62 BIOTECHNOLOGY
AUTOMATED WORKFLOW for CANCER RESEARCH
Brad Larson & Glauco Souza on automated, label-free image- based methods to monitor inhibition of metastatic cell migration in 3D colorectal cancer models
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olorectal cancer (CRC) is a devastating disease, not only for the person afflicted with it but also for the patient’s entire support system, and society in general. Te cancer is known to resist treatment therapies and metastasize, often to the liver, thus considerably reducing survival rates for those in advanced stages. Research into the mechanisms of CRC tumorigenesis and metastasis indicate that two distinct complexes of the downstream serine/threonine kinase ‘mechanistic target of rapamycin’ (mTOR), known as mTORC1 (containing RAPTOR) and mTORC2 (containing RICTOR), are overexpressed. Terefore, therapies that incorporate mTOR inhibitors may reduce CRC metastasis and improve survival rates. One limitation when
developing therapies or characterising CRC tumorigenesis and metastasis is culturing the cells in vitro. In the body, CRC tumours grow as three dimensional (3D) masses, whereas many culture methods rely on single-layer, or two dimensional (2D), cell attachment to a solid surface. Such artificial 2D culture
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conditions prevent formation of complex cell-cell or cell- extracellular matrix (ECM) communication networks, thereby not allowing cells to replicate in vivo morphology and function. In contrast, 3D cell culture methods enable cells to organise in a manner that forms complex communication networks, which exist as part of in vivo microenvironments. Automating imaging and analysis of kinetic label-free assays performed with cells cultured in 3D increases laboratory throughput, while also providing robust, accurate and repeatable results. Here, we demonstrate how automation and a 3D cell culture workflow are applied to characterisation of CRC cell migration.
Automated 3D cell culture workflow As part of the BiO Assay Kit and protocol from Nano3D Biosciences and Greiner Bio-One, HCT116 epithelial colorectal carcinoma cells were incubated with a nontoxic magnetic nanoparticle assembly, known as NanoShuttle-PL. Tis magnetised the cells without inducing an inflammatory cytokine response, and allowed the cells to levitate and induce ECM formation during incubation. After incubation, cell aggregates were broken up to create a single cell suspension, and transferred to a separate 384-well assay plate, along with titrated inhibitor compounds known to inhibit cell signalling pathways leading to mTOR activation. Ten magnetic 3D bioprinting was used, where the plate was placed atop a 384-well magnet drive so that cells are
magnetically patterned into a dot/spheroid configuration at the well bottom. Next, the magnet was removed, and automated kinetic brightfield imaging was performed over 48 hours to track cell and ECM movement away from the original HCT116 spheroid pattern. Automated image analysis and processing, along with automated quantitative
A
determinations were performed in the modular Cytation 5 Cell Imaging Multi- Mode Reader with integrated Gen5 Microplate Reader and Imager Software from BioTek Instruments.
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Automated 3D cell culture analysis Prior to phenotypic analysis, the images were subject to a preprocessing step using Gen5 software. By automatically applying preprocessing parameters, overall image quality is improved by reducing or eliminating uneven contrast, background noise and artifacts without the time and subjectivity inherent to manual methods (Fig. 1). Tis also allows appropriate placement of object masks (Fig. 1C) around the complete 3D structure, composed of cells and ECM, to accurately assess migration rates.
C
Fig. 1. Image background signal removal via pre- processing. Representative 2.5x brightfield image (A) before; and (B) after pre- processing; (C) object masks automatically placed around cells using Gen5 Software
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