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PANCREAS Publication-derived

Pancreatic Ductal Adenocarcinoma Tumouroids for Biobanking

Full SOP with Academic or a plan

Source Driehuis et al., 2019 · Hubrecht Institute and University Medical Center Utrecht · 10.1073/pnas.1911273116

Authors: Else Driehuis, Arne van Hoeck, Kat Moore, Sigrid Kolders, Hayley E. Francies, M. Can Gulersonmez, Edwin C. A. Stigter, Boudewijn Burgering, Veerle Geurts, Ana Gracanin, Gergana Bounova, Folkert H. Morsink, Robert Vries, Sylvia Boj, Johan van Es, G. Johan A. Offerhaus, Onno Kranenburg, Mathew J. Garnett, Lodewyk Wessels, Edwin Cuppen, Lodewijk A. A. Brosens, Hans Clevers Duration: 180 days 7 phases Cell source: Patient-Derived tumor tissue from surgical resections and biopsies (pancreatic cancer, distal cholangiocarcinoma)

Abstract

This protocol describes the derivation and culture of patient-derived organoids (PDOs) from pancreatic cancer and distal cholangiocarcinoma tumors. PDOs recapitulate tumor histology and genetic alterations typical of pancreatic ductal adenocarcinoma (PDAC). The established PDO biobank is characterized by whole-genome sequencing, RNA sequencing, and histology, and is used for high-throughput drug screening with a panel of 76 therapeutic agents to identify personalized treatment sensitivities.

Cell source
Patient-Derived tumor tissue from surgical resections and biopsies (pancreatic cancer, distal cholangiocarcinoma)
Application
Disease modeling and personalized drug screening for pancreatic cancer

Protocol overview

35 steps across 7 phases

Phase 1 · Tissue Collection and Processing Day 0-1
  1. a Tissue Collection and Storage
  2. b Tissue Enzymatic Digestion
  3. c Cell Suspension Preparation
  4. d BME Preparation and Plating
  5. e Initial Medium Addition and ROCK Inhibitor Supplementation
Phase 2 · Organoid Culture in Parallel Media Day 1-Day 21+
  1. a Establish Tumor Organoids in Two Media Types
  2. b Monitor Organoid Outgrowth
  3. c Organoid Passaging
  4. d Maintenance of Parallel Cultures
Phase 3 · Functional Selection and Genetic Characterization Day 21-Day 60+
  1. a SMAD4 Status Detection via Growth Factor Withdrawal
  2. b RNA Extraction for Molecular Characterization
  3. c qPCR for KRAS and Gene Expression Validation
  4. d DNA Extraction and Whole-Genome Sequencing Library Preparation
  5. e RNA Sequencing Library Preparation and Sequencing
Phase 4 · Histological and Immunohistochemical Analysis Day 30-Day 60
  1. a Sample Preparation for H&E and Immunohistochemistry
  2. b TP53 Immunohistochemical Staining
  3. c SMAD4 Immunohistochemical Staining
  4. d MTAP Immunohistochemical Staining
  5. e Morphological Comparison of PDOs and Primary Tumor Tissue
Phase 5 · High-Throughput Drug Screening Day 60-Day 120
  1. a Organoid Dissociation and Single-Cell Plating
  2. b Compound Library Preparation and Addition
  3. c Incubation with Compounds
  4. d Cell Viability Assay
  5. e Data Processing and IC50 Calculation
  6. f Drug Sensitivity Profile Analysis
Phase 6 · Functional Validation: PRMT5 Inhibitor and MTAP Status Day 100-Day 150
  1. a MTAP and CDKN2A Genomic Status Determination
  2. b PRMT5 Inhibitor (EZP015556) Dose-Response Screening
  3. c MTAP Expression Induction via Lentiviral Vector
  4. d Validation of MTAP Expression and EZP015556 Sensitivity
  5. e MTA Level Quantification via Mass Spectrometry
  6. f Correlation of MTA Levels with EZP015556 Sensitivity
Phase 7 · Clinical Correlation and Biomarker Analysis Day 120-Day 180
  1. a Collection of Clinical Treatment Data
  2. b Comparison of PDO In Vitro Response with Clinical Patient Outcome
  3. c Integrated Genomic and Drug Response Analysis
  4. d Biomarker Validation and Clinical Translation Planning

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Attribution

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

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