Name: laboratory-engineered glioblastoma organoid culture and drug screening
Uploaded: 2025-01-10T14:50:00.000+00:00
Duration: 10 min 49 s
Description: 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 ...

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 f&#252;r Translationale Krebsforschung (DKTK) grant AIM2GO (H-K.L.).

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 EF1&#945; promoter driving Luc2, along with a puromycin-resistant cassette, were used.
<ol>
	<li>When the cells reach ~70% confluency, digest iPSCs with passaging reagents A and B (see Table of Materials), respectively, for 4 min at 37 &#176;C, centrifuge at 120 x g for 5 min, and resuspend into a single cell suspension by gentle pipetting.</li>
	<li>Count the cells and seed 1 x 104 single cells of iPSC in each well of a basement matrix membrane (BMM)-coated 48 well plate, culture for 24 h with human embryonic cell qualified culture medium containing 10 &#181;M rho kinase (ROCK) inhibitor (see Table of Materials).</li>
	<li>Infect the cells with lentivirus-containing luciferase-expressing plasmids (MOI around 10) and 8 &#181;g/mL Polybrene (see Table of Materials). Replace the medium with a fresh human embryonic cell-qualified culture medium 6 h later.</li>
	<li>After 24 h, add 150 &#181;g/mL Luciferase Substrate (see Table of Materials) to the cell culture medium and check the bioluminescence (BLI) signal with bioluminescence imaging equipment (see Table of Materials) with a 5 s exposure.</li>
	<li>Add 2 &#181;g/mL puromycin to the culture medium for 2 days, then change the medium every other day for 1 week to expand the cells.</li>
	<li>When the cells reach ~70% confluency, digest iPSCs with passaging reagents A and B (see Table of Materials), respectively, for 4 min at 37 &#176;C, centrifuge at 120 x g for 5 min, and resuspend into a single cell suspension by gentle pipetting.</li>
	<li>Count the cells and seed the single cells at a concentration of 50 cells per 10 cm dish for 2 dishes. Change the medium every other day until the clones reach 3-5 mm in diameter (Figure 1B).</li>
	<li>Check the BLI signal as described above and pick the clones with strong BLI signals under the microscope with sterilized 10 &#181;L pipette tips.</li>
	<li>Expand the cells for further usage.</li>
</ol>
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.
<ol>
	<li>Design gRNAs targeting the respective genes of interest with online web tools (see Table of Materials). Select the gRNAs targeting the exons presented in all splicing variants and locate the closest to the transcription starting site.</li>
	<li>Anneal gRNA oligos (95 &#176;C for 5 min, cool down to 25 &#176;C at a speed of 1 &#176;C/s) and linearize pX330A-1x2 (see Table of Materials) and pX330S-2 (see Table of Materials) plasmids with Bbs1 enzyme.</li>
	<li>Ligate one of the gRNAs with pX330A-1x2 or pX330S-2, respectively, with T4 ligase at room temperature (RT) for 4 h.</li>
	<li>Digest the two resulting plasmids with BsaI enzyme (37 &#176;C for 1 h) and purify the products by gel extraction.</li>
	<li>Ligate the gRNA scaffolds from the pX330S-2-gRNA plasmid to the linearized pX330A-1x2-gRNA plasmid with T4 ligase at RT for 4 h.</li>
	<li>Clone puromycin resistance gene (PuroR) from the pX459 (see Table of Materials) plasmid to the resulting plasmids by EcoRI digestion (37 &#176;C for 1 h) and subsequential T4 ligation at RT for 4 h.</li>
