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Real time assays for quantifying cytotoxicity with single cell resolution.

ABSTRACT: A new live cell-based assay platform has been developed for the determination of complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC), and overall cytotoxicity in human whole blood. In these assays, the targeted tumor cell populations are first labeled with fluorescent Cell Tracker dyes and immobilized using a DNA-based adhesion technique. This allows the facile generation of live cell arrays that are arranged arbitrarily or in ordered rectilinear patterns. Following the addition of antibodies in combination with serum, PBMCs, or whole blood, cell death within the targeted population can be assessed by the addition of propidium iodide (PI) as a viability probe. The array is then analyzed with an automated microscopic imager. The extent of cytotoxicity can be quantified accurately by comparing the number of surviving target cells to the number of dead cells labeled with both Cell Tracker and PI. Excellent batch-to-batch reproducibility has been achieved using this method. In addition to allowing cytotoxicity analysis to be conducted in real time on a single cell basis, this new assay overcomes the need for hazardous radiochemicals. Fluorescently-labeled antibodies can be used to identify individual cells that bear the targeted receptors, but yet resist the CDC and ADCC mechanisms. This new approach also allows the use of whole blood in cytotoxicity assays, providing an assessment of antibody efficacy in a highly relevant biological mixture. Given the rapid development of new antibody-based therapeutic agents, this convenient assay platform is well-poised to streamline the drug discovery process significantly.

SUBMITTER: Hsiao SC

PROVIDER: S-EPMC3691166 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Real time assays for quantifying cytotoxicity with single cell resolution.

Hsiao Sonny C SC Liu Hong H Holstlaw Taylor A TA Liu Cheng C Francis Catherine Y CY Francis Matthew B MB

PloS one 20130624 6

A new live cell-based assay platform has been developed for the determination of complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC), and overall cytotoxicity in human whole blood. In these assays, the targeted tumor cell populations are first labeled with fluorescent Cell Tracker dyes and immobilized using a DNA-based adhesion technique. This allows the facile generation of live cell arrays that are arranged arbitrarily or in ordered rectilinear patterns. Fo ...[more]

PMID: 23826123

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Project description:A growing understanding of the molecular interactions between immune effector cells and target tumor cells, coupled with refined gene therapy approaches, are giving rise to novel cancer immunotherapeutics with remarkable efficacy in the clinic against both solid and liquid tumors. While immunotherapy holds tremendous promise for treatment of certain cancers, significant challenges remain in the clinical translation to many other types of cancers and also in minimizing adverse effects. Therefore, there is an urgent need for functional potency assays, in vitro and in vivo, that could model the complex interaction of immune cells with tumor cells and can be used to rapidly test the efficacy of different immunotherapy approaches, whether it is small molecule, biologics, cell therapies or combinations thereof. Herein we report the development of an xCELLigence real-time cytolytic in vitro potency assay that uses cellular impedance to continuously monitor the viability of target tumor cells while they are being subjected to different types of treatments. Specialized microtiter plates containing integrated gold microelectrodes enable the number, size, and surface attachment strength of adherent target tumor cells to be selectively monitored within a heterogeneous mixture that includes effector cells, antibodies, small molecules, etc. Through surface-tethering approach, the killing of liquid cancers can also be monitored. Using NK92 effector cells as example, results from RTCA potency assay are very well correlated with end point data from image-based assays as well as flow cytometry. Several effector cells, i.e., PBMC, NK, CAR-T were tested and validated as well as biological molecules such as Bi-specific T cell Engagers (BiTEs) targeting the EpCAM protein expressed on tumor cells and blocking antibodies against the immune checkpoint inhibitor PD-1. Using the specifically designed xCELLigence immunotherapy software, quantitative parameters such as KT50 (the amount of time it takes to kill 50% of the target tumor cells) and % cytolysis are calculated and used for comparing the relative efficacy of different reagents. In summary, our results demonstrate the xCELLigence platform to be well suited for potency assays, providing quantitative assessment with high reproducibility and a greatly simplified work flow.

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Real time assays for quantifying cytotoxicity with single cell resolution.

Publications

Real time assays for quantifying cytotoxicity with single cell resolution.

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