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Validation of an Image-Based 3D Natural Killer Cell Mediated Cytotoxicity Assay
Validation of an Image-Based 3D Natural Killer Cell Mediated Cytotoxicity Assay
Submitted on 11 Jul 2017

Brad Larson1, Lubna Hussain2 and Jenny Schroeder3
1BioTek Instruments, Inc. Winooski, VT USA • 2Lonza Walkersville Inc. Walkersville, MD USA • 3Lonza GmbH Cologne DE
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Poster Abstract
Natural killer (NK) cells are a type of cytotoxic lymphocyte found in peripheral blood that play a role in host defense and immune regulation. Most recently, NK cells have become of particular interest in the field of immunotherapy due to their potential to target and destroy specific cancer cells, while leaving non-target healthy cells intact. The anti-cancer activity of NK cells has been shown to be associated with a better prognosis in several cancers such as colorectal cancer1, non-small cell lung cancer2, and clear cell renal cell carcinoma3.

In order to properly study the interaction between NK cells and target tumor cells, an appropriate in vitro model system must be established. However, much of the data published to date has used cancer cells plated as a two dimensional (2D) monolayer on the bottom of microplate wells. A growing amount of data has shown that cells cultured in this manner lack the cell:cell and cell:matrix communication, metabolic gradients, and polarity demonstrated in vivo4. The ability to perform matrix infiltration studies is also eliminated with the use of 2D cell culture. By embedding cancer cells into a three dimensional (3D) matrix and allowing the formation of tumor spheroids, or tumoroids, the shortcomings of using 2D cultured cells can be overcome as communication networks and cellular gradients observed within in vivo tumors are reestablished.

With the incorporation of 3D cultured cells, however, traditional methods to monitor target and NK cell interactions, and subsequent target cell killing can become problematic. Microplate reader assays designed to detect signal from cell monolayers lack the sensitivity to quantify signal from spheroids surrounded by non-cell containing areas in the well with no signal generation. By incorporating microscopy-based detection and cellular analysis, signal emanating solely from spheroids is quantified, providing a highly robust method to detect induced toxicity within target cancer cells.

Here we describe a novel 3D NK cell mediated cytotoxicity (CMC) assay. HCT116 colorectal cancer cells were embedded within a collagen hydrogel of defined concentration and thickness, mimicking in vivo extracellular matrix (ECM). Following cell propagation to create tumoroids within the matrix, HCT116 and NK cells were labeled with individual cell tracking dyes, followed by NK cell addition. Fluorescent apoptosis and necrosis probes were also added to track cytotoxic events within the tumoroids. Cellular imaging and analysis were performed at regular intervals over a seven day period to monitor NK cell binding to the tumoroids and induced apoptosis and necrosis of the HCT116 cells making up each tumoroid. Experimental testing validated that the combined assay technique provides a sensitive, accurate, and repeatable in vitro method to determine the ability of NK cells to target and kill tumor cells.

1. The RAFT 3D cell culture system provides an ideal method to prepare and study co-cultures of tumoroids and immune cells.
2. Peripheral blood CD56+ CD16+ natural killer cells can be used with 3D and 2D cultured target cells to assess potential CMC activity.
3. IL-2 activation increases the cytotoxic effect of NK cells on 3D and 2D cultured target HCT116 cells.
4. The BioSpa 8 and Cytation 5 can be combined to provide an automated method, with proper environmental conditions, to perform long-term CMC assays.
5. The combination of appropriate 3D cell models, assay methodology, and walk away automation provide a robust process to generate accurate in vitro NK cell mediated cytotoxicity results.
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