Laboratory-Engineered Glioblastoma Organoid Culture and Drug Screening

Summary

Here, we outline a comprehensive protocol for generating and utilizing laboratory-engineered glioblastoma organoids (LEGO) to investigate genotype-phenotype dependencies and screen potential drugs for glioblastoma treatment.

Abstract

Glioblastoma (GBM) is described as a group of highly malignant primary brain tumors and stands as one of the most lethal malignancies. The genetic and cellular characteristics of GBM have been a focal point of ongoing research, revealing that it is a group of heterogeneous diseases with variations in RNA expression, DNA methylation, or cellular composition. Despite the wealth of molecular data available, the lack of transferable pre-clinic models has limited the application of this information to disease classification rather than treatment stratification. Transferring the patients' genetic information into clinical benefits and bridging the gap between detailed descriptions of GBM, genotype-phenotype associations, and treatment advancements remain significant challenges. In this context, we present an advanced human GBM organoid model, the Laboratory Engineered Glioblastoma Organoid (LEGO), and illustrate its use in studying the genotype-phenotype dependencies and screening potential drugs for GBM. Utilizing this model, we have identified lipid metabolism dysregulation as a critical milestone in GBM progression and discovered that the microsomal triglyceride transfer protein inhibitor Lomitapide shows promise as a potential treatment for GBM.

Introduction

Glioblastoma (GBM) accounts for more than 60% of all diagnosed primary brain tumors in adults, with a median survival of less than two years^1,2,3. Despite considerable efforts in deciphering the underlying complexity of this disease, continuous attempts with targeted therapies or immunotherapies, and screening for potential anti-cancer drugs, the treatment strategy for newly diagnosed GBM patients remains to be the maximal safe surgical resection followed by radiotherapy (RT) combined with temozolomide (TMZ) and then adjuvant TMZ⁴.

Protocol

1. Luciferase labeling of the iPSCs

NOTE: For detailed instructions on culturing iPSCs, refer to the protocol provided by the institutions or the companies from which the cells were obtained. In all experiments described in this protocol, the iPSCs at passages 3-10 post-recovery were used, and any cells showing signs of differentiation were discarded. For the virus production, plasmids containing an EF1α promoter driving Luc2, along with a puromycin-resistant cassette, were used.

1. When the cells reach ~70% confluency, digest iPSCs with passaging reagents A and B (see Table of Materials), res....

Results

In our hands, LEGOs derived from mutant iPSCs displayed increased expansion compared to the WT isogenic control (Figure 1C) and showed atypical nuclear after 4 weeks of culture (Figure 1D), which supports the potential malignant transformation of the cells in mutant organoids¹⁸. The tumorigenic potential of the organoids can be further verified with xenografted experiments if required, as we have shown previously¹².......

Discussion

The lack of personalized treatment in human GBM could largely be attributed to the fact that many GBM models, such as human cell lines or mouse models, cannot faithfully recapitulate the human GBM. Consequently, the treatment strategies selected based on these model systems cannot be transferred into clinical applications. Instead, organoids can tackle these translational problems with the presence of human physiological conditions. To this end, we have generated LEGO and shown that LEGOS can faithfully recapitulate many.......

Materials

Name	Company	Catalog Number	Comments
Accutase	A6964	Sigma-Aldrich	Passaging reagent B
Antibiotic-Antimycotic	15240096	Life-Technologies
B27	17504044	Life Technologies
B27 without vitamin A	12587010	Life Technologies
Bbs1-HF	R3539S	New England Biolabs
Bsa1-HF	R3535S	New England Biolabs
CHIR99021	4423	Tocris Bioscience
CloneR	#05889	STEMCELL Technologies	Reagent to facilitate single-cell survival
D-Luciferin	L2916	Invitrogen	Luciferase Substrate
DMEM/F-12	11330032	Life Technologies
EcoRl-HF	R3101T	New England Biolabs
Fetal Bovine Serum	30-2020	ATCC
FGF-2	100-18B	PeproTech
GlutaMAX	35050038	Life Technologies
Heparin	H3149	Sigma-Aldrich
hES-quality Fetal Bovine Serum	10270106	Life Technologies
Insulin Solution	I9278	Sigma-Aldrich
IVIS Lumina II	NA	PerkinElmer
Knockout serum replacement	10828-028	Life Technologies
L-Ascorbic Acid	A4544-25G	Sigma-Aldrich
Matrigel® hES qualified	354277	Corning	Basement matrix membrane
MEM-NEAA	11140050	Life Technologies
mTeSR Plus	100-0276	STEMCELL Technologies	Human embryonic cell qualified culture medium
N2	17502048	Life Technologies
Neon Electroporation System	NA	Thermo Fisher Scientific	Electroporation system
Neon Transfection System 100 µL Kit	MPK10096	Invitrogen	Electroporation kit
Neurobasal medium	21103049	Life Technologies
Online knockout efficiency analysis	NA		http://shinyapps.datacurators.nl/tide/
Organoid Embedding Sheet	# 08579	STEMCELL Technologies
Penicillin-Streptomycin	15140122	Life-Technologies
Polybrene (10mg/mL)	TR-1003-50UL	Merck Millipore
Puromycin	P8833	Sigma-Aldrich
px330-A-1x2	https://www.addgene.org/58766/	Addgene
px330-S-2	https://www.addgene.org/58778/	Addgene
px459	https://www.addgene.org/48139/	Addgene
ReleSR	5872	STEMCELL Technologies	Passaging reagent A
Rock Inhibitor	72304	STEMCELL Technologies
sgRNA design	NA		https://zlab.bio/guide-design-resources
sgRNA design	NA	NA	www.benchling.com
T4 DNA ligase	M0202S	New England Biolabs
β-Mercaptoethanol	M3148	Sigma-Aldrich