CENTER-DRIVEN RESEARCH PROJECT 1: 3D HIGH-CONTENT SCREENING A. INTRODUCTION &SPECIFIC AIMS High-throughput cellular screens interrogate more biologically relevant processes than do cell-free screens, but they grow in complexity proportionally to their ability to mimic the in vivo state. While most high-content (cellimage- based) screening (HCS) is performed on standard cell culture models, it is well known that many normal and malignant cells lose key phenotypic and functional characteristics when grown in monolayer culture. Introduction of organotypic 3-dimensional (3D) culture and screening systems into mainstream small-molecule and drug discovery processes is increasingly discussed but severely limited by complex methodological requirements and a lack of sophisticated biological model systems, miniaturized screening methods, and 3D image analysis and instrumentation (21, 22). Burnham's Center-driven component will apply our multidisciplinary expertise in cell biology, cellular imaging, and high throughput microscopy (HTM) algorithm and instrumentation development to address this fundamental unmet need in chemical genomics and drug discovery. The broad objectives are to: (1) develop validated 3D model cellular systems for screening;(2) develop software analysis and instrumentation tools for 3D visualization and screening of the model systems;and (3) utilize the 3D culture protocols and 3D image analysis tools to perform screens and disseminating the tools and screens to the scientific community. Realization of these 3D culture models and HTM screening tools will fill the gap between traditional monolayer cultures and in vivo animal models, allowing all MLPCN users to generate their own 3D-HCS primary and secondary assays to enhance and expand the development of biologically meaningful Probes. We will pursue these goals through the following Specific Aims:
Aim 1. Develop a toolbox of model organotypic 3D systems for screening. We propose to develop a wide variety of 3D culture systems, including spheroids, multi-layered cultures, and co-cultures of interacting cell types, which are of high interest to the scientific community, in 384- and 1536-well formats amenable to medium and high throughput screening.
Aim 2. Create image acquisition and analysis tools for screening of 3D models. A range of image acquisition and analysis tools will be developed to enable primary and secondary 3D-HCS ~ from fixed endpoint 3D imaging for fast primary screens to high-resolution 3D time-lapse analyses for in-depth secondary screens. The following approaches will enable completion of this Aim: 2.1. Development and validation of 3D image acquisition using existing confocal and non-confocal HCS instruments, including deconvolution protocols. 2.2. Development and implementation of 2D assay read-outs from optical sections of 3D images. 2.3. Development and implementation of 3D assay read-outs directly from the 3D images. 2.4. Development and implementation of parallel confocal microscopy instrumentation to speed 3D image acquisition. 2.5. Implementation of 3D time-lapse image acquisition and analysis tools for living cells.
Aim 3. Integrate and validate tools from Aims 1 and 2 for primary and/or secondary 3D-HCS screens. Here we will integrate the tools developed in Aims 1 and 2 into assays that will be screened within the MLPCN, thus validating the technology for wider use in the scientific community.
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