	<li>Transform the plasmids in DH5alpha-competent cells and extract the plasmids after expanding.</li>
</ol>
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.
<ol>
	<li>Digest the iPSCs with passaging reagents A and B (see Table of Materials) for 4 min at 37 &#176;C each, and spin down the cells for 5 min at 120 x g and resuspend in human embryonic cell qualified culture medium (see Table of Materials) to generate single-cell suspensions.</li>
	<li>Count the cells and divide them into several tubes to achieve 1.2 x 106 cells per sample and spin down the cells at 120 x g for 5 min.</li>
	<li>Meanwhile, mix 15 &#181;g of the plasmids in 135 &#181;L of resuspension buffer from the transfection kit (see Table of Materials).</li>
	<li>Use 120 &#181;L of the mixture to resuspend the cell pellet.</li>
	<li>Electroporate the cells for two pulses at 1200 V for 20 ms with an electroporation system (see Table of Materials) and plate the resulting cells in one 6 well plate well with a human embryonic cell qualified culture medium containing reagent to facilitate single-cell survival (see Table of Materials).</li>
	<li>Add 2 &#181;g/mL puromycin to the culture medium every 12 h for 2 days. Then, expand the cells for 1 week before harvest.</li>
	<li>Check the efficiency of the knockout by western blotting or webtool analysis (see Table of Materials) and select the knockout population with the highest efficiency.</li>
	<li>Digest the iPSCs with the highest knockout efficiency with passaging reagents A and B for 4 min at 37 &#176;C each, and spin down the cells for 5 min at 120 x g. Resuspend in human embryonic cell qualified culture medium to generate single-cell suspensions.</li>
	<li>Count the cells and seed approximately 100 cells in two 10 cm dishes (50 cells in each dish) containing pre-warmed human embryonic cell qualified culture medium supplied with a reagent to facilitate single-cell survival.</li>
	<li>Change the medium every other day and culture the cells for 2 weeks.</li>
	<li>When the single-cell colonies are 3-5 mm in diameter, pick the colonies with 10 &#181;L pipette tips under the microscope and expand the cells in two wells of 48 well plates.</li>
	<li>When the cells reach 30%-50% confluency, extract the DNA from one well of the 48 well plate and perform PCR.</li>
	<li>Sequence the PCR products and analyze the results with the web tool (see Table of Materials).</li>
	<li>Select 5 positive clones to expand and further validate the knockout results with TA cloning and western blots.</li>
</ol>
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.
<ol>
	<li>On day 0, digest iPSCs with passaging reagents A and B (see Table of Materials) respectively for 4 min at 37 &#176;C, centrifuge at 120 x g for 5 min, and resuspend into a single cell suspension using low basic fibroblast growth factor (bFGF) hES media (Table 1).</li>
	<li>Count the cells and add an appropriate number of cells to reach a concentration of 6 &#215; 104 cells/mL.</li>
	<li>Add 50 &#181;M ROCK inhibitor and 6 ng/mL bFGF to the low bFGF hES medium-containing cells.</li>
	<li>Seed 150 &#181;L of cell suspension (9000 cells) into each well of the 96-well ultra-low attachment plate for both control (WT organoids) and experimental (knockout organoids) groups.</li>
