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BREAST Publication-derived

Modeling Breast Cancer Using CRISPR-Cas9–Mediated Engineering of Human Breast Organoids

Source Dekkers et al., 2020 · The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia · 10.1093/jnci/djz196

👤 Johanna F. Dekkers, James R. Whittle, François Vaillant, Huei-Rong Chen, Caleb Dawson, Kevin Liu, Maarten H. Geurts, Marco J. Herold, Hans Clevers, Geoffrey J. Lindeman, Jane E. Visvader ⏱ 20 days 📋 10 phases 🧫 Human breast epithelial cells from reduction mammoplasties

Abstract

This protocol demonstrates the generation of human breast epithelial organoids from normal reduction mammoplasty tissue and their genetic engineering using CRISPR-Cas9 to knockout tumor suppressor genes (P53, PTEN, RB1, NF1). Mutant organoids develop long-term culturing capacity and, when transplanted into mice, form estrogen-receptor positive luminal tumors that respond to endocrine therapy and chemotherapy, providing a tool to study the clonal evolution and molecular events of breast cancer.

Cell source
Human breast epithelial cells from reduction mammoplasties
Application
Disease modeling; Breast cancer oncogenesis and drug screening

Protocol overview

60 steps across 10 phases

Organoid Generation from Normal Breast Tissue Day 0–14
  1. 1 Tissue collection and donor consent
  2. 2 Flow cytometry sorting of epithelial subsets
  3. 3 Combine basal and luminal progenitor populations
  4. 4 Culture organoids in appropriate growth medium
  5. 5 Assess organoid formation and morphology
Lentiviral Cas9 Introduction and Selection Week 2–3 post-organoid formation
  1. 1 Prepare lentivirus expressing Cas9
  2. 2 Dissociate organoids to single cells
  3. 3 Transduce organoid-derived cells with Cas9 lentivirus
  4. 4 Expand Cas9-expressing organoids
Sequential CRISPR-Cas9 Gene Editing Week 3–8 post-Cas9 introduction
  1. 1 Prepare single-guide RNA–expressing lentiviruses
  2. 2 First round: Transduce with P53 sgRNA lentivirus
  3. 3 Apply Nutlin-3a selection pressure for P53 editing
  4. 4 Verify P53 editing by immunoblot
  5. 5 Second round: Transduce P53-edited organoids with PTEN sgRNA
  6. 6 Verify PTEN editing by immunoblot
  7. 7 Third round: Transduce P53/PTEN-edited organoids with RB1 sgRNA
  8. 8 Verify RB1 editing by immunoblot
  9. 9 Optional fourth round: Transduce with NF1 sgRNA
  10. 10 Verify NF1 editing by immunoblot (if performed)
Next-Generation Sequencing and Clonal Analysis Week 4–8 (concurrent with gene editing)
  1. 1 Collect organoid samples for DNA extraction
  2. 2 Extract genomic DNA
  3. 3 Prepare samples for targeted or whole-genome sequencing
  4. 4 Perform next-generation sequencing (miSeq)
  5. 5 Analyze sequencing data
In Vitro Proliferation Assessment Weeks 4–20 post-editing (ongoing)
  1. 1 Establish proliferation measurement protocol
  2. 2 Passage control and mutant organoids weekly
  3. 3 Measure cell viability and proliferation rate
  4. 4 Continue culture until passage 20 or growth cessation
  5. 5 Perform statistical comparison
In Vivo Xenotransplantation and Tumor Development Week 8 onwards
  1. 1 Prepare immunodeficient mice
  2. 2 Prepare organoid–fibroblast cell suspensions
  3. 3 Perform subcutaneous (SQ) transplantation
  4. 4 Perform mammary fat pad (MFP) transplantation
  5. 5 Monitor tumor growth
  6. 6 Harvest tumors at endpoint
  7. 7 Document transplantation outcomes
Tumor Histological and Immunohistochemical Analysis Following tumor harvest
  1. 1 Prepare tumor tissue for histology
  2. 2 Perform hematoxylin & eosin (H&E) staining
  3. 3 Perform immunohistochemistry for estrogen receptor (ER)
  4. 4 Perform immunohistochemistry for progesterone receptor (PR)
  5. 5 Perform immunohistochemistry for HER2
  6. 6 Perform immunohistochemistry for pan-cytokeratin
  7. 7 Assess proliferative index by Ki67 immunostaining
  8. 8 Classify tumor subtype
Tumor Sequencing and Clonal Analysis Following tumor harvest
  1. 1 Extract genomic DNA from tumor tissue
  2. 2 Perform next-generation sequencing (miSeq) on tumor DNA
  3. 3 Characterize P53 indel spectrum
  4. 4 Analyze clonal selection patterns
Tumor-Derived Organoid Generation and Drug Response Testing Following tumor harvest
  1. 1 Dissociate tumor tissue to single cells
  2. 2 Culture tumor-derived cells as organoids
  3. 3 Prepare drug-response test plates
  4. 4 Treat with tamoxifen across a concentration series
  5. 5 Treat with docetaxel across a concentration series
  6. 6 Culture for 7 days with drug treatment
  7. 7 Assess organoid viability using CellTiter-Glo
  8. 8 Analyze drug response data
Serial Transplantation (Confirmation of Tumorigenicity) Optional; performed on selected tumors
  1. 1 Prepare primary tumor-derived organoids for retransplantation
  2. 2 Transplant into secondary NSG mice
  3. 3 Monitor secondary tumors
  4. 4 Harvest secondary tumors and confirm phenotype

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Dekkers et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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