Spheroid Drug Sensitivity Screening in Glioma Stem Cell Lines

Summary

In vitro drug sensitivity screens are important tools for discovering anti-cancer drug combinations. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening for spheroid lines.

Abstract

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens are performed on cells grown in a monolayer. However, these two-dimensional (2D) models are considered less accurate compared to three-dimensional (3D) spheroid cell models; this is especially true for glioma stem cell lines. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening of spheroid lines; mouse and human glioma stem cell lines are used as an example. This protocol describes a 3D spheroid drug sensitivity and synergy assay that can be used to determine if a drug or drug combination induces cell death and if two drugs synergize. Glioma stem cell lines are modified to express RFP. Cells are plated in low attachment round well bottom 96 plates, and spheres are allowed to form overnight. Drugs are added, and the growth is monitored by measuring the RFP signal over time using the Incucyte live imaging system, a fluorescence microscope embedded in the tissue culture incubator. Half maximal inhibitory concentration (IC50), median lethal dose (LD50), and synergy score are subsequently calculated to evaluate sensitivities to drugs alone or in combination. The three-dimensional nature of this assay provides a more accurate reflection of tumor growth, behavior, and drug sensitivities in vivo, thus forming the basis for further preclinical investigation.

Introduction

Glioblastoma is a devastating, high-grade neoplasm of the brain with a 5% five-year overall survival¹. High-grade gliomas (HGG) like glioblastoma represent the leading cause of cancer-related mortality in the pediatric population² and are one of the most recalcitrant tumors to treat in adults as well³. Despite significant advances in our understanding of the molecular drivers of HGG, treatment options remain limited³, emphasizing the need for drug screening methods that more accurately predict therapeutic sensitivities in the clinic.

3D cell cultures have primarily been used for the modeling of physiologically relevant cell behavior⁴. Furthermore, the 3D architecture of the tumor microenvironment can be recapitulated in vitro by establishing 3D growth assays⁵. Spheroid growth also activates different signaling pathways and is hence considered more representative of in vivo models^6,7 compared to 2D culture. Pediatric HGG stem cell and our mouse NF1 glioma stem cell lines naturally grow as neurospheres, and used these mouse NF1 glioma cell lines in a medium throughput drug screen⁸. The pediatric lines used here were derived from hemispheric, midline, and cerebellar pediatric HGG and were acquired from and fully characterized by the Children's Brain Tumor Network (mutation and gene expression profiles)⁹. These lines were modified to express a nuclear red fluorescent protein (RFP), which allows for monitoring of proliferation and survival using the Incucyte live imaging system. The intensity of the RFP signal is representative of the number of cells present. Other fluorophores, like green fluorescent protein (GFP), could be used as well.

Combination chemotherapy for childhood acute lymphoblastic leukemia, lymphomas, epithelial malignancies, and many other cancers is an effective way to eradicate tumors and prevent drug resistance to single agents^10,11. However, there is limited information on which agents to combine to achieve therapeutic sensitivities in HGG, encouraging the use of more accurate spheroid models in in vitro drug testing.

Protocol

All protocol procedures were approved by the Children's Hospital of Philadelphia Institutional Review Board (IRB). 1. 3D spheroid cell plating Prepare glioma stem cell media: To make the base media, add 50 mL of the proliferation supplement and 5 mL of a 100x penicillin-streptomycin solution (10,000 U/mL) to the basal medium (500 mL). To make glioma stem cell media, add epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) to a final conc…

Representative Results

As an example, the synergy of Trametinib (MEK inhibitor) and GDC-0941 (PI3K inhibitor), which inhibit two RAS downstream effector pathways in mouse glioma stem cell line 5746 (RFP expressing) was evaluated (Figure 4). Figure 4A shows the same sphere at 0 h and 72 h treated with a combination of Trametinib and GDC-0941. These images were exported directly from the live imager software. IC50 and LD50 were calculated as described in GraphPad (<strong class="xf…

Discussion

This protocol describes the 3D drug screening assays that have been effectively used to assess drug vulnerabilities in spheroid models of glioma⁸. This 3D spheroid assay system was specifically designed to allow for a more accurate preclinical investigation of combinatorial chemotherapies for glioma cell lines grown in spheres. For HGG, this method provides a framework for identifying prospective drug vulnerabilities for this devastating disease. However, the potential applications of this system …

Materials

15 mL centrifugation tubes	CELLTREAT	229411	15 mL polypropylene centrifuge tubes, sterile
96- well plate	S-bio	MS9096UZ	96-well round-bottom ultra-low attachment plate
Accutase	STEMCELL Technologies	7922	Cell detachment solution
Acridine Orange/Propidium Iodide Stain	Logos Biosystems	F23001	Live/dead stain for cell counting
bFGF	STEMCELL Technologies	78003.2	Human recombinant bFGF
Cell Counter	Logos Biosystems	L20001	LUNA-FL Dual Fluorescence Cell Counter
Centrifuge	Eppendorf	5810R	Centrifuging cells and plates
DMSO	Pierce	20688	solvent for compounds
EGF	STEMCELL Technologies	78006.2	Human recombinant EGF
Eppendorf tubes	Costar	07-200-534	Microcentrifuge tubes
Excel	Microsoft		Microsoft excel
GDC-0941	Selleckchem	S1065	Drug 1
GraphPad	GraphPad	GraphPad Prism 9	Calculation of IC50 and LD50
Hemocytometer	Logos Biosystems	LGBD10008	Luna PhotonSlide
Incucyte	Sartorius	S3	Fluorescence microscope embedded in the tissue culture incubator that images every well at specific time intervals.
Incucyte software	Sartorius	Incucyte 2022B	Analysis of proliferation data
Media	STEMCELL Technologies	5702	NeuroCult (Mouse and Rat) proliferation kit containging Basal Medium and growth supplement
Penicillin-Streptomycin	Gibco	15140122	Antibiotics to add to media
Trametinib	Selleckchem	S2673	Drug 2