Real-time Cytotoxicity Assays in Human Whole Blood

Summary

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

Abstract

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex vivo condition.

Introduction

Recent advances in the pharmaceutical industry have led to an increased interest in realizing the specific identifications of tumor cell antibodies and personalized cancer treatments; however, several obstacles are encountered in the process. Only 5% of agents that have anticancer activity in preclinical development are licensed after showing sufficient efficacy in phase II-III testing^1,2. The preclinical strategies (both in vitro and in vivo) are suboptimal as many examples have shown that antitumor drugs behave differently in human and in laboratory animals mainly due to their different blood components^3-5.

Protocol

1. Target Cell Preparation

1. Maintain target cells (e.g. Raji lymphoma cells) in growth media (RPMI 1640 culture medium, with 10% heat-inactivated fetal bovine serum (FBS), 4nM L-glutamine, and 500 IU/ml penicillin/ streptomycin) at 37^oC in a 5% CO₂ incubator.
2. Centrifuge the sample to pellet the target cells in a 15 ml tube. Centrifugation time varies with different cell types; centrifuge for 3 min at 468 x g for Raji cells.
3. First aspirate any bubbles formed on surface of the supernatant, and remove the entire supernatant.
4. Add 10 ml of PBS to the tube. Re-suspend the cells by pipetting the solution....

Results

Anti-CD20 antibodies and lymphoma cells (Raji cells and MC/CAR) were chosen as a model system to demonstrate the whole blood cytotoxicity assay (WCA)^7,8. Raji cells had high copy number of CD20 on the cell surface, while MC/CAR cells had low copy number of CD20 on their membrane. Targeted cells were first stained green with green fluorescence cytosolic dyes and arrayed on the 96-well plate. 10,000 - 50,000 target lymphoma cells were immobilized in each well. 180 µl of freshly drawn human whole blood was added .......

Discussion

WCA is a critical in vitro anti-cancer screening tool with single cell resolution^12-16, ideally utilized after traditional target screenings such as CDC and ADCC assays^9-11, and before preclinical animal tests. Currently, primary target screening assays such as CDC or ADCC assays are all performed in a simplified media or a buffer system. However, drug candidates that show efficacy in these simplified buffer system are not always effective in the more complex whole blood system. Therefore, .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cell attachment 96 well plate kits	Adheren	AP9601
Suspension Single cell array 8 well chamber slide	Adheren	SS0801
Adherent Single cell array 8 well chamber slide	Adheren	SS0802
Lymphoma cell line CD20+	ATCC	CCL-86	Raji cells
Lymphoma cell line CD20-	ATCC	CRL-8083	MC/CAR
RPMI 1640 with L-glutamine	Life Technologies	11875-119
Fetal Bovine Serum	Thermo	SH30070.01HI
Peni/Strep	Life Technologies	15070063
Cytosolic dye	Life Technologies	C7025	Cell Tracker Green
Rituxan (Biosimilar)	Eureka Therapeutics
Human whole blood	Allcells	WB001
Propidium Iodide	Sigma	P4170-10MG
Automatic imaging system	Molecular Devices	Contact Vendor	Cell Reporter
Cell counting program	Molecular Devices	Contact Vendor	Cell Reporter