	<li>On day 3, carefully remove 80 &#181;L of old culture medium from each well and add 150 &#181;L fresh low bFGF hES medium.</li>
	<li>On day 5, when embryonic bodies (EBs) begin to brighten and have smooth edges, remove as much old culture medium as possible from each well and add 150 &#181;L to 200 &#181;L of fresh neural induction medium (Table 1).</li>
	<li>On day 7, remove as much old culture medium as possible from each well and add 150-200 &#181;L of neural induction medium.</li>
	<li>On day 9, observe the EBs under the tissue culture microscope and select the EBs that are brighter around the outside.</li>
	<li>Thaw BMM at 4 &#176;C or on ice for 30 min before starting the following steps.</li>
	<li>Use a cut 200 &#181;L wide pipette tip to transfer the EBs to the organoid embedding sheet (see Table of Materials), remove the excessive medium, drop 30 &#181;L of BMM on the organoids, position the EBs in the middle of the droplet with a 10 &#181;L pipette tip, and let it solidify in the 37 &#176;C incubator for 20 min.</li>
	<li>Wash off the BMM droplets to a 6-well plate containing 3 mL of NeuroDMEM - A medium (Table 1) and 3 &#181;M CHIR99021 (see Table of Materials).</li>
	<li>On day 11, remove as much of the old culture medium from each well and add 3 mL of fresh NeuroDMEM -A medium containing 3 &#181;M CHIR99021.</li>
	<li>On day 13, remove as much of the old culture medium from each well as possible and add 3 mL of NeuroDMEM + A culture medium (Table 1). Place the 6-well plate on a shaker rotating at 75 rpm in the incubator.</li>
	<li>Refresh NeuroDMEM + A medium every 3 days until organoids are used.</li>
	<li>Use the organoids for multi-omic analysis following the standard protocols or drug screen as performed in the previous publication12.</li>
</ol>
5. Drug screen with LEGOs
NOTE: The exposure time for iPSCs or organoids BLI should be evaluated using various equipment or laboratory environments.
<ol>
	<li>Transfer organoids at the desired age to a 24-well plate containing 1 mL of NeuroDMEM + A medium, one organoid per well, 2 days before the experiment.</li>
	<li>Add 150 &#181;g/mL D-luciferin and incubate on the shaker for 15-30 min.</li>
	<li>Perform BLI as described before with an exposure time of 1 s-5 s.</li>
	<li>Select the organoids with similar signal strengths for subsequent experiments (at least 3 organoids for each group).</li>
	<li>On day 0, repeat steps 5.2 and 5.3. Then apply dimethyl sulfoxide (DMSO) or interested drugs to the culture medium of the organoids. Start with an initial drug concentration of 10 &#181;M, then perform an IC50 assay or adjust the concentration further according to different purposes.</li>
	<li>Repeat step 5.5 every 2 days. Treat the organoids for 6-10 days. 
	NOTE: The duration can be changed according to different purposes or the different nature of the drugs.</li>
	<li>Add 100 &#181;M BrdU to the medium and incubate it in a CO2 incubator for 2 h before harvesting the organoids, if needed, for subsequent proliferation analysis.</li>
	<li>Normalize the BLI signal to the DMSO control measured on the same day and then compare it with before drug treatment to evaluate the drug treatment effect. Consider drugs with a P value less than 0.05 and a signal decrease of more than 50% effective.</li>
</ol>

