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GASTRIC Publication-derived

Gastric Adenocarcinoma Tumouroids

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: 5 days 11 phases Cell source: 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

Phase 1 · Tissue acquisition and processing Day 0
  1. a Obtain gastric cancer tissue
  2. b Digest tissue for organoid initiation
Phase 2 · Human organoid culture establishment Day 1 onwards
  1. a Initiate human gastric organoid culture
  2. b Maintain organoid cultures
  3. c Cryopreservation of organoid lines
Phase 3 · Media component dependency analysis Week 1-5
  1. a Test growth factor dependency
  2. b Evaluate 2D culture potential
Phase 4 · Xenograft transplantation Day variable
  1. a Prepare organoids for transplantation
  2. b Perform subcutaneous xenograft injection
  3. c Verify human origin of xenografts
Phase 5 · Immunohistochemical characterisation Day variable
  1. a Prepare samples for immunohistochemistry
  2. b Perform immunohistochemical staining
Phase 6 · Chemotherapeutic drug response testing Day 1-3 or Day 1-4
  1. a Prepare organoids for drug treatment
  2. b Treat with conventional chemotherapeutics
  3. c Assess viability by cell viability assay
  4. d Quantify apoptosis by flow cytometry
  5. e Observe morphological changes
  6. f Calculate IC50 values
Phase 7 · Genomic and transcriptomic analysis Day variable
  1. a Extract DNA from organoids and normal tissue
  2. b Perform whole genome sequencing
  3. c Extract RNA from organoids
  4. d Perform RNA sequencing
  5. e Sequence primary tumour tissue
  6. f Perform bioinformatic processing
Phase 8 · Pathway enrichment and classification analysis Day variable
  1. a Perform gene set enrichment analysis
  2. b Visualise pathway data
  3. c Classify organoid lines by TCGA subtype
Phase 9 · Targeted drug testing Day 1-3
  1. a Identify targetable mutations
  2. b Treat organoids with HER2-targeted therapy
  3. c Assess HER2 signalling by Western blot
  4. d Test combination therapy
  5. e Treat with CDK4/6 inhibitor
  6. f Perform EdU proliferation assay
  7. g Test c-KIT inhibition
Phase 10 · Mouse organoid generation and characterisation Variable
  1. a Establish normal gastric organoids from mouse corpus
  2. b Induce genetic alterations using Cre-GFP adenovirus
  3. c Culture CIN model organoids
  4. d Culture GS-WNT model organoids
  5. e Characterise tumour organoid morphology
  6. f Perform immunohistochemistry on mouse organoids
  7. g Assess growth factor dependency
  8. h Test single-cell clonogenicity
  9. i Test targeted drug response in GS-WNT model
  10. j Perform quantitative RT-PCR
Phase 11 · Statistical analysis Variable
  1. a Perform statistical comparisons
  2. b Generate graphs using Prism
  3. c Quantify Western blot data

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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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