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REPRODUCTIVE Publication-derived

Patient-Derived High-Grade Serous Ovarian Cancer Organoid Generation and DNA Repair Profiling

Source Hill et al., 2018 · Dana-Farber Cancer Institute and Brigham and Women's Hospital, Harvard Medical School · 10.1158/2159-8290.cd-18-0474

👤 S.J. Hill, E.A. Roberts, N.S. Horowitz, M.G. Muto, M.J. Worley Jr, C.M. Feltmate, C. Yang, R. Morizane, K.T. Do, B. Decker, B.S. Kochupurakkal, H. Nguyen, P.A. Konstantinopoulos, J.F. Liu, U.A. Matulonis, G.I. Shapiro, E.M. Swisher, M.R. Nucci, C.P. Crum, R.S. Berkowitz, J.V. Bonventre, A.D. D'Andrea ⏱ 30 days 📋 8 phases 🧫 Patient-Derived Tumor Cells (High-Grade Serous Ovarian Cancer)

Abstract

This protocol describes the generation of short-term patient-derived organoid cultures from high-grade serous ovarian cancers (HGSC) and their use for functional profiling of DNA repair capacity. The organoids are generated within 7-10 days, maintain genetic concordance with parent tumors, and enable rapid assessment of homologous recombination (HR) competency and replication fork stability to predict sensitivity to DNA-damaging agents and repair-targeting drugs (PARP inhibitors, CHK1/ATR inhibitors, platinum agents).

Cell source
Patient-Derived Tumor Cells (High-Grade Serous Ovarian Cancer)
Application
Disease modeling and drug screening for DNA repair defects and therapeutic sensitivity

Protocol overview

46 steps across 8 phases

Tumor Tissue Collection and Processing Day 0 (on day of surgery)
  1. 1 Collect Fresh Tumor Tissue
  2. 2 Prepare Basal Culture Media
  3. 3 Dice and Digest Tumor Tissue
  4. 4 Dilute and Filter Digested Tissue
  5. 5 Harvest and Wash Cell Pellet
Organoid Plating and Initial Culture Days 0-7
  1. 1 Mix Cells with Matrigel
  2. 2 Plate Matrigel-Cell Suspension
  3. 3 Add General Culture Medium
  4. 4 Incubate Organoid Culture
  5. 5 Monitor Organoid Growth
Organoid Expansion and Passage Days 7-30
  1. 1 Assess Organoid Maturation
  2. 2 Perform Organoid Passage
  3. 3 Collect Dissociated Cells
  4. 4 Re-plate Organoid Cells
  5. 5 Continue Culture Maintenance
RAD51 Focus Assay for HR Competency Assessment Days 10-14
  1. 1 Prepare Organoid Culture on Coverslips
  2. 2 Treat Organoids with Ionizing Radiation (0 or 10 Gy)
  3. 3 Fix Organoids
  4. 4 Block and Immunostain
  5. 5 Microscopy and Scoring
DNA Fiber Assay for Replication Fork Stability Assessment Days 10-14
  1. 1 Pulse Organoid Cells with CldU
  2. 2 Pulse with IdU
  3. 3 Treat with Hydroxyurea (HU) Fork Stalling Agent
  4. 4 Harvest and Prepare DNA Fibers
  5. 5 Immunofluorescence Detection and Measurement
  6. 6 Calculate Fork Stability Ratio
Drug Sensitivity Testing Days 10-21
  1. 1 Prepare Organoid Cultures for Drug Testing
  2. 2 Prepare Drug Dilutions
  3. 3 Treat Organoids with Drugs
  4. 4 Assess Cell Viability
  5. 5 Generate Dose Response Curves
Western Blot Analysis for DNA Damage and Signaling Days 10-21
  1. 1 Treat Organoid Cells with Drugs or Drug Combinations
  2. 2 Harvest Cells and Prepare Lysates
  3. 3 Quantify Protein Concentration
  4. 4 Prepare and Run SDS-PAGE Gel
  5. 5 Transfer Proteins to Membrane
  6. 6 Block and Immunostain Membrane
  7. 7 Detect Proteins by Chemiluminescence
  8. 8 Quantify Band Intensity and Normalize
Whole-Exome Sequencing (WES) and Genomic Analysis Days 0-21 (sample collection through sequencing)
  1. 1 Collect Samples for WES
  2. 2 Extract DNA
  3. 3 Prepare DNA Libraries
  4. 4 Perform Sequencing
  5. 5 Perform Variant Calling and Mutation Analysis
  6. 6 Analyze Copy Number Alterations (CNA)
  7. 7 Assess HRD Mutational Signature

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Hill et al., 2018. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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