LEGO: A Human GBM Organoid Model Revealing Lipid Dysregulation and Drug Targets

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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioblastoma+in+adults:+A+Society+for+Neuro-Oncology+(SNO)+and+European+Society+of+Neuro-Oncology+(EANO)+consensus+review+on+current+management+and+future+directions.">Glioblastoma in adults: A Society for Neuro-Oncology (SNO) and European Society of Neuro-Oncology (EANO) consensus review on current management and future directions.</a> Neuro Oncol. 22 (8), 1073-1113 (2020).</li><li>Ohgaki, H., Kleihues, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+pathways+to+primary+and+secondary+glioblastoma.">Genetic pathways to primary and secondary glioblastoma.</a> Am J Pathol. 170 (5), 1445-1453 (2007).</li><li>Cancer Genome Atlas Research. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+genomic+characterization+defines+human+glioblastoma+genes+and+core+pathways.">Comprehensive genomic characterization defines human glioblastoma genes and core pathways.</a> Nature. 455 (7216), 1061-1068 (2008).</li><li>Brennan, C. 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The authors have no conflicts of interest to disclose

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 aspects of the molecular and cellular pathology of human GBM12. With LEGO, we can identify the major molecular consequences driven by genetic mutations and perform genotype-specific drug screens to enable personalized treatment of human GBM12.
In this protocol, we generated and conducted a GBM drug screen using gene-mutated organoids. The first critical step involved ensuring the pluripotency of the iPSCs after knockout generation and thoroughly verifying the knockouts. We observed that NF1 knockout leads to increased signs of differentiation in the iPSCs. To maintain the pluripotency, we applied 3 &#181;M of CHIR99021 and 1 &#181;M of PD032590119 to these clones during iPSC culture and the embryonic body culture stages. It is essential to frequently check the pluripotency of the iPSCs using staining or flow cytometry.
Controls are crucial in this protocol. Mutations are generated on the isogenic controls and can be incrementally increased. Therefore, the controls included not only WT controls but also those with fewer mutations. These controls should be carefully assessed before initiating the protocol.
The organoid drug screen relies on BLI. Given that organoids are 3D structures requiring more time for luciferase penetration, we tested different incubation times. A trial experiment is recommended to determine the optimal incubation time for different facilities. In our experience, the BLI signal increases during the first 15 min of incubation, reaching its maximum and stabilizing for about 1 h. This step is crucial for obtaining a stable signal for drug effect comparison.
Before the approach described in this protocol, previous studies have utilized electroporation to deliver plasmids to healthy brain organoids10,11 or co-culture healthy brain organoids with GBM cells10,20,21 to induce tumors. These approaches would be ideal for studying tumor invasion, migration, and interaction with surrounding normal brain cells. However, in these GBM organoid models, the tumor cells only occupy a small portion of the entire tumor-bearing organoid, which leads to a limited ability to study tumor intrinsic properties. Hence, constructing the mutational spectra at the iPSC level and inducing organoids from the knockout iPSCs would be preferable to enrich the tumor cells and allow for more detailed characterization. 
GBM is a highly heterogeneous disease. The approach described here decreases complexity by studying each subclone individually. However, the complexity can easily be increased by assembling pieces of different mutant organoids as a concept, like building blocks, as in the concept of LEGO. Also, the heterogeneity of GBM is also underlined by the presence of stromal cells, such as brain resident microglia, blood-derived immune cells, or myeloid cells. These cell types could be incorporated into the present model by adding cells to the culture medium or by co-embedding them in the matrigel to allow investigation of genetic heterogeneity determining immune cell behaviors. Once the experimental parameters are set, organoids with stromal cells can also be used for evaluating immune therapy responses. Further experiments should also aim to improve the general growth conditions of the organoids, for example, with the constant addition of fresh medium by bioengineering22 or by co-culture with blood vessel organoids23.

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).

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

laboratory-engineered glioblastoma organoid culture and drug screening

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.

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 LEGOs with different mutations could harbor different phenotypes; this can be assessed with different approaches, such as staining, single-cell RNA sequencing, metabolomics, lipidomics, etc. We have shown that LEGOs with NF1 mutation highly simulate the mesenchymal subtype of GBM12.
Ideally, with appropriate steps, the BLI signal of the organoids would increase as the LEGOs grow larger. When reaching the desired age, drug screening can be performed. Normally, effective drugs would have simultaneous signal drops in all the organoids in the experimental group compared to the control (Figure 1E).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67593/67593fig01.jpg" /> 
Figure 1: Representative results. (A) Overview of the protocol. (B) A representative image illustrates the appearance of the single-cell colonies under bioluminescence imaging in a 10 cm plate. (C) Representative images12 showing the morphology of organoids at indicated time points in different groups. Scale bars, 500 &#181;m. (D) Representative hematoxylin and eosin (HE) staining images12 of the organoids at different stages. The red arrows indicate the cells with atypical nuclei. Scale bar, 50 &#181;m. (E) Representative bioluminescence images (BLI) of effective and non-effective drug treatments on LEGOs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67593/67593fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Homemade cell culture medium composition. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67593/Table 1.xlsx" target="_blank">Please click here to download this Table.</a>

Laboratory-Engineered Glioblastoma Organoid Culture and Drug Screening

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 ...

laboratory-engineered-glioblastoma-organoid-culture-and-drug-screening

Nanyang Technological University

Research

JoVE Journal

Cancer Research

470 Views.  German Cancer Research Center (DKFZ). 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.

Video: Laboratory-Engineered Glioblastoma Organoid Culture and Drug Screening

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

Medium-Throughput Drug- and Radiotherapy Screening Assay using Patient-Derived Organoids

