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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.
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.
Glioblastoma (GBM) accounts for more than 60% of all diagnosed primary brain tumors in adults, with a median survival of less than two years1,2,3. 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.
The highly heterogeneous mutational nature of glioblastoma within and among tumors makes it extremely difficult to dissect the molecular properties of this tumor, which eventually leads to treatment failure. Thus, it is essential to decomplexify the disease by reducing the variety of genetic mutations to the modules of mutations that occur most often in GBM patients5,6,7,8 and to investigate the molecular consequences of each mutational subclone individually.
Recently, organoids have emerged as a promising model for cancer research. Organoids surpass the canonical cancer models since they exhibit a human microenvironment and complex cellular components while being easy to generate and expand with a relatively low cost9. There were also pioneers of genetically engineered organoids with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to study glioma10,11. Following this direction, we generated a set of induced pluripotent stem cell (iPSC)-based human GBM organoid models (LEGO: Laboratory Engineered Glioblastoma Organoid) based on CRISPR/Cas9 genetic engineering of the frequent mutations identified in GBM patients. With detailed characterization of the single-cell transcriptome, DNA methylome, metabolome, lipidome, proteome, and phospho-proteome of LEGOs, we have demonstrated the high resemblance of LEGOs with human GBM and discovered major milestones during the progression of GBM, and with luciferase-based drug screen, we have identified several potential drugs for GBM treatment, the details of such results have been published previously12. This article describes the detailed protocol for the generation and drug screening of LEGOs (Figure 1A).
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.
2. gRNA design and plasmid cloning
NOTE: These steps can be carried out simultaneously with Section 1. CRISPR/Cas9 plasmids with a proper selection system should work. These specific plasmids were used because they enable simultaneous knockout of several target genes. This section demonstrates how to clone a plasmid with two gRNA scaffolds; more gRNA scaffolds can be cloned in one plasmid following the same principle. The original protocol was previously published13.
3. Knockout generation and validation on iPSCs
NOTE: Follow the standard CRISPR/Cas9 knockout protocols published before13,14. This section describes the method used to perform the knockouts via electroporation.
4. Organoid Culture
NOTE: The organoids were generated following previously published protocols with minor adaptations15,16,17. The optimal age of the organoids (the culture duration) should be determined based on the specific purpose of the study. For instance, when investigating genotype-to-phenotype associations or observing organoid growth patterns, analyzing multiple time points ranging from 1-4 months or longer is recommended. Organoids aged 2-2.5 months were used for drug screening, as they tended to develop a necrotic core in the center due to nutrient limitations at larger sizes, which could impact the accuracy of drug screening results.
5. Drug screen with LEGOs
NOTE: The exposure time for iPSCs or organoids BLI should be evaluated using various equipment or laboratory environments.
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...
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...
The authors have no conflicts of interest to disclose
This work is supported by Deutsche Forschungsgemeinschaft grant SFB1389 (H-K.L.), Deutsche Krebshilfe grant 110227 (H-K.L.), European Research Council (ERC) grant 647055 (H-K.L.), Deutschen Konsortium für Translationale Krebsforschung (DKTK) grant AIM2GO (H-K.L.).
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 |
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