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

1. 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 concentration of 20 ng/mL and 10 ng/mL, respectively, as per the manufacturer description.
   NOTE: Glioma stem cell media can be used for 1 week when stored at 4 °C.
2. Dissociate RFP-expressing cells:
   NOTE: In this example, the mouse NF1 HGG stem cell line 5746 is used.
   1. Transfer the glioma spheres to a 15 mL conical tube, and spin down glioma spheres in a centrifuge (150 x g for 5 min). Remove the supernatant and add 300 µL of accutase to dissociate spheres (incubate for 5 min at 37 °C).
   2. Add 1 mL of glioma stem cell media and disrupt the spheres by gentle pipetting. Centrifuge the dissociated cells for 5 min at 150 x g, aspirate supernatant, and dissolve the pellet in 1 mL of glioma stem cell media.
3. Measure the concentration of cells using a cell counter or hemocytometer. When using a fluorescence cell counter, add 18 µL of the cell suspension to 2 µL of Acridine Orange/Propidium Iodide Stain. Load 12 µL of this suspension in a hemocytometer to count the number of live/dead cells present in the suspension.
4. Dilute the cells to a final concentration of 2,000 cells per 100 µL of glioma stem cell media.
   NOTE: Modifications to cell concentration may be cell line-dependent and can be adjusted accordingly.
5. Using a multichannel pipette, dispense 100 µL of the cell suspension (2,000 cells per 100 µL) in each well of a 96-well round bottom low attachment plate by reverse pipetting.
   NOTE: Use round bottom low attachment plates; these plates will stimulate the formation of a single glioma sphere in each well. Reverse pipetting avoids the formation of bubbles, which will interfere with image capture of the live-cell analysis system.
6. Centrifuge the plates at 150 x g for 5 min.
   ​NOTE: Centrifugation is a critical step to initiate 3D neurosphere formation.
7. Incubate overnight to allow a single sphere to form in each well.

2. Adding drugs for synergy assay (Figure 1)

1. Ensure each well of the 96-well plate receives a specific concentration of both drugs 1 and 2. Create a grid where each concentration of drug 1 is combined with each concentration of drug 2 in duplicate.
2. Make a serial dilution of the two drugs for which the synergy will be determined (5 dilutions of drug 1 and 7 dilutions of drug 2) using the glioma stem cell media (Figure 1A).
   NOTE: In this study, a 50% serial dilution series is used; however, any dilution series can be utilized.
   1. The dilution series should be 7x more concentrated than the desired final concentration in each well. For example, if the highest final concentration for drug 1 is 1 µM, make the first concentration of the dilution series for drug 1, 7 µM (1 mL, Tube B or I in Figure 1A).
   2. Add 500 µL of glioma stem cell media to each additional tube of the dilution series (Tubes C-H and J-M, as shown in Figure 1).
   3. To create the dilution series, aspirate 500 µL of the drug mixture from Tube B or Tube I and add it to the next tube in the dilution series Tube C or Tube J, respectively (containing 500 µL of glioma stem cell media). Mix well and repeat to the next tube in the dilution series. Make a DMSO control (Tube A) and ensure that the DMSO concentration is the same across conditions.
      NOTE: Modifications to drug concentrations and dilution series can be made. Make sure to list the correct drug concentrations in Supplementary File 1 if the dilution series is changed.
3. To each well of the 96 well plates, add 20 µL of drug 1 and 20 µL of drug 2 by reverse pipetting, as shown in Figure 1B, for a final volume of 140 µL per well; drug combinations are evaluated in duplicate. If using the provided downstream analysis for IC50 and LD50, use the drug layout as shown in Figure 1B.
   1. In this assay, the effect of each drug by itself (without the other drug being present) is also measured. Make sure to add 20 µL of DMSO control media (Tube A in Figure 1B) to those single drug wells to bring the final volume of each well to 140 µL.
   2. Ensure the wells do not contain bubbles, as this will affect the imaging. 
      NOTE: Modifications to the final volume can be made; however, make sure to adjust the concentrations in point 2.2.
4. Place the plates in the live imager and monitor the growth of each sphere in each well using the spheroid module, single sphere setting, imaging both brightfield and RFP at 4x magnification.
5. Monitor plates for 72 h, imaging each well of the plate at a regular time interval, typically 2 h.
   ​NOTE: Upload a correct plate map; this will assure proper downstream analysis (Figure 1C).

3. Calculation of IC50, LD50, and synergy score (Figure 2 and Figure 3)

1. Analyze the RFP intensity for each well and each time point using the live imager software. The analysis parameters used are listed in the supplementary files (Supplementary File 2).
   NOTE: Different analysis parameters can be used as preferred.
2. Export the RFP data as Total Red Integrated RFP Intensity for each well, making sure to export the raw non-grouped data (Figure 2A).
   NOTE: If using the provided template (Supplementary File 1) to calculate IC50 and LD50, it is essential to adhere to point 3.2
3. Determine the average and standard deviation for each drug combination of (1) the fold change compared to DMSO only (for this calculation, the 0 h values are not needed) and (2) the Log2 of fold change compared to 0 h by pasting the raw data into the provided excel sheet (Figure 2B and see example in Supplementary File 1).
4. Adjust the maximum concentration of drugs 1 and 2 in the spreadsheet (Figure 2B).
   NOTE: Make sure to adjust the concentrations of drugs 1 and 2; otherwise, incorrect LD50 and IC50 values will be calculated.
5. Calculate IC50 values using an appropriate data analysis software application (here, GraphPad is used). For the IC50 value calculation, use the average fold change and standard deviation fold change compared to DMSO only determined in step 3.4, and copy the IC50 tables of drugs 1 and 2 to the data analysis software (Log(inhibitor) vs. normalized response, variable slope) (Figure 3A).
   1. Only use data from wells to which a single compund was added for the IC50 calculations. Use the Log drug concentration values in the data analysis software, which are automatically calculated in the provided spreadsheet.
6. Calculate synergy score:
   1. Use the average fold change compared to DMSO only of every combination to calculate the synergy score using a software package or website. For example, https://synergyfinder.fimm.fi/¹² (parameters: LL4 curve fit, ZIP method for synergy score calculation). Supplementary File 1 automatically calculates the values needed to create the synergy input table that can be uploaded to SynergyFinder (Figure 3B). An example of the synergy input table is provided in Supplementary File 3.
      NOTE: A score above 10 denotes synergy, one below -10 denotes antagonism and a value between -10 and 10 shows additive effects. 
7. Calculate the LD50 score:
   1. For each concentration of drug 1, calculate the LD50 of drug 2. The supplementary file automatically calculates the Log2 value and standard deviation for each concentration (Figure 3C).
   2. Enter the average and standard deviation of the Log2 fold change compared to 0 h, as well as the number of replicates in the data analysis software (in this study, 2,) and calculate the LD50 value and LD50 standard deviation by determining the concentration at -1 (Log2 = -1 represents the log2 value for which 50% of the signal is lost compared to 0 h). In these calculations, use the Log(inhibitor) vs. response, variable slope 4 parameters model.
      NOTE: The data analysis software does not automatically calculate the drug concentration corresponding to a log2 = -1 value; however, this value can be added to the reported calculations of the data analysis software as a user-defined equation (Figure 3C). Make sure to use the Log concentration values in the data analysis software, which are automatically calculated in the provided spreadsheet. Supplementary File 4 shows the IC50 and LD50 panels in the GraphPad software.

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 (

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

Name	Company	Catalog Number	Comments
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