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. The development of more sophisticated cell culture techniques and their further adaption to multi-well formats has enabled the search for new drugs in 3D settings. In contrast to cells grown under 2D conditions, 3D models of tumors range in complexity from layered cellular systems⁵ to single-cell-type spheres of different sizes, to complex multi-cell-type spheres^6,7,8. The discovery of novel compounds or biologics that potently induce cell death in these 3D model systems is, therefore, of high interest in drug development campaigns. Endpoints of these assays are often identical to those performed in 2D cultures to assess changes in cell proliferation, but when conducted in a more physiologically relevant setting, they may reveal the true level of dependency of the target gene or pathway being interrogated.

As introduced above, a variety of model systems have been developed to study drug responses in 3D culture systems, but the majority use either 96 or 384-well microtiter plates and are not easily adaptable to the high-throughput screening (HTS) formats often used in drug discovery screening campaigns. Such systems include the use of hanging droplet technologies, spheroid cultures, pulsating cells with magnetic particles to induce levitation, and cultures incorporating natural or synthetic gels such as collagen, Matrigel, or polyethylene glycol (PEG)². Here, we present the detailed methods of a previously developed technique to produce 3D spheroid cultures from established cancer cell lines in a 1536-well plate format. In this protocol, a highly defined medium is used which prevents the attachment of normally adherent cell lines⁹. This system has limitations (i.e., it cannot fully recapitulate a complex model system of cancer), but nevertheless, these assays enable a high-throughput screening of large collections of small molecules and crosswise comparisons of drug response between 2D and 3D cultures against a variety of cell lines and compounds.

The cell lines selected to demonstrate the methods in this article all harbor mutations in genes related to the MAPK signaling pathway, a pathway which is highly dysregulated in cancer, and for which many therapeutics are available. Many of the lines have activating oncogenic mutations in the Kirsten Rat Sarcoma virus also referred to as KRAS, the Neuroblastoma RAS or NRAS, the Harvey Rat Sarcoma virus oncogene or HRAS, and the associated kinases Rapidly Accelerated Fibrosarcomas, also known as RAFs. Recent literature suggests that the inhibitors of different nodes of this pathway are uniquely more efficacious in a subset of the cell lines when grown under 3D conditions^9,10. One study found that when cancer cells with active RAS were cultured in 3D, they demonstrated an increased sensitivity to MAPK inhibitors, and further, that this approach could identify pathways and targeted inhibitors that might be missed in the traditional 2D setting. The goal of this study is to present the methods used to culture these cell lines, and further, to demonstrate the differential responses to these inhibitors which can be observed only when using 3D cell culture systems.

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. Detach the cancer cell lines, which have been growing according to recommended routine culture conditions, from the traditional cell-culture flasks by washing them 3x with 1x phosphate buffered saline (PBS) and adding an appropriate amount of 0.25% trypsin (1 mL per 5 cm²) for 5 min to create a thin layer over the cells. Neutralize the trypsin with 5x the amount of the medium containing the serum and proceed to count the cells. For example, use 25 mL of medium for 5 mL of trypsin.
4. Adjust the concentration of the cell solution to 62.5 x 10⁴ cells/mL in order to seed a total of 500 cells per well in 8 µL of spheroid medium into the 1536-well tissue-culture-treated plates.
5. In a biological safety cabinet, seed the cells using a sterilized, small stainless-steel-tipped cassette with a peristaltic-pump-based system. Between different cell lines, flush the tubing of the cassette with 1x PBS and 70% Ethanol for 10 s.
6. Alternatively, seed the cells using a liquid handler located within a HEPA-filtered room using either a sterilized, small stainless-steel-tipped cassette and a peristaltic pump or an attached syringe pump, depending on the number of plates per cell line needed.
7. Seal the plates using a breathable adhesive plate seal either manually or using a plate sealer.
8. Place the plates in a spinning incubator set at 10 rpm and 37 °C with 5% carbon dioxide (CO₂) and 95% relative humidity. Allow the spheroids to form for 3 d.

