Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Summary

Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

Abstract

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin.

In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Introduction

In the past decade, more and more studies have implemented the use of 3D cell culture models to understand concepts that are not fully recapitulated by growing cells in 2D on plastic. Examples of these concepts include the alternations in normal epithelial cell polarity¹ where the spatial orientation of apical and basal layers of cells are lost, as well as the role of the extracellular matrix in regulating survival and cell fate. Oncology research, in particular, has used 3D models to understand the basic biology of cancer cells and the differences between 2D and 3D cell culture systems^3,4

Protocol

1. Culturing of 1536-well 3D Tumor Spheroids

1. Prepare a fresh 3D tumor spheroid medium stem (cell medium + knockout serum replacement + insulin-transferrin-selenium) by adding the following reagents to 500 mL of Dulbecco's Modified Eagles Medium (DMEM/F12): 1x penicillin/streptomycin, 10 ng/mL of human basic fibroblast growth factor (bFGF), 20 ng/mL of human epidermal growth factor (EGF), 0.4% bovine serum albumin (BSA) (Fraction V), 1x insulin-transferrin-selenium, and 1% knockout (KO) serum replacement (do not freeze-thaw).
2. Filter-sterilize the entire medium with the supplements through a 0.4 µm bottle-top filter system.
3. Detac....

Results

A variety of established cancer cell lines known to grow well under 2D culture conditions were tested using the methods outlined here. Representative images from a variety of MAPK mutant cancer cell lines (Calu-6:KRAS, NCI-H1299:NRAS, SK-MEL-30:NRAS, and KNS-62:HRAS) are seen in Figure 1. These images demonstrate that although the cell lines have various morphologies, each formed 3D structures in the 1536-well assay plates. Figure 2

Discussion

The methods presented here demonstrate a detailed protocol on how to produce tumor spheroids in 1536-well plates for large-scale compound screenings. These methods were initially adapted from work at the National Cancer Institute where tumor spheroids were grown in low- throughput assays in 6-well and 96-well plates to ask questions about genetic dependencies and compound sensitivity^13,14,15. A critical and unique feature of thi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
DMEM Dulbecco`s Modified Eagle Media (DMEM)/F12	ATCC	30-2006	Purchased as a prepared solution. This reciepe was found to be preferred compared to other vendors.
DMEM Dulbecco`s Modified Eagle Media (DMEM)/F12	Lonza	12-604F	Purchased as a prepared solution.
Roswell Park Memorial Institute (RPMI)	Lonza	12-115Q	Purchased as a prepared solution.
Fetal Bovine Serum (FBS)	Seradigm	1500-500	Purchased as a prepared solution.
1x 0.25% Trypsin with Ethylenediaminetetraacetic acid (EDTA)	Hyclone	SH30042.01	Purchased as a prepared solution.
1x Penicillin/Streptomycin	Gibco	15140	Purchased as a prepared solution.
10 ng/mL Human basic Fibrobast Growth Factor (bFGF)	Sigma	F0291	Resuspend in sterile water.
20 ng/mL Human Epidermal Growth Factor (EGF)	Sigma	E9644	Resuspend in sterile water.
0.4% Bovine Serum Albumin (BSA)	Sigma	A9418	Resuspend in sterile PBS and filter.
1x Insulin Transferrin Selenium (ITS)	Gibco	51500056
1% KnockOut (KO) Serum Replacement	Gibco	10828-028	Add fresh right before use.
100% Dimethyl sulfoxide (DMSO)	Sigma	276855-100ML
CellTiter-Glo	Promega	G7573	Purchased as a prepared solution.
Consumables
Small Combi Cassette	ThermoFisher	24073295
Small Multiflow Cassette	BioTek	294085
1536-well sterile TC plates (white)	Corning	3727
1536-well sterile TC plates (black)	Corning	3893
Scivax Plates	MBL International	NCP-LH384-10
Equipment
Adhesives seals	ThermoFisher	AB0718
Spinning Incubator	LiCONiC
Stainless Steel Lids	The Genomics Institute of Novartis Research Foundation (GNF)
ATS Acoustic Dispensor	EDS Biosystems
Echo Acoustic Dispensor	Labcyte
Luminometer	Envision-Perkin Elmer
Peristaltic Pump- Multidrop Combi	ThermoFisher
Liquid Handler-Multiflow FX	BioTek