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INTESTINAL Publication-derived

Colorectal Cancer Colonic Tumouroids for Transplantation

Full SOP with Academic or a plan

Source Schütte et al., 2017 · Max Planck Institute for Molecular Genetics and Bayer AG · 10.1038/ncomms14262

Authors: Moritz Schütte, David Henderson, Hans Lehrach, Marie-Laure Yaspo Duration: 90 days 14 phases Cell source: 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

Phase 1 · Patient Sample Collection and Tissue Processing Day 0 (tissue receipt)
  1. a Resected Tissue Reception and Macroscopic Preparation
  2. b Tissue Mincing
  3. c Enzymatic Digestion
  4. d Cell Suspension Pelleting and Filtration
  5. e Red Blood Cell Depletion
Phase 2 · Patient-Derived Organoid (PDO) Establishment Days 0-7 (initial culture)
  1. a Cell-Matrigel Mixture Preparation
  2. b Culture Medium Overlay
  3. c Addition of Growth Factors and Inhibitors (Days 0-7)
  4. d Culture Maintenance and Monitoring (Days 1-7)
  5. e Mycoplasma Testing
Phase 3 · PDO Passaging and Expansion Days 7-14 onwards (continuous culture)
  1. a Monitoring Organoid Size for Passaging
  2. b Organoid Release from Matrigel
  3. c Enzymatic Cell Dissociation
  4. d Trypsinization Quenching
  5. e Re-plating of Cell Clusters
Phase 4 · Patient-Derived Xenograft (PDX) Establishment in Mice Days 0-90 (initial engraftment)
  1. a Xenograft Recipient Mouse Preparation
  2. b Subcutaneous Tumour Fragment Transplantation
  3. c Post-operative Recovery and Monitoring
  4. d Tumour Engraftment Monitoring (Weeks 1-12)
  5. e Passage Tumour Excision and Cryo-preservation
  6. f Tumour Histology Confirmation
Phase 5 · Nucleic Acid Extraction and Quality Control Days 0-7 (varies by sample type)
  1. a DNA and RNA Extraction from Tissue/Cells
  2. b DNA Extraction from Blood (Patient Germline Reference)
  3. c Nucleic Acid Concentration Determination
  4. d RNA Integrity Assessment
Phase 6 · High-Throughput Drug Sensitivity Testing in PDOs Days 0-10 (assay duration)
  1. a PDO Culture Dissociation to Single-Cell Suspension
  2. b Seeding Cells into 384-Well Plates with Matrigel
  3. c Pre-Treatment Incubation (Days 0-4)
  4. d Compound Addition (Dose-Response Testing)
  5. e Post-Treatment Incubation
  6. f Cell Viability Measurement by Luminescence
  7. g Data Analysis and Response Classification
Phase 7 · In Vivo Drug Response Testing in PDX Models Days 0-56 (treatment period + follow-up)
  1. a PDX Tumour Fragment Preparation and Transplantation
  2. b Tumour Growth Monitoring (Pre-treatment, Days 0-14)
  3. c Randomization and Treatment Group Assignment
  4. d Treatment Initiation (Day 0)
  5. e Tumour Volume and Body Weight Monitoring (Days 0-56)
  6. f End-of-Study Euthanasia and Sample Collection
  7. g Drug Sensitivity Classification
Phase 8 · Whole Genome Sequencing (WGS) and Copy Number Variant Analysis Library prep: 2-3 days; Sequencing: 5-7 days
  1. a WGS Library Preparation (High Coverage)
  2. b WGS Low-Coverage Library Preparation
  3. c High-Coverage WGS Sequencing
  4. d Low-Coverage WGS Sequencing
  5. e DNA Read Alignment
  6. f Copy Number Variant Detection
Phase 9 · Whole Exome Sequencing (WES) and Somatic Variant Calling Library prep: 2-3 days; Sequencing: 5-7 days
  1. a WES Library Preparation (SOLiD Platform Option)
  2. b WES Library Preparation (Illumina Platform Option)
  3. c WES Sequencing (SOLiD)
  4. d WES Sequencing (Illumina)
  5. e Somatic Single Nucleotide Variant (SNV) Detection
  6. f Somatic Insertion/Deletion (Indel) Detection
  7. g Microsatellite Status Analysis
Phase 10 · Whole Transcriptome Sequencing (RNA-seq) and Gene Expression Analysis Library prep: 2-3 days; Sequencing: 5-7 days
  1. a RNA-seq Library Preparation (Option 1: Strand-Specific TruSeq)
  2. b RNA-seq Library Preparation (Option 2: TruSeq Stranded)
  3. c Ribosomal RNA Depletion (for total RNA samples)
  4. d RNA-seq Sequencing
  5. e RNA Read Alignment and Gene Expression Quantification
  6. f Gene Fusion Detection
  7. g Gene Fusion Validation by RT-PCR
Phase 11 · Targeted Sequencing for Mutation Validation Library prep: 1-2 days; Sequencing: 3-5 days
  1. a Targeted Sequencing Library Preparation
  2. b Custom Amplicon Panel Design
  3. c Targeted Sequencing
  4. d Sanger Sequencing Validation of Selected Mutations
Phase 12 · DNA Methylation Profiling 3-5 days (bisulfite conversion, hybridization, scanning)
  1. a Bisulfite Conversion of Genomic DNA
  2. b Bisulfite Conversion Quality Control
  3. c Hybridization to Infinium 450K BeadChips
  4. d Data Pre-processing and Quality Control
  5. e Methylation Data Normalization
  6. f Methylation Clustering and Classification
Phase 13 · Immunohistochemistry and Immunofluorescence of PDO Cultures Days 4-7 (culture to staining)
  1. a PDO Culture Fixation and FFPE Embedding
  2. b Immunohistochemistry Staining
  3. c Immunofluorescence Staining and Confocal Microscopy
  4. d Live Imaging and Time-Lapse Microscopy
Phase 14 · WNT Reporter Assay and FACS-Based Cell Sorting Days 0-7 (transduction to RNA extraction)
  1. a Lentiviral Transduction with WNT Reporter
  2. b Post-Transduction Culture and Selection
  3. c Organoid Dissociation to Single Cells
  4. d Fluorescence-Activated Cell Sorting (FACS)
  5. e RNA Extraction from Sorted Cell Fractions
  6. f RNA-seq Analysis of Sorted Populations

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Attribution

This SOP was written by Organthis based on the published method in Schütte et al., 2017. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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