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PANCREAS Publication-derived

Organoid profiling identifies common responders to chemotherapy in pancreatic cancer

Source Tiriac et al., 2018 · Cold Spring Harbor Laboratory · 10.1158/2159-8290.cd-18-0349

👤 Hervé Tiriac, Pascal Belleau, Dannielle D. Engle, David A. Tuveson, Steven Gallinger ⏱ 90 days 📋 10 phases 🧫 Patient-Derived iPSC (Pancreatic Cancer)

Abstract

This protocol describes the generation, molecular characterization, and pharmacotyping of pancreatic cancer patient-derived organoids (PDOs). The workflow enables deep genomic and transcriptomic profiling of PDOs and establishes a drug-testing pipeline (pharmacotyping) to predict individual patient chemotherapy responses and identify alternative treatment strategies for chemotherapy-refractory disease.

Cell source
Patient-Derived iPSC (Pancreatic Cancer)
Application
Disease modeling, Drug screening, Precision medicine

Protocol overview

57 steps across 10 phases

Tissue Sample Acquisition and Preparation Day 0
  1. 1 Source human pancreatic tissues
  2. 2 Mince tissue samples
Organoid Isolation and Culture Initiation Day 0–1
  1. 1 Enzymatic digestion of tissue
  2. 2 Plate cells with Matrigel
  3. 3 Add Human complete Feeding Medium
  4. 4 Incubate organoid cultures
Organoid Expansion and Quality Control Days 1–14
  1. 1 Monitor organoid growth
  2. 2 Passage organoids
  3. 3 Test for Mycoplasma contamination
  4. 4 Prepare organoid samples for sequencing
DNA Extraction and Whole Exome/Genome Sequencing Days 14–30
  1. 1 Extract genomic DNA from organoids
  2. 2 Prepare Whole Exome Sequencing (WES) libraries
  3. 3 Sequence WES libraries
  4. 4 Prepare Whole Genome Sequencing (WGS) libraries for matched tumor-normal pairs
  5. 5 Sequence WGS libraries
RNA Extraction and RNA-sequencing Days 14–30
  1. 1 Extract total RNA from organoids
  2. 2 Construct RNA-seq libraries
  3. 3 Sequence RNA-seq libraries
Genomic Data Processing and Variant Calling Days 30–60
  1. 1 Preprocess sequencing reads
  2. 2 Call somatic single nucleotide variants (SNVs)
  3. 3 Call somatic indels
  4. 4 Detect structural variants (SVs)
  5. 5 Call copy number variants (CNVs)
  6. 6 Calculate purity and ploidy estimates
  7. 7 Determine organoid-tumor SNV concordance
  8. 8 Compare CNV between organoids and primary tumors
RNA-sequencing Data Processing and Analysis Days 30–60
  1. 1 Assess RNA-seq read quality
  2. 2 Trim sequencing reads
  3. 3 Align reads to reference genome
  4. 4 Quantify gene expression
  5. 5 Normalize gene expression counts
  6. 6 Perform principal component analysis (PCA)
  7. 7 Identify PDAC molecular subtypes (Classical vs. Basal-like)
  8. 8 Perform non-negative matrix factorization (NMF) clustering
  9. 9 Perform differential gene expression analysis
  10. 10 Perform gene set enrichment analysis (GSEA)
Pharmacotyping (Drug Sensitivity Testing) Days 5–10 (after organoid expansion)
  1. 1 Dissociate organoids into single cells
  2. 2 Plate dissociated cells in ultra-low attachment 96-well plates with Matrigel
  3. 3 Add chemotherapeutic agents
  4. 4 Add targeted agents (optional alternative therapy screening)
  5. 5 Incubate with drugs for 5 days
  6. 6 Assess cell viability
  7. 7 Fit dose-response curves
  8. 8 Calculate area under the curve (AUC)
  9. 9 Classify PDOs by drug sensitivity
  10. 10 Validate pharmacotyping stability across passages
Pharmacotranscriptomic Analysis Days 60–90
  1. 1 Correlate PDO drug sensitivity with gene expression
  2. 2 Filter genes for drug-specific sensitivity signatures
  3. 3 Cluster genes by correlation coefficient
  4. 4 Score PDO cultures using drug sensitivity signatures
  5. 5 Validate signatures in independent patient cohorts
  6. 6 Correlate signature enrichment with patient clinical outcomes
  7. 7 Assess signature performance in treatment-naïve controls
Quality Control and Data Interpretation Throughout protocol
  1. 1 Verify PDO tumor identity
  2. 2 Check pharmacotyping curve quality
  3. 3 Validate concordance between primary tumor and PDO
  4. 4 Document all QC metrics

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Tiriac et al., 2018. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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