panel as a strategy to improve the predictive accuracy of the screen. 3D cultures more closely resemble tumors than monolayer cultures with tighter cell-cell contact and nutrient and oxygen gradients between the periphery and the center. We optimized the NCI60 cell line panel for generating 3D spheroids of a prespecified diameter (300–500 µm) in ultra-low attachment (ULA) plates. Spheroids were classified into four categories based on imaging, and concentration response of select agents in 2D and 3D models is presented.

A High-Throughput Screening Model of the Tumor Microenvironment for Ovarian Cancer Cell Growth
Abstract:

The tumor microenvironment plays an important role in the processes of tumor growth, metastasis, and drug resistance. We have used a multilayered 3D primary cell culture model that reproduces the human ovarian cancer metastatic microenvironment to study the effect of the microenvironment on the pharmacological responses of different classes of drugs on cancer cell proliferation. A collection of oncology drugs was screened to identify compounds that inhibited the proliferation of ovarian cancer cells growing as monolayers or forming spheroids, on plastic and on a 3D microenvironment culture model of the omentum metastatic site, and also cells already in preformed spheroids. Target-based analysis of the pharmacological responses revealed that several classes of targets were more efficacious in cancer cells growing in the absence of the metastatic microenvironment, and other target classes were less efficacious in cancer cells in preformed spheres compared to forming spheroid cultures. These findings show that both the cellular context of the tumor microenvironment and cell adhesion mode have an essential role in cancer cell drug resistance. Therefore, it is important to perform screens for new drugs using model systems that more faithfully recapitulate the tissue composition at the site of tumor growth and metastasis.
A 1536-Well 3D Viability Assay to Assess the Cytotoxic Effect of Drugs on Spheroids
Abstract:

Evaluation of drug cytotoxicity traditionally relies on use of cell monolayers, which are easily miniaturized to the 1536-well plate format. Three-dimensional (3D) cell culture models have recently gained popularity thanks to their ability to better mimic the complexity of in vivo systems. Despite growing interest in these more physiologically relevant and highly predictive cell-based models for compound profiling and drug discovery, 3D assays are currently performed in a medium- to low-throughput format, either in 96-well or 384-well plates. Here, we describe the design and implementation of a novel high-throughput screening (HTS)–compatible 1536-well plate assay that enables the parallel formation, size monitoring and viability assessment of 3D spheroids in a highly consistent manner. Custom-made plates featuring an ultra-low-attachment surface and round-bottom wells were evaluated for their compatibility with HTS requirements through a luminescence-based cytotoxicity pilot screen of ~3300 drugs from approved drug and National Cancer Institute (NCI) collections. As anticipated, results from this screen were significantly different from a parallel screen performed on cell monolayers. With the ability to achieve an average Z′ factor greater than 0.5, this automation-friendly assay can be implemented to either profile lead compounds in a more economical plate format or to interrogate large compound libraries by ultra-HTS (uHTS).
RNAi High-Throughput Screening of Single- and Multi-Cell-Type Tumor Spheroids: A Comprehensive Analysis in Two and Three Dimensions
Abstract:

The widespread use of two-dimensional (2D) monolayer cultures for high-throughput screening (HTS) to identify targets in drug discovery has led to attrition in the number of drug targets being validated. Solid tumors are complex, aberrantly growing microenvironments that harness structural components from stroma, nutrients fed through vasculature, and immunosuppressive factors. Increasing evidence of stromally-derived signaling broadens the complexity of our understanding of the tumor microenvironment while stressing the importance of developing better models that reflect these interactions. Three-dimensional (3D) models may be more sensitive to certain gene-silencing events than 2D models because of their components of hypoxia, nutrient gradients, and increased dependence on cell-cell interactions and therefore are more representative of in vivo interactions. Colorectal cancer (CRC) and breast cancer (BC) models composed of epithelial cells only, deemed single-cell-type tumor spheroids (SCTS) and multi-cell-type tumor spheroids (MCTS), containing fibroblasts were developed for RNAi HTS in 384-well microplates with flat-bottom wells for 2D screening and round-bottom, ultra-low-attachment wells for 3D screening. We describe the development of a high-throughput assay platform that can assess physiologically relevant phenotypic differences between screening 2D versus 3D SCTS, 3D SCTS, and MCTS in the context of different cancer subtypes. This assay platform represents a paradigm shift in how we approach drug discovery that can reduce the attrition rate of drugs that enter the clinic.
Exploring Drug Dosing Regimens in Vitro using Real-Time 3D Spheroid Tumor Growth Assays
Abstract:

Two-dimensional monolayer cell proliferation assays for cancer drug discovery have made the implementation of large-scale screens feasible but only seem to reflect a simplified view that oncogenes or tumor suppressor genes are the genetic drivers of cancer cell proliferation. However, there is now increased evidence that the cellular and physiological context in which these oncogenic events occur play a key role in how they drive tumor growth in vivo and, therefore, in how tumors respond to drug treatments. in vitro 3D spheroid tumor models are being developed to better mimic the physiology of tumors in vivo, in an attempt to improve the predictability and efficiency of drug discovery for the treatment of cancer. Here we describe the establishment of a real-time 3D spheroid growth, 384-well screening assay. The cells used in this study constitutively expressed green fluorescent protein (GFP), which enabled the real-time monitoring of spheroid formation and the effect of chemotherapeutic agents on spheroid size at different time points of sphere growth and drug treatment. This real-time 3D spheroid assay platform represents a first step toward the replication in vitro of drug dosing regimens being investigated in vivo. We hope that further development of this assay platform will allow the investigation of drug dosing regimens, efficacy, and resistance before preclinical and clinical studies.