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BRAIN Publication-derived

NPI-0052 and γ-radiation synergistic apoptotic treatment in medulloblastoma cells and organoids

Source Frisira et al., 2019 · Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London · 10.1038/s41419-019-2026-y

👤 Eleni Frisira, Fatima Rashid, Swastina Nath Varma, Sara Badodi, Valentine Ayodele Benjamin-Ombo, David Michod, Maria Victoria Niklison-Chirou ⏱ 28 days 📋 10 phases 🧫 Patient-Derived iPSC (Medulloblastoma G3/G4)

Abstract

This protocol demonstrates the use of NPI-0052, a proteasome inhibitor, alone and in combination with γ-radiation to induce apoptosis in patient-derived medulloblastoma cells and three-dimensional tumor organoids. The treatment synergistically activates apoptosis through ROS generation and p53/p73 stabilization, providing a preclinical model for testing adjuvant therapies in aggressive medulloblastoma subtypes.

Cell source
Patient-Derived iPSC (Medulloblastoma G3/G4)
Application
Disease modeling and drug screening for medulloblastoma treatment

Protocol overview

37 steps across 10 phases

Cell culture preparation and maintenance Day 0 onwards
  1. 1 Prepare DMEM + GlutaMAX culture medium
  2. 2 Prepare DMEM:F12 culture medium with growth factors
  3. 3 Culture cells in humidified CO2 incubator
NPI-0052 preparation and cell treatment Day 0-1
  1. 1 Dissolve NPI-0052 in DMSO
  2. 2 Prepare DMSO vehicle control
  3. 3 Treat cells with NPI-0052 at indicated concentrations
γ-radiation treatment Day 1
  1. 1 Irradiate cells or organoids with γ-radiation
  2. 2 Return cells to incubator post-irradiation
Combination treatments and timing schemes Day 1-2
  1. 1 Pre-treatment protocol: NPI-0052 followed by γ-radiation
  2. 2 Post-treatment protocol: γ-radiation followed by NPI-0052
p73 knockdown via siRNA Day 0-3
  1. 1 Prepare siRNA transfection complex
  2. 2 Incubate transfected cells for 48 hours
Tumor organoid generation and culture Day 0-28
  1. 1 Prepare tumor cell-Matrigel suspension
  2. 2 Plate Matrigel-cell droplets on parafilm mould
  3. 3 Allow Matrigel droplets to set
  4. 4 Transfer organoids to tissue culture dish with medium
  5. 5 Static culture phase (4 days)
  6. 6 Transfer organoids to orbital shaker
  7. 7 Continued organoid culture with medium changes
Apoptosis detection via Annexin V-FITC Day 1-2
  1. 1 Harvest treated cells
  2. 2 Perform Annexin V-FITC staining
  3. 3 Analyze by flow cytometry
Cell viability assessment Day 1-2
  1. 1 Treat cells with NPI-0052 and/or radiation
  2. 2 Harvest treated cells
  3. 3 Perform CellTiter-Glo Luminescent Cell Viability Assay
  4. 4 Record luminescence
Oxidative stress assessment Day 1
  1. 1 Measure mitochondrial membrane potential via DiOC6(3) staining
  2. 2 Measure hydrogen peroxide levels
  3. 3 Measure total glutathione and GSH/GSSG ratio
  4. 4 Antioxidant rescue experiment
Immunostaining and histological analysis of organoids Day 1-3
  1. 1 Treat tumor organoids
  2. 2 Harvest organoids
  3. 3 Fix organoids for paraffin embedding
  4. 4 Section paraffin-embedded organoids
  5. 5 Perform immunostaining
  6. 6 Digitize histological slides
  7. 7 Quantify immunostaining

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Frisira et al., 2019. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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