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GASTRIC Publication-derived

Human gastric cancer modelling using organoids

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

👤 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 ⏱ 5 days 📋 11 phases 🧫 Patient-Derived iPSC (Gastric cancer)

Abstract

This protocol describes the establishment and culture of human and mouse gastric cancer organoids derived from patient surgical specimens and normal tissue. The organoids recapitulate histological, immunohistochemical, and molecular characteristics of primary gastric adenocarcinomas and enable assessment of chemotherapeutic response, mutation profiling via whole genome sequencing, and targeted drug testing for personalised therapy approaches.

Cell source
Patient-Derived iPSC (Gastric cancer)
Application
Disease modeling and drug screening

Protocol overview

47 steps across 11 phases

Tissue acquisition and processing Day 0
  1. 1 Obtain gastric cancer tissue
  2. 2 Digest tissue for organoid initiation
Human organoid culture establishment Day 1 onwards
  1. 1 Initiate human gastric organoid culture
  2. 2 Maintain organoid cultures
  3. 3 Cryopreservation of organoid lines
Media component dependency analysis Week 1–5
  1. 1 Test growth factor dependency
  2. 2 Evaluate 2D culture potential
Xenograft transplantation Day variable
  1. 1 Prepare organoids for transplantation
  2. 2 Perform subcutaneous xenograft injection
  3. 3 Verify human origin of xenografts
Immunohistochemical characterisation Day variable
  1. 1 Prepare samples for immunohistochemistry
  2. 2 Perform immunohistochemical staining
Chemotherapeutic drug response testing Day 1–3 or Day 1–4
  1. 1 Prepare organoids for drug treatment
  2. 2 Treat with conventional chemotherapeutics
  3. 3 Assess viability by cell viability assay
  4. 4 Quantify apoptosis by flow cytometry
  5. 5 Observe morphological changes
  6. 6 Calculate IC50 values
Genomic and transcriptomic analysis Day variable
  1. 1 Extract DNA from organoids and normal tissue
  2. 2 Perform whole genome sequencing
  3. 3 Extract RNA from organoids
  4. 4 Perform RNA sequencing
  5. 5 Sequence primary tumour tissue
  6. 6 Perform bioinformatic processing
Pathway enrichment and classification analysis Day variable
  1. 1 Perform gene set enrichment analysis
  2. 2 Visualise pathway data
  3. 3 Classify organoid lines by TCGA subtype
Targeted drug testing Day 1–3
  1. 1 Identify targetable mutations
  2. 2 Treat organoids with HER2-targeted therapy
  3. 3 Assess HER2 signalling by Western blot
  4. 4 Test combination therapy
  5. 5 Treat with CDK4/6 inhibitor
  6. 6 Perform EdU proliferation assay
  7. 7 Test c-KIT inhibition
Mouse organoid generation and characterisation Variable
  1. 1 Establish normal gastric organoids from mouse corpus
  2. 2 Induce genetic alterations using Cre-GFP adenovirus
  3. 3 Culture CIN model organoids
  4. 4 Culture GS-WNT model organoids
  5. 5 Characterise tumour organoid morphology
  6. 6 Perform immunohistochemistry on mouse organoids
  7. 7 Assess growth factor dependency
  8. 8 Test single-cell clonogenicity
  9. 9 Test targeted drug response in GS-WNT model
  10. 10 Perform quantitative RT-PCR
Statistical analysis Variable
  1. 1 Perform statistical comparisons
  2. 2 Generate graphs using Prism
  3. 3 Quantify Western blot data

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Seidlitz 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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