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BREAST Publication-derived

Breast Cancer Tumouroids for Biobanking

Full SOP with Academic or a plan

Source Sachs et al., 2018 · Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and University Medical Center Utrecht · 10.1016/j.cell.2017.11.010

Authors: Norman Sachs, Joep de Ligt, Oded Kopper, Ewa Gogola, Gergana Bounova, Fleur Weeber, Anjali Vanita Balgobind, Karin Wind, Ana Gracanin, Harry Begthel, Jeroen Korving, Ruben van Boxtel, Alexandra Alves Duarte, Daphne Lelieveld, Arne van Hoeck, Robert Frans Ernst, Francis Blokzijl, Isaac Johannes Nijman, Marlous Hoogstraat, Marieke van de Ven, David Anthony Egan, Vittoria Zinzalla, Jurgen Moll, Sylvia Fernandez Boj, Emile Eugene Voest, Lodewyk Wessels, Paul Joannes van Diest, Sven Rottenberg, Robert Gerhardus Jacob Vries, Edwin Cuppen, Hans Clevers Duration: 14 days 12 phases Cell source: Human primary and metastatic breast cancer tissue

Abstract

This protocol describes the establishment and long-term culture of human breast cancer organoids from primary and metastatic tumors. The method enables generation of >100 BC organoid lines that capture disease heterogeneity, retain histological and genetic features of original tumors, and allow high-throughput drug sensitivity screening for functional prediction of patient treatment response.

Cell source
Human primary and metastatic breast cancer tissue
Application
Disease modeling, drug screening, and personalized therapy assessment

Protocol overview

74 steps across 12 phases

Phase 1 · Tissue Processing and Cell Isolation Day 0-1
  1. a Prepare tissue for processing
  2. b Enzymatic tissue digestion
  3. c Sequential shearing and filtration
  4. d Wash and concentrate cells
  5. e Remove erythrocytes (if necessary)
  6. f Prepare cells for seeding
Phase 2 · BC Organoid Culture Initiation and Expansion Day 1-14
  1. a Plate organoid drops
  2. b Add organoid culture medium
  3. c Perform media changes
  4. d Passage cystic organoids (mechanical dissociation)
  5. e Passage dense organoids (enzymatic dissociation)
  6. f Wash and concentrate dissociated organoids
  7. g Passage frequency and timing
Phase 3 · Quality Control and Cryopreservation Week 2 onwards
  1. a Test for mycoplasma contamination
  2. b Implement cross-contamination prevention
  3. c Cryopreserve organoid lines
Phase 4 · Histopathological and Immunohistochemical Characterization Throughout culture (5-7 days post-passage)
  1. a Prepare samples for histology
  2. b Perform H&E staining
  3. c Perform immunohistochemistry for hormone and HER2 receptors
  4. d Image and analyze sections
Phase 5 · Genomic Characterization (Karyotyping) Throughout culture expansion
  1. a Prepare organoids for karyotyping
  2. b Fix cells and prepare metaphase spreads
  3. c Image and count chromosomes
  4. d Data visualization
Phase 6 · Whole Genome Sequencing and Genomic Analysis Throughout culture (parallel to expansion)
  1. a Extract genomic DNA
  2. b Generate DNA libraries and perform sequencing
  3. c Process sequencing data and map reads
  4. d Detect somatic mutations
  5. e Analyze mutational signatures
  6. f Detect copy number alterations
  7. g Validate organoid identity
Phase 7 · RNA Sequencing and Gene Expression Analysis Throughout culture (4-6 days post-passage)
  1. a Extract total RNA
  2. b Prepare RNA-seq libraries
  3. c Perform high-throughput sequencing
  4. d Align reads to reference genome
  5. e Count reads per feature
  6. f Normalize and batch-correct expression data
  7. g Perform subtype classification
  8. h Perform hierarchical clustering and differential expression analysis
Phase 8 · Gene Editing (Optional: TP53 Mutation Generation) Week 3-4
  1. a Prepare organoids for electroporation
  2. b Prepare electroporation mixture
  3. c Perform electroporation
  4. d Post-electroporation recovery
  5. e Select for edited clones
  6. f Pick and clone individual organoids
  7. g Validate mutations by sequencing
Phase 9 · High-Throughput Drug Screening Week 2-3 post-passage
  1. a Prepare organoids for drug screening
  2. b Prepare organoid suspension
  3. c Add drug compounds
  4. d Incubate organoids with drugs
  5. e Measure cell viability with CellTiter-Glo 3D
  6. f Read luminescence
  7. g Calculate IC50 values
  8. h Compile and visualize results
  9. i Perform PARPi drug screening (BRCA-related organoids)
  10. j Measure PARPi response and validate predictions
Phase 10 · In Vivo Xeno-Transplantation and Comparative Drug Response Studies Week 4-8
  1. a Select and prepare organoid lines for transplantation
  2. b Harvest and resuspend organoids
  3. c Perform orthotopic transplantation
  4. d Monitor tumor growth and randomize treatment groups
  5. e Administer drug treatment
  6. f Continue tumor monitoring during treatment
  7. g Determine humane endpoints and euthanize mice
  8. h Analyze and compare in vitro and in vivo drug responses
Phase 11 · Clinical Correlation Studies (Patient-Derived Metastatic Organoids) Throughout study
  1. a Collect needle biopsies from metastatic BC patients
  2. b Generate BC organoid lines from metastatic biopsies
  3. c Perform drug screening on metastatic organoid lines
  4. d Compare in vitro organoid responses with clinical patient responses
  5. e Calculate predictive accuracy metrics
Phase 12 · Biobanking and Ethical Management Ongoing
  1. a Catalog and annotate organoid lines
  2. b Establish ethics and governance protocols
  3. c Maintain patient confidentiality
  4. d Register organoid lines in central biobank repository
  5. e Manage long-term organoid storage and distribution

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Attribution

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

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