Recursos - Pôsteres Científicos
Validation of an Image-Based 3D Natural Killer Cell Mediated Cytotoxicity AssayDownload
February 13, 2017
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 cancer, non-small cell lung cancer, and clear cell renal cell carcinoma.
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 vivo. 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.