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REPRODUCTIVE Publication-derived

High-Grade Serous Ovarian Cancer Reproductive Tumouroids

Full SOP with Academic or a plan

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

Authors: 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 Duration: 30 days 8 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Hill et al., 2018. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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