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INTESTINAL Publication-derived

Patient-Derived Colorectal Cancer Organoid and Xenograft Models for Drug Sensitivity Testing and Molecular Characterization

Source Schütte et al., 2017 · Max Planck Institute for Molecular Genetics (correspondence: lehrach@molgen.mpg.de; yaspo@molgen.mpg.de) and Bayer AG (henderson@bayer.com) · 10.1038/ncomms14262

👤 Moritz Schütte, David Henderson, Hans Lehrach, Marie-Laure Yaspo ⏱ 90 days 📋 14 phases 🧫 Patient-Derived CRC (Primary tumours and metastases, stages I-IV)

Abstract

This protocol establishes patient-derived organoid (PDO) and patient-derived xenograft (PDX) models from colorectal cancer (CRC) patient tumours for comparative genomic, transcriptomic, and drug sensitivity analysis. The models are comprehensively characterized by whole genome/exome sequencing and RNA-seq, and tested against a panel of 16 clinically relevant therapeutic compounds to identify biomarkers predicting drug response.

Cell source
Patient-Derived CRC (Primary tumours and metastases, stages I-IV)
Application
Disease modeling and drug sensitivity screening for colorectal cancer

Protocol overview

79 steps across 14 phases

Patient Sample Collection and Tissue Processing Day 0 (tissue receipt)
  1. 1 Resected Tissue Reception and Macroscopic Preparation
  2. 2 Tissue Mincing
  3. 3 Enzymatic Digestion
  4. 4 Cell Suspension Pelleting and Filtration
  5. 5 Red Blood Cell Depletion
Patient-Derived Organoid (PDO) Establishment Days 0-7 (initial culture)
  1. 1 Cell-Matrigel Mixture Preparation
  2. 2 Culture Medium Overlay
  3. 3 Addition of Growth Factors and Inhibitors (Days 0–7)
  4. 4 Culture Maintenance and Monitoring (Days 1–7)
  5. 5 Mycoplasma Testing
PDO Passaging and Expansion Days 7–14 onwards (continuous culture)
  1. 1 Monitoring Organoid Size for Passaging
  2. 2 Organoid Release from Matrigel
  3. 3 Enzymatic Cell Dissociation
  4. 4 Trypsinization Quenching
  5. 5 Re-plating of Cell Clusters
Patient-Derived Xenograft (PDX) Establishment in Mice Days 0–90 (initial engraftment)
  1. 1 Xenograft Recipient Mouse Preparation
  2. 2 Subcutaneous Tumour Fragment Transplantation
  3. 3 Post-operative Recovery and Monitoring
  4. 4 Tumour Engraftment Monitoring (Weeks 1–12)
  5. 5 Passage Tumour Excision and Cryo-preservation
  6. 6 Tumour Histology Confirmation
Nucleic Acid Extraction and Quality Control Days 0–7 (varies by sample type)
  1. 1 DNA and RNA Extraction from Tissue/Cells
  2. 2 DNA Extraction from Blood (Patient Germline Reference)
  3. 3 Nucleic Acid Concentration Determination
  4. 4 RNA Integrity Assessment
High-Throughput Drug Sensitivity Testing in PDOs Days 0–10 (assay duration)
  1. 1 PDO Culture Dissociation to Single-Cell Suspension
  2. 2 Seeding Cells into 384-Well Plates with Matrigel
  3. 3 Pre-Treatment Incubation (Days 0–4)
  4. 4 Compound Addition (Dose-Response Testing)
  5. 5 Post-Treatment Incubation
  6. 6 Cell Viability Measurement by Luminescence
  7. 7 Data Analysis and Response Classification
In Vivo Drug Response Testing in PDX Models Days 0–56 (treatment period + follow-up)
  1. 1 PDX Tumour Fragment Preparation and Transplantation
  2. 2 Tumour Growth Monitoring (Pre-treatment, Days 0–14)
  3. 3 Randomization and Treatment Group Assignment
  4. 4 Treatment Initiation (Day 0)
  5. 5 Tumour Volume and Body Weight Monitoring (Days 0–56)
  6. 6 End-of-Study Euthanasia and Sample Collection
  7. 7 Drug Sensitivity Classification
Whole Genome Sequencing (WGS) and Copy Number Variant Analysis Library prep: 2–3 days; Sequencing: 5–7 days
  1. 1 WGS Library Preparation (High Coverage)
  2. 2 WGS Low-Coverage Library Preparation
  3. 3 High-Coverage WGS Sequencing
  4. 4 Low-Coverage WGS Sequencing
  5. 5 DNA Read Alignment
  6. 6 Copy Number Variant Detection
Whole Exome Sequencing (WES) and Somatic Variant Calling Library prep: 2–3 days; Sequencing: 5–7 days
  1. 1 WES Library Preparation (SOLiD Platform Option)
  2. 2 WES Library Preparation (Illumina Platform Option)
  3. 3 WES Sequencing (SOLiD)
  4. 4 WES Sequencing (Illumina)
  5. 5 Somatic Single Nucleotide Variant (SNV) Detection
  6. 6 Somatic Insertion/Deletion (Indel) Detection
  7. 7 Microsatellite Status Analysis
Whole Transcriptome Sequencing (RNA-seq) and Gene Expression Analysis Library prep: 2–3 days; Sequencing: 5–7 days
  1. 1 RNA-seq Library Preparation (Option 1: Strand-Specific TruSeq)
  2. 2 RNA-seq Library Preparation (Option 2: TruSeq Stranded)
  3. 3 Ribosomal RNA Depletion (for total RNA samples)
  4. 4 RNA-seq Sequencing
  5. 5 RNA Read Alignment and Gene Expression Quantification
  6. 6 Gene Fusion Detection
  7. 7 Gene Fusion Validation by RT-PCR
Targeted Sequencing for Mutation Validation Library prep: 1–2 days; Sequencing: 3–5 days
  1. 1 Targeted Sequencing Library Preparation
  2. 2 Custom Amplicon Panel Design
  3. 3 Targeted Sequencing
  4. 4 Sanger Sequencing Validation of Selected Mutations
DNA Methylation Profiling 3–5 days (bisulfite conversion, hybridization, scanning)
  1. 1 Bisulfite Conversion of Genomic DNA
  2. 2 Bisulfite Conversion Quality Control
  3. 3 Hybridization to Infinium 450K BeadChips
  4. 4 Data Pre-processing and Quality Control
  5. 5 Methylation Data Normalization
  6. 6 Methylation Clustering and Classification
Immunohistochemistry and Immunofluorescence of PDO Cultures Days 4–7 (culture to staining)
  1. 1 PDO Culture Fixation and FFPE Embedding
  2. 2 Immunohistochemistry Staining
  3. 3 Immunofluorescence Staining and Confocal Microscopy
  4. 4 Live Imaging and Time-Lapse Microscopy
WNT Reporter Assay and FACS-Based Cell Sorting Days 0–7 (transduction to RNA extraction)
  1. 1 Lentiviral Transduction with WNT Reporter
  2. 2 Post-Transduction Culture and Selection
  3. 3 Organoid Dissociation to Single Cells
  4. 4 Fluorescence-Activated Cell Sorting (FACS)
  5. 5 RNA Extraction from Sorted Cell Fractions
  6. 6 RNA-seq Analysis of Sorted Populations

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Schütte et al., 2017. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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