2. Culturing of 1536-well 2D Cancer Lines

NOTE: The 1536-well 2D cancer lines should be cultured 24 h before the addition of the compound to the 3D plates.

1. Prepare a 2D cancer cell medium by adding the following reagents to 500 mL of an appropriate growth medium for the selected lines: 1x penicillin/streptomycin and 10% fetal bovine serum (FBS).
2. Filter-sterilize the entire medium with the supplements through a 0.4 µm bottle-top filter system.
3. Detach the cancer cell lines, which have been growing according to recommended routine culture conditions, from the traditional cell-culture flasks by washing them 3x with 1x PBS and adding an appropriate amount of 0.25% trypsin (1 mL per 5 cm²) for 5 min to create a thin layer over the cells. Neutralize the trypsin with 5x the amount of the medium containing the serum and proceed to count the cells. For example, use 25 mL of medium for 5 mL of trypsin.
4. Adjust the concentration of the cell solution to 62.5 x 10⁴ cells/mL in order to seed a total of 500 cells per well in 8 µL of complete medium into 1536-well tissue-culture-treated plates.
5. In a biological safety cabinet, seed the cells using a sterilized, small stainless-steel-tipped cassette with a peristaltic-pump-based system. Between the different cell lines, flush the tubing of the cassette with 1x sterile PBS and 70% Ethanol for 10 s.
6. Alternatively, seed the cells using a liquid handler located within a HEPA-filtered robotics room using either a sterilized, small stainless-steel-tipped cassette and a peristaltic pump or the attached syringe pump, depending on the volume of the plates per cell line needed.
7. Seal the plates using a breathable adhesive plate seal either manually or using a plate sealer.
8. Place the plates in a spinning incubator set at 10 rpm and 37 °C for 24 h prior to the compound addition, with 5% CO₂ and 95% relative humidity.

3. Compound Addition

1. Prepare 8-point, 3-fold serial dilutions of MAPK inhibitors in 100% dimethyl sulfoxide (DMSO) on a liquid-handling robot with a 10 mM top concentration. The proteasome inhibitor Bortezomib is used as a positive control for a complete cell killing to determine the dynamic range of the assay.
2. Quick-spin all assay plates and compound plates at 100 x g to collect any condensation. Remove the adhesive seal from each plate and place a plate on an acoustic dispenser set-up to add a total of 8 nL of each compound/dilution representing a final concentration of 0.1% DMSO per well and a top compound dose of 10 µM.
3. After the addition of the compound is completed, place a custom-made, stainless-steel cell culture lid which prevents evaporation on the plate and place the plate into a spinning incubator at 37 °C for 5 d, with 5% CO₂ and 95% relative humidity.

4. Detection Reagent Addition and the Acquirement of Raw Data

1. Pre-warm an adequate volume of a cell lysis reagent containing luciferin to detect changes in adenine triphosphate (ATP) and, thus, changes in the cell proliferation at room temperature or in a 37 °C water bath. Add a total of 3 µL of the detection reagent to each well using a peristaltic pump and incubate the plate at room temperature for at least 15 min.
2. Capture the luminescence on a plate luminometer.

5. Data Processing

1. Extract the raw data from the instrument and normalize all fields containing test compounds to the average of all wells containing DMSO alone as the neutral control. From this value, calculate the percent growth inhibition of each compound. This is completed using the formula function in Microsoft Excel where f(x)={(sample value relative light units (RLUs)/(average DMSO value RLUs)*100)}.
2. Generate the dose-response curves and inhibitory IC50s by graphing the values of the normalized data from step 5.1 against the compound concentration using a graphing program.
   NOTE: We analyzed the data using Helios, an internal NIBR software tool. To complete this function, we selected the nonparametric curve analysis tool.

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