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PANCREAS Publication-derived

Pancreatic Ductal Adenocarcinoma Organoids for Drug Screening

Full SOP with Academic or a plan

Source Seino et al., 2018 · Keio University School of Medicine, Department of Gastroenterology, Tokyo · 10.1016/j.stem.2017.12.009

Authors: Takashi Seino, Shintaro Kawasaki, Mariko Shimokawa, Hiroki Tamagawa, Kohta Toshimitsu, Masayuki Fujii, Yuki Ohta, Mami Matano, Kosaku Nanki, Kenta Kawasaki, Sirirat Takahashi, Shinya Sugimoto, Eisuke Iwasaki, Junichi Takagi, Takao Itoi, Minoru Kitago, Yuko Kitagawa, Takanori Kanai, Toshiro Sato Duration: 24 days 8 phases Cell source: Patient-Derived PDAC

Abstract

This protocol describes the establishment and culture of patient-derived pancreatic ductal adenocarcinoma (PDAC) organoids from surgical specimens, fine-needle aspirates, or ascites samples, followed by functional characterization of Wnt/R-spondin niche dependencies. The method enables generation of a comprehensive tumor organoid library for investigating niche factor requirements and transcriptional programs underlying PDAC heterogeneity.

Cell source
Patient-Derived PDAC
Application
Disease modeling; Drug screening

Protocol overview

34 steps across 8 phases

Phase 1 · Sample Processing and Tissue Dissociation Day 1
  1. a Tissue Washing and Mincing
  2. b Enzymatic Digestion with Liberase TH
  3. c Secondary Digestion with TrypLE Express
  4. d Enzyme Inactivation
Phase 2 · Organoid Culture Initiation Day 1-14
  1. a Matrigel Embedding
  2. b Basal Culture Medium Preparation
  3. c Initial Culture Establishment
Phase 3 · Enrichment of PDAC Organoids Week 2-4
  1. a EGF Removal Selection
  2. b Sequential Niche Factor Removal Selection
Phase 4 · Functional Characterization of Niche Dependencies Week 4-12
  1. a Systematic Niche Factor Withdrawal
  2. b Classification into Wnt Niche Subtypes
  3. c Porcupine Inhibitor Treatment to Validate Wnt Production
  4. d ICG001 Treatment to Assess Wnt-Signal Dependency
Phase 5 · Molecular Analysis and Quality Control Week 4-24
  1. a Whole-Exome Sequencing
  2. b Copy-Number Analysis by CGH/SNP Microarray
  3. c Gene-Expression Microarray Analysis
  4. d DNA Methylation Analysis
  5. e Real-Time Quantitative PCR Validation
Phase 6 · CAF Co-culture and In Vivo Validation Week 4-16
  1. a Cancer-Associated Fibroblast (CAF) Establishment
  2. b PDAC Organoid–CAF Aggregation
  3. c Transfer to Collagen–Matrigel Embedding
  4. d Assessment of CAF-Mediated Wnt Support
  5. e Xenotransplantation of Organoids with or without CAFs
  6. f In Vivo Porcupine Inhibitor Treatment
Phase 7 · GATA6 Functional Analysis Week 8-20
  1. a GATA6 Knockdown and Knockout Generation
  2. b Assessment of Wnt Niche Adaptation upon GATA6 Loss
  3. c GATA6 Overexpression in W+ Organoids
  4. d Clinical Validation: GATA6 Expression Correlation with Epithelial Wnt
Phase 8 · CRISPR-Cas9 Engineering of Pancreatic Organoids Week 8-24
  1. a Generation of Engineered Organoids with Driver Mutations
  2. b Transcriptome Validation of Engineered Organoids
  3. c Subcutaneous Xenotransplantation of Engineered Organoids
  4. d Assessment of Wnt-Niche Independency Acquisition during Tumorigenesis
  5. e Gene-Expression and Epigenetic Analysis of Adapted Organoids
  6. f Validation of Porcupine-Inhibitor Sensitivity in Adapted Organoids

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Attribution

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

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