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BREAST Publication-derived

Breast Cancer Organoids with Gene Editing

Full SOP with Academic or a plan

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

Authors: 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 Duration: 20 days 10 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Dekkers et al., