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BRAIN Publication-derived

Meningioma Brain Organoids

Full SOP with Academic or a plan

Source Jungwirth et al. · Division of Experimental Neurosurgery, Department of Neurosurgery, Heidelberg University · 10.1101/2024.11.26.625347

Authors: Gerhard Jungwirth, Junguo Cao, Catharina Lotsch, Rolf Warta, Mahmoud Moustafa, Maximilian Knoll, Tao Yu, Viktor Braun, Lena Jassowicz, Philip Dao Trong, Alexander Younsi, Moritz Scherer, Martin Bendszus, Sandro M. Krieg, Andreas von Deimling, Juergen Debus, Felix Sahm, Amir Abdollahi, Andreas Unterberg, Christel Herold-Mende Duration: 45 days 11 phases Cell source: Patient-Derived Meningioma Tissue

Abstract

This protocol establishes a standardized patient-derived tumor organoid (TO) model from meningioma tissue that preserves genotype, phenotype, and tumor microenvironment. The protocol includes large-scale drug screening of 107 targeted anticancer drugs on meningioma cell lines, followed by validation in TOs from 60 patients, identification of panobinostat as a lead compound targeting HDAC1/2, and molecular characterization of resistance mechanisms via HDAC8-TGFβ-EMT axis.

Cell source
Patient-Derived Meningioma Tissue
Application
Disease modeling, Drug screening, Personalized medicine

Protocol overview

76 steps across 11 phases

Phase 1 · In Silico Drug Target Identification and Library Preparation Prior to screening
  1. a Extract target genes from transcriptome data
  2. b Filter drugs by clinical development status
  3. c Limit inhibitors per target gene
Phase 2 · Cell Line-Based Drug Screening (Step 1: Single-Dose) Days 1-2
  1. a Culture meningioma cell lines
  2. b Seed cells for single-dose screening
  3. c Treat cells with drug library at single dose
  4. d Assess cell viability by CellTiter-Glo 2.0
  5. e Select hits based on viability reduction
Phase 3 · Cell Line-Based Drug Screening (Step 2: Dose-Response) Days 3-5
  1. a Culture four meningioma cell lines
  2. b Seed cells for dose-response screening
  3. c Prepare dose-response series
  4. d Treat cells with dose-response series
  5. e Measure cell viability by CellTiter-Glo 2.0
  6. f Calculate IC50, AUC, and Emax values
  7. g Select top drugs based on z-AUC and z-Emax
Phase 4 · Tumor Organoid Generation Days 1-3
  1. a Obtain meningioma tissue samples
  2. b Mechanically mince tissue
  3. c Enzymatic digestion of tissue
  4. d Filter cell suspension
  5. e Remove red blood cells
  6. f Wash and centrifuge cells
  7. g Incubate cells with magnetic particles (NanoShuttle)
  8. h Seed cells into anti-adhesive plates and form organoids
  9. i Monitor organoid formation (days 2-3)
Phase 5 · Characterization of Tumor Organoids Days 3-7
  1. a Assess organoid viability by CellTiter-Glo 3D
  2. b Perform live/dead staining
  3. c Extract RNA and DNA
  4. d Perform bulk RNA-seq
  5. e Perform panel sequencing for mutation analysis
  6. f Perform methylation classification
  7. g Perform H&E histology
  8. h Perform immunofluorescence staining for microenvironment markers
Phase 6 · Large-Scale Drug Screening in Tumor Organoids Days 3-6
  1. a Standardize organoid cell number
  2. b Prepare drug concentration series
  3. c Treat organoids with drugs
  4. d Measure viability by CellTiter-Glo 3D
  5. e Record luminescence
  6. f Normalize data and calculate IC50/AUC
  7. g Calculate Z-factor for assay quality control
  8. h Perform hierarchical clustering and stratification
Phase 7 · Target Validation: MELK Inhibitor Studies Days 1-5
  1. a Test alternative MELK inhibitors in organoids
  2. b Perform RNAi-mediated MELK knockdown in cell lines
  3. c Validate MELK knockdown by qRT-PCR
  4. d Assess viability of MELK-knockdown cells
  5. e Perform RNAi-mediated EZH2 knockdown
  6. f Interpret results for OTSSP167 target validation
Phase 8 · Target Validation: HDAC Inhibitor Studies Days 1-7
  1. a Test alternative HDAC inhibitors in organoids
  2. b Perform RNAi-mediated HDAC1 knockdown
  3. c Perform RNAi-mediated HDAC2 knockdown
  4. d Perform RNAi-mediated HDAC1/2 double knockdown
  5. e Measure viability of HDAC1/2-knockdown cells
  6. f Validate HDAC1/2 double knockdown in tumor organoids
  7. g Assess HDAC1/2 mRNA expression in primary tissues
Phase 9 · In Vivo Validation: Orthotopic Xenograft Model Days 1-45
  1. a Prepare animals and obtain approval
  2. b Prepare tumor cells for intracranial injection
  3. c Perform intracranial tumor inoculation
  4. d Post-operative care
  5. e Allow tumor establishment and randomization
  6. f Initiate drug treatment (Cycle 1)
  7. g Monitor animals during treatment (Days 7-21)
  8. h Complete three treatment cycles and discontinue therapy
  9. i Monitor survival until endpoint
  10. j Harvest tumors for histology
  11. k Perform Ki-67 immunohistochemistry on tumor tissues
  12. l Analyze survival data
Phase 10 · Resistance Mechanism Studies: Differential Gene Expression Analysis Ongoing analysis
  1. a Select panobinostat-sensitive and -resistant organoids
  2. b Extract RNA from primary tissue samples
  3. c Perform bulk RNA sequencing
  4. d Perform PROGENy pathway analysis
  5. e Perform gene set enrichment analysis (GSEA)
  6. f Identify differentially expressed HDAC genes
Phase 11 · Target Validation: HDAC8 Depletion Studies Days 1-5
  1. a Design and validate HDAC8 siRNA
  2. b Transfect meningioma cell lines with HDAC8 siRNA
  3. c Validate HDAC8 knockdown by qRT-PCR
  4. d Treat HDAC8-knockdown cells with panobinostat
  5. e Calculate IC50 values and compare curves

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Attribution

This SOP was written by Organthis based on the published method in Jungwirth et al.. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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