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.

To address the necessity of an antitumor drug screening platform and to provide a check-point before costly animal experiments and clinical trials, a human whole blood cytotoxicity assay (WCA) is proposed for evaluating antitumor drug efficacy in a more relevant biological environment. The whole blood cytotoxicity assay can be used to evaluate the response of individual cells to antibodies and other drug candidates in human whole blood.

The WCA is developed by incorporating high-throughput cell positioning technology with high-throughput and high-content imaging⁶. By utilizing an automated imaging system, the number of both living and dead cells can be determined with a high degree of precision. Due to the fact that target cells are immobilized on the same focal plane, WCA is able to provide quantitative cytotoxicity analysis in real time without the removal of red blood cells. Moreover, the automatic imaging system provides several advantages such as that only target cells that have reached the specified criteria (e.g., fluorescently labeled cells, and cell morphology) are gated and processed. Also, it allows the production of 144 plates per day. Consequently, this imaging capability and throughput allows running of high-content and high-throughput experiments simultaneously. By combining WCA and the automated imaging system, high throughput quantitative cell cytotoxicity analysis can be achieved within a more biological relevant environment.

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 up and down several times to break up the cell clump.
5. Centrifuge the sample for 3 min at 468 x g.
6. Aspirate any bubbles formed on the surface of the supernatant, and remove the entire supernatant.

2. Preparation of Cell Microarrays

1. Obtain cell attachment 96 well plate kits or single cell array 8 well chamber slides (see table of materials/reagents).
2. Apply 1.2 μl of the Activator to the DNA reagent from the kit. Cap the vial, invert it, and gently shake to mix the solution.
3. Allow the solution to react for 20 min at RT.
4. Apply the mixed solution to the target cells pellet (in the 15 ml tube).
5. Add green fluorescence cytosolic dyes at a final concentration of 500 nM.
6. Incubate the target cells with the mixed solution and the dye for 30 min at RT on an orbital shaker.
7. Wash the cells twice with 5 ml PBS, and resuspend the cells in 10 ml growth media.
8. Apply 100 µl of the solution from step 2.7 to each well. Count the cell number by using a hemacytometer. Make sure cell numbers range between 5×10⁴ to 1×10⁶ cells per well.
9. After 10-15 min of incubation at RT, gently wash the samples twice with 200-300 µl growth media to remove any unbound cells.
   NOTE: About 5-10 µl growth media remains in each well after washing.

3. Whole Blood Cytotoxicity Assays

1. Add 180 μl of human whole blood to each sample well.
2. Obtain Anti-CD20 antibody solution at 5 mg/ml, and perform serial dilution to make 50, 20, 10, 5, 2, 1, 0.5 μg/ml of Anti-CD20 antibody solution in 1.5 ml tubes. Add 20 µl of each concentration of Anti-CD20 antibody to each sample well making triplicates.
3. Add Propidium iodide at a final concentration of 500 nM to each well.
4. Incubate the resulting plate at 37°C with 5% CO₂ for 16 hr.
5. Image the cells on the slides or 96 well plates with an automatic imaging system with 8X magnification.
6. Use the cell counting program of the automatic imaging system to quantify the results.
   NOTE: The software of the automatic imaging system provides simple step-by-step operations for cell counting. The protocol of using this software can be found at provider’s website.
7. Select [Review Results] from the main page of the software.
8. Select [The File Saved for The Samples].
9. First gate FITC mean intensity >50, then gate TxRed mean intensity >20.
10. Select [Plate Overview].
11. Select [Export Tables].
    NOTE: The percentage cell killing formula of the program is shown below:
    % target cell killing = (# of cells stained both green and red/ # of cells stained green)*100%

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 a...

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

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