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GASTRIC Publication-derived

Gastric Adenocarcinoma Tumouroids for Drug Screening

Full SOP with Academic or a plan

Source Seidlitz et al., 2019 · University Hospital Carl Gustav Carus, Technische Universität Dresden · 10.1136/gutjnl-2017-314549

Authors: Therese Seidlitz, Sebastian R. Merker, Alexander Rothe, Falk Zakrzewski, Clàre von Neubeck, Konrad Grützmann, Ulrich Sommer, Christine Schweitzer, Sebastian Schölch, Heike Uhlemann, Anne-Marlene Gaebler, Kristin Werner, Mechthild Krause, Gustavo B. Baretton, Thilo Welsch, Bon-Kyoung Koo, Daniela E. Aust, Barbara Klink, Jürgen Weitz, Daniel E. Stange Duration: 35 days 12 phases Cell source: Human gastric cancer tissue (patient-derived)

Abstract

This protocol describes the establishment and culture of human gastric cancer organoids from surgical resection specimens, followed by characterization via immunohistochemistry, whole genome sequencing, and drug sensitivity testing to conventional chemotherapeutics and targeted therapies. The organoids maintain primary tumor characteristics and enable personalized therapy response assessment.

Cell source
Human gastric cancer tissue (patient-derived)
Application
Disease modeling and drug screening

Protocol overview

41 steps across 12 phases

Phase 1 · Human Gastric Tissue Acquisition and Processing Day 0
  1. a Surgical tissue collection
  2. b Tissue optimization for organoid initiation
Phase 2 · Human Organoid Culture Initiation and Maintenance Day 1 onwards
  1. a Seed organoid culture in standard organoid medium
  2. b Passage organoid cultures
  3. c Cryopreserve organoid lines
Phase 3 · Growth Factor Dependency Analysis Day 1-35
  1. a Deplete media components individually
  2. b Assess phenotypical response to media modifications
Phase 4 · Two-Dimensional Culture Assessment Day 1
  1. a Attempt 2D culture in plastic flasks
Phase 5 · Xenograft Transplantation Day 0–variable (follow tumor growth curve)
  1. a Prepare organoids for subcutaneous injection
  2. b Inject organoids subcutaneously into mouse hind legs
  3. c Harvest and verify xenograft tumors
Phase 6 · Immunohistochemical Characterization Day 1-3 (tissue processing)
  1. a Prepare organoid samples for immunohistochemistry
  2. b Perform immunohistochemical staining
  3. c Evaluate immunohistochemical results
Phase 7 · Conventional Chemotherapy Drug Sensitivity Testing Day 1-4
  1. a Prepare chemotherapeutic agents
  2. b Seed organoids into multi-well assay plates
  3. c Treat organoids with chemotherapeutic agents (24 hours post-seeding)
  4. d Assess cell viability via viability assays
  5. e Perform apoptosis detection (optional confirmation)
  6. f Calculate IC50 values and define active drugs
Phase 8 · Genomic Characterization - Whole Genome Sequencing Day 1-5
  1. a Extract genomic DNA from organoids and normal tissue
  2. b Prepare DNA library and perform whole genome sequencing
  3. c Sequence primary tumor tissue
  4. d Perform bioinformatic processing
Phase 9 · Transcriptomic Characterization - RNA Sequencing Day 1-5
  1. a Extract RNA from organoid cultures
  2. b Prepare stranded RNA-Seq library
  3. c Perform RNA-Seq on Illumina NextSeq500
  4. d Perform bioinformatic analysis of RNA-Seq data
Phase 10 · Pathway Analysis and Molecular Classification Day 1-7
  1. a Perform gene set enrichment analysis (GSEA)
  2. b Visualize pathway alterations
  3. c Classify organoid lines according to TCGA gastric cancer subtypes
Phase 11 · Targeted Therapy Testing Day 1-5
  1. a Identify druggable mutations and activated pathways
  2. b Test Anti-HER2 Therapy (Trastuzumab)
  3. c Assess downstream signaling in HER2-targeted organoids
  4. d Test combination therapy (Trastuzumab + Chemotherapy)
  5. e Test CDK4/6 Inhibition (Palbociclib)
  6. f Test Tyrosine Kinase Inhibition (Imatinib)
Phase 12 · Statistical Analysis and Data Reporting Day 1-7
  1. a Perform statistical testing
  2. b Generate graphs and visualizations
  3. c Calculate and report IC50 values
  4. d Quantify and analyze Western blot results

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Attribution

This SOP was written by Organthis based on the published method in Seidlitz et al., 2019. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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