Brad Larson and Glauco Souza on automated, label-free image- based methods to monitor inhibition of metastatic cell migration in 3D colorectal cancer models
Colorectal 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.
The 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. Therefore, therapies that incorporate mTOR inhibitors may reduce CRC metastasis and improve survival rates.
One limitation when developing therapiesor 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 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. This 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. Then 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 determinations were performed in the modular Cytation 5 Cell Imaging Multi- Mode Reader with integrated Gen5 Microplate Reader and Imager Software from BioTek Instruments.
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). This also allows appropriate placement of object masks (Fig. 1C) around the complete 3D structure, composed of cells and ECM, to accurately assess migration rates.
In this manner, each captured image of wells containing cells and ECM treated with various concentrations of the inhibitor compound KU-0063794, or untreated wells, over the 48-hour imaging period, was subject to preprocessing prior to placing object masks over the area (images not shown). Over time, cells and ECM in untreated wells are seen to migrate away from the original printed area to cover an increasing portion of the microplate well. This same migration is completely inhibited by the highest tested concentrations of KU-0063794, confirming the compound’s ability to inhibit cell signalling pathways leading to mTOR activation.
Additionally, the area covered by each 3D combination of cells and ECM was automatically calculated by Gen5 software, and returned as a calculated metric at each timepoint for the various compound concentrations tested. This data, along with fold change values compared to original coverage area at time 0, were then graphed as seen in Fig. 2. The data aligns with the phenotypic image findings, and confirms the dose-dependent effect of KU- 0063794 on the migratory ability of HCT116 cells.
Incorporating an automated 3D cell culture workflow in colorectal cancer research allows increased throughput and reduced human subjectivity in an environment that more closely resembles that found in vivo. A magnetic 3D bioprinting process, such as the BiO Assay Kit, provides a biomimetic 3D structure with essential cell-cell and cell-ECM communication pathways, while a multi-mode microplate reader with automated digital imaging and advanced analysis capabilities, such as the Cytation 5, simplifies the experimental process by generating and analysing reproducible data in a single instrument.
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Brad Larson is with BioTek Instruments and Glauco Souza is with Nano3D Biosciences.