Patient-derived organoid (PDO) models allow for long-term expansion and maintenance of primary epithelial cells grown in three dimensions and a near-native state. When derived from resected or biopsied tumor tissue, organoids closely recapitulate in vivo tumor morphology and can be used to study therapy response in vitro. Biobanks of tumor organoids reflect the vast variety of clinical tumors and patients and therefore hold great promise for preclinical and clinical applications. This paper presents a method for medium-throughput drug screening using head and neck squamous cell carcinoma and colorectal adenocarcinoma organoids. This approach can easily be adopted for use with any tissue-derived organoid model, both normal and diseased. Methods are described for in vitro exposure of organoids to chemo- and radiotherapy (either as single-treatment modality or in combination). Cell survival after 5 days of drug exposure is assessed by measuring adenosine triphosphate (ATP) levels. Drug sensitivity is measured by the half-maximal inhibitory concentration (IC50), area under the curve (AUC), and growth rate (GR) metrics. These parameters can provide insight into whether an organoid culture is deemed sensitive or resistant to a particular treatment.

We describe detailed protocols to use patient-derived organoids for medium-throughput therapy sensitivity screenings. Therapies tested include chemotherapy, radiotherapy, and chemo-radiotherapy. Adenosine triphosphate levels are used as a functional readout.

Patient-derived organoid (PDO) models allow for long-term expansion and maintenance of primary epithelial cells grown in three dimensions and a ...

Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in  Clinically Relevant Animal Models and 3D Immunochemistry

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor.
Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol&#160;and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

Protocol for propagation of dissociated high grade glioma surgical specimens in serum-free neurosphere medium to select for cells with cancer stem cell phenotype. For specimens that fail to grow as neurospheres, an alternative protocol is suggested. A paraffin embedding technique for maintaining the 3D neurosphere architecture for immunocytochemistry is described.

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell ...

Medicine

Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture

Glioblastoma (GBM) is the most commonly occurring primary malignant brain cancer with an extremely poor prognosis. Intra-tumoral cellular and molecular diversity, as well as complex interactions between tumor microenvironments, can make finding effective treatments a challenge. Traditional adherent or sphere culture methods can mask such complexities, whereas three-dimensional organoid culture can recapitulate regional microenvironmental gradients. Organoids are a method of three-dimensional GBM culture that better mimics patient tumor architecture, contains phenotypically diverse cell populations, and can be used for medium-throughput experiments. Although three-dimensional organoid culture is more laborious and time-consuming compared to traditional culture, it offers unique benefits and can serve to bridge the gap between current in vitro and in vivo systems. Organoids have established themselves as invaluable tools in the arsenal of cancer biologists to better understand tumor behavior and mechanisms of resistance, and their applications only continue to grow. Here, details are provided about methods for generating and maintaining GBM organoids. Instructions of how to perform organoid sample embedding and sectioning using both frozen and paraffin-embedding techniques, as well as recommendations for immunohistochemistry and immunofluorescence protocols on organoid sections, and measurement of total organoid cell viability, are all also described.

Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture

Here, we describe a method of generating glioblastoma (GBM) organoids from primary patient specimens or patient-derived cell cultures and maintaining them to maturity. These GBM organoids contain phenotypically diverse cell populations and recreate tumor microenvironments ex vivo.

Glioblastoma (GBM) is the most commonly occurring primary malignant brain cancer with an extremely poor prognosis. Intra-tumoral cellular and molecular ...

