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BRAIN Publication-derived

Targeting the Mitochondrial NADH Shuttle in Group 3 Medulloblastoma under Hypoxic Conditions

Source Contenti et al., 2023 · Université Côte d'Azur, INSERM U1065, C3M, Nice, France · 10.1038/s41419-023-06275-0

👤 J. Contenti, Y. Guo, A. Mazzu, M. Irondelle, M. Rouleau, C. Lago, G. Leva, L. Tiberi, I. Ben-Sahra, F. Bost, N. M. Mazure ⏱ 92 days 📋 11 phases 🧫 Human iPSC (cerebellar organoids); Human medulloblastoma cell lines (HDMB-03, D-458)

Abstract

This protocol establishes a comprehensive wet-lab methodology for investigating mitochondrial NADH shuttle pathways (glycerol-3-phosphate shuttle and malate-aspartate shuttle) as therapeutic targets in Group 3 medulloblastoma cells under hypoxic conditions (1% O₂, 6% O₂, and 21% O₂). The protocol encompasses cell culture, pharmacological inhibition studies, metabolic profiling, and validation in human cerebellar organoid-derived tumor models to identify synergistic drug combinations that induce apoptosis in treatment-resistant Group 3 medulloblastoma.

Cell source
Human iPSC (cerebellar organoids); Human medulloblastoma cell lines (HDMB-03, D-458)
Application
Disease modeling and drug screening for Group 3 medulloblastoma metabolic vulnerabilities

Protocol overview

52 steps across 11 phases

Cell Culture Preparation and Maintenance Days −7 to 0
  1. 1 Culture HDMB-03 and D-458 medulloblastoma cells
  2. 2 Prepare hypoxic and normoxic culture conditions
Pharmacological Inhibitor Treatment Days 0 to 3
  1. 3 Treat cells with iGP-1 (mGPDH inhibitor)
  2. 4 Treat cells with AOAA (MAS inhibitor)
  3. 5 Treat cells with GNE-140 (LDHA inhibitor)
  4. 6 Treat cells with Phenformin (mitochondrial complex I inhibitor)
  5. 7 Treat cells with drug combinations
  6. 8 Prepare control and vehicle-treated samples
Cell Viability and Proliferation Assessment Days 1 to 3
  1. 9 Count cells and measure viability at 24 h and 72 h
  2. 10 Record total cell count and viability percentage
Metabolic Profiling and Respirometry Days 1 to 2
  1. 11 Measure two parameters on the analyzer. Record the oxygen consumption rate, abbreviated OCR. Record the extracellular acidification rate, abbreviated ECAR.
  2. 12 Perform sequential metabolic inhibitor injection protocol
  3. 13 Normalize OCR and ECAR to protein content
  4. 14 Perform Phenotype MicroArray analysis on OmniLog system
  5. 15 Measure tetrazolium dye reduction kinetically in OmniLog analyzer
  6. 16 Analyze OmniLog data and perform pathway analysis
Metabolomic Analysis (Steady-State Metabolite Profiling) Days 1 to 7
  1. 17 Culture cells for hypoxic metabolite extraction
  2. 18 Extract metabolites using 80% methanol on dry ice
  3. 19 Perform two sequential re-extractions of the pellet
  4. 20 Dry metabolite extracts under nitrogen gas
  5. 21 Resuspend dried metabolites and prepare for LC-MS/MS
  6. 22 Quantify 300 endogenous water-soluble metabolites by selected reaction monitoring (SRM)
  7. 23 Process metabolomics data and perform statistical analysis
Enzymatic Assays (Optional Supporting Assays) Days 1 to 3
  1. 24 Measure intracellular glycerol-3-phosphate levels
  2. 25 Measure NAD/NADH ratio
  3. 26 Measure intracellular malate levels
Protein Expression Analysis (Immunoblotting) Days 1 to 3
  1. 27 Harvest and lyse cells for immunoblotting
  2. 28 Determine protein concentration by BCA assay
  3. 29 Separate proteins by SDS-PAGE
  4. 30 Transfer proteins to PVDF membrane
  5. 31 Block and incubate with primary antibodies
  6. 32 Incubate with secondary antibodies
  7. 33 Visualize bands using ECL and quantify
Generation and Maintenance of Human Cerebellar Organoids Days −35 to 0 (or Days 1 to 85 from initial differentiation)
  1. 34 Culture and maintain human iPSCs
  2. 35 Differentiate iPSCs into cerebellar organoids (Days 0–35)
  3. 36 Electroporate organoids to introduce oncogenic alleles (Day 35)
  4. 37 Continue organoid culture post-electroporation (Days 35–71/79)
Pharmacological Treatment of Tumor Organoids Days 71–85 (from initial differentiation)
  1. 38 Prepare organoids for drug treatment
  2. 39 Treat organoids with single-drug and combination therapies
  3. 40 Monitor organoid response to treatment
Immunofluorescence and Apoptosis Detection in Organoids Days 14–21 of treatment (days 85–92 of differentiation)
  1. 41 Fix and permeabilize organoids
  2. 42 Block non-specific binding
  3. 43 Incubate with primary antibody against cleaved Caspase-3
  4. 44 Incubate with secondary antibody
  5. 45 Stain with Annexin V for apoptosis confirmation
  6. 46 Mount organoids and perform confocal microscopy
  7. 47 Generate 3D reconstructions and quantify apoptosis
  8. 48 Generate merged color images for publication
Colony-Forming Assay Days 0 to 7
  1. 49 Plate cells for colony formation
  2. 50 Fix and stain colonies
  3. 51 Wash, dry, and count colonies
  4. 52 Calculate plating efficiency and survival

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Contenti et al., 2023. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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