Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery

Summary

Here, we present a method to identify compounds that modulate the ADCC mechanism, an important cancer cell-killing mechanism of antitumor antibodies. The cytotoxic effect of NK cells is measured in breast cancer cell spheroids in the presence of Trastuzumab. Image analysis identifies live and dead killer and target cells in spheroids.

Abstract

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of action of the antibodies is antibody-dependent cellular cytotoxicity (ADCC), where the antibody binds to the cancer cells and engages the cellular component of the immune system, e.g., natural killer (NK) cells, to kill the tumor cells. The effectiveness of these therapies could be improved by identifying adjuvant compounds that increase the sensitivity of the cancer cells or the potency of the immune cells. In addition, undiscovered drug interactions in cancer patients co-medicated for previous conditions or cancer-associated symptoms may determine the success of the antibody therapy; therefore, such unwanted drug interactions need to be eliminated. With these goals in mind, we created a cancer ADCC model and describe here a simple protocol to find ADCC-modulating drugs. Since 3D models such as cancer cell spheroids are superior to 2D cultures in predicting in vivo responses of tumors to anticancer therapies, spheroid co-cultures of EGFP-expressing HER2+ JIMT-1 breast cancer cells and the NK92.CD16 cell lines were set up and induced with Trastuzumab, a monoclonal antibody clinically approved against HER2-positive breast cancer. JIMT-1 spheroids were allowed to form in cell-repellent U-bottom 96-well plates. On day 3, NK cells and Trastuzumab were added. The spheroids were then stained with Annexin V-Alexa 647 to measure apoptotic cell death, which was quantitated in the peripheral zone of the spheroids with an automated microscope. The applicability of our assay to identify ADCC-modulating molecules is demonstrated by showing that Sunitinib, a receptor tyrosine kinase inhibitor approved by the FDA against metastatic cancer, almost completely abolishes ADCC. The generation of the spheroids and image acquisition and analysis pipelines are compatible with high-throughput screening for ADCC-modulating compounds in cancer cell spheroids.

Introduction

Multicellular tumor spheroids (MCTS) are widely used three-dimensional (3D) models that form due to the tendency of adherent cells to aggregate and represent an important tool for gaining mechanistic insight into cancer cell biology. They can be generated from a broad range of cell types by numerous techniques, such as liquid-based and scaffold-based 3D cultures¹. Their main advantage over monolayer 2D models is that they recapitulate the main features of in vivo tumors, namely structural organization and hypoxia, by mimicking the biological behavior of tumor cells, especially the mechanisms leading to therapeutic escape and drug resis....

Protocol

1. Setting up the JIMT-1-enhanced fluorescent protein (EGFP) spheroid model

1. To form a U-shaped cell-repellent bottom, coat the 96-well plate with 0.5% agarose-PBS solution (30 µL/well). Incubate the plate at room temperature for approximately 30-45 min.
2. Wash the JIMT-1-EGFP cells twice with 2 mL of sterile PBS (generation of JIMT1-EGFP cell line was reported in a previous publication¹⁷). Use T25 tissue culture flasks and JIMT-1 media (DMEM/F-12 medium supplemented with 20% fetal bovine serum (FBS), 0.3 U/mL insulin (100 IU/mL, Humulin R), and 1% penicillin-streptomycin) for the cell culture.

Results

EGFP expressing JIMT-1 cells were generated, and spheroids were grown from these cells. Sunitinib was used as a test compound as it was previously shown to affect the course of ADCC¹⁷. Spheroids were allowed to clump for 72 h. On day 3, 10 µg/mL of Trastuzumab (or equimolar 6.6 µg/mL TR-F(ab')₂¹⁹) and NK cells (20:1) were added to the spheroids in the presence or the absence of 20 µM Sunitinib (1 h pre-treatment), for a total time of 24 h. JIMT.......

Discussion

Despite significant improvements in treating BC over the past several decades, patients still regularly develop medication resistance or experience negative side effects²⁴. The high morbidity and mortality linked to BC demand a continuing investigation into the underlying molecular mechanisms, just as robust screening platforms to identify new molecules actionable for therapeutic development²⁵. These strategies require cell culture-based translational assays. 3D tumor cultu.......

Materials

Name	Company	Catalog Number	Comments
96-well glass bottom Cell Carrier Ultra microplates	PerkinElmer, Waltham, MA, USA	LLC 6055302	for spheroids measurements
96-well tissue culture plates	TPP	92096	for cell seeding
α-MEM medium	Sigma	M8042	in NK medium
Agarose	Sigma	A9539	for spheroids seeding
Annexin V-Alexa Fluo 647 conjugate	Invitrogen-ThermoFisher Scientific	A23204	for apoptosis measurement with HCS
CD16.176 V.NK-92 cells	Dr. Kerry S. Campbell (the Fox Chase Cancer Center, Philadelphia, PA on behalf of Brink Biologics, Inc. San Diego, CA)	ATCC CRL-2407	for cell culture
Cell Tracker Blue	Invitrogen-Thermo Scientific, Waltham, MA, USA)	C2110	for staining of NK cells
DMEM/F-12 medium	Sigma	D8437	in JIMT1-EGFP medium
Dimethyl sulfoxide (DMSO)	Sigma	D8418	for coating HCS plate before transfering the spheroids
Fetal bovine serum (FBS)	Biosera	FB-1090/500	JIMT-1-EGFP and NK medium
Glutamine	Gibco	35,050–061	in NK medium
GraphPad Prism 8.0.1	GraphPad Software Inc., San Diego, CA, USA		for statistical analysis
Harmony software	PerkinElmer, Waltham, MA, USA		for HCA
IL-2	Proleukin, Novartis Hungária Kft., Budapest, Hungary	PHC0026	in NK medium
Insulin (Humulin R)	Eli Lilly	HI0219	JIMT-1-EGFP medium
JIMT-1 breast cancer cells			for cell culture
MEM Non-essential Amino Acids (MEM-NEAA)	Gibco	11,140–050	in NK medium
Na-pyruvate	Lonza	BE13-115E	in NK medium
Opera Phenix High-Content Analysis equipment	PerkinElmer, Waltham, MA, USA	HH14001000	for HCA
PBS (Posphate buffered saline)	Lonza	BE17-517Q	for washing the cells
Penicillin-Streptomycin	Biosera	LM-A4118	JIMT-1-EGFP and NK medium
pLP-1, pLP-2, pLP-VSV-G, pWOXEGFP	Invitrogen, (Prof. József Tzsér, University of Debrecen)		for JIMT-1-EGFP cell line
Pluronic-F127	Sigma	P2443	for coating HCS plate before transfering the spheroids
Sunitinib malate	SigmaAldrich	PZ0012	for treatments
Trastuzumab Ab (humanized anti-HER2 monoclonal antibody)	Herzuma®, EGIS Pharmaceuticals, Budapest, Hungary	NDC-63459-303-43	for treatments
Trastuzumab-F(ab')2	Gift from Prof. György Vereb and Árpád Ször	Department of Biophysics and Cell Biology, University of Debrecen	for treatments
Trypan blue 0.4% solution	Sigma	T8154	for cell counting
Trypsin-EDTA 1X in PBS	Biosera	LM-T1706	for cells detachment