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based drug screening assays do not capture the complexity of a drug response (cytostatic or cytotoxic) and intratumor heterogeneity that has been shown to be retained in PDTOs due to a bulk readout. Live-cell imaging is a powerful tool to overcome this issue and visualize drug responses more in-depth. However, image analysis software is often not adapted to the three-dimensionality of PDTOs, requires fluorescent viability dyes, or is not compatible with a 384-well microplate format. This paper describes a semi-automated methodology to seed, treat, and image PDTOs in a high-throughput, 384-well format using conventional, widefield, live-cell imaging systems. In addition, we developed viability marker-free image analysis software to quantify growth rate-based drug response metrics that improve reproducibility and correct growth rate variations between different PDTO lines. Using the normalized drug response metric, which scores drug response based on the growth rate normalized to a positive and negative control condition, and a fluorescent cell death dye, cytotoxic and cytostatic drug responses can be easily distinguished, profoundly improving the classification of responders and non-responders. In addition, drug-response heterogeneity can by quantified from single-organoid drug response analysis to identify potential, resistant clones. Ultimately, this method aims to improve the prediction of clinical therapy response by capturing a multiparametric drug response signature, which includes kinetic growth arrest and cell death quantification.

This protocol describes a semi-automated method for medium- to high-throughput organoid drug screenings and microscope-agnostic, automated image analysis software to quantify and visualize multiparametric, single-organoid drug responses to capture intratumor heterogeneity.

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex ...

Derivation of 3D Glioma Organoids from Chopper-Processed Patient Tumors

Cultivating Ex Vivo Patient-Derived Glioma Organoids Using a Tissue Chopper

Glioblastoma, IDH-wild type, CNS WHO grade 4 (GBM) is a primary brain tumor associated with poor patient survival despite aggressive treatment. Developing realistic ex vivo models remain challenging. Patient-derived 3-dimensional organoid (PDO) models offer innovative platforms that capture the phenotypic and molecular heterogeneity of GBM, while preserving key characteristics of the original tumors. However, manual dissection for PDO generation is time-consuming, expensive and can result in a number of irregular and unevenly sized PDOs. This study presents an innovative method for PDO production using an automated tissue chopper. Tumor samples from four GBM and one astrocytoma, IDH-mutant, CNS WHO grade 2 patients were processed manually as well as using the tissue chopper. In the manual approach, the tumor material was dissected using scalpels under microscopic control, while the tissue chopper was employed at three different angles. Following culture on an orbital shaker at 37 &#176;C, morphological changes were evaluated using bright field microscopy, while proliferation (Ki67) and apoptosis (CC3) were assessed by immunofluorescence after 6 weeks. The tissue chopper method reduced almost 70% of the manufacturing time and resulted in a significantly higher PDOs mean count compared to the manually processed tissue from the second week onwards (week 2: 801 vs. 601, P = 0.018; week 3: 1105 vs. 771, P = 0.032; and week 4:1195 vs. 784, P &lt; 0.01). Quality assessment revealed similar rates of tumor-cell apoptosis and proliferation for both manufacturing methods. Therefore, the automated tissue chopper method offers a more efficient approach in terms of time and PDO yield. This method holds promise for drug- or immunotherapy-screening of GBM patients.

Author Spotlight: Enhanced Generation of Patient-Derived 3D Organoids for Glioblastoma and Glioma

This study introduces an automated method to generate patient-derived glioblastoma 3-dimensional organoids utilizing a tissue chopper. The method provides a suitable and effective approach to obtain such organoids for therapeutical testing.

Glioblastoma, IDH-wild type, CNS WHO grade 4 (GBM) is a primary brain tumor associated with poor patient survival despite aggressive treatment. Developing ...

Spheroid Drug Sensitivity Screening in Glioma Stem Cell Lines

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&#160;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,&#160;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&#160;provides a more accurate reflection of tumor growth, behavior, and drug sensitivities in vivo, thus forming the basis for further preclinical investigation.

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.

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...

Laboratory-Engineered Glioblastoma Organoid Culture and Drug Screening

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A 3D Spheroid Model for Glioblastoma

Medium-Throughput Drug- and Radiotherapy Screening Assay using Patient-Derived Organoids

Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in Clinically Relevant Animal Models and 3D Immunochemistry

Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Cultivating Ex Vivo Patient-Derived Glioma Organoids Using a Tissue Chopper

Spheroid Drug Sensitivity Screening in Glioma Stem Cell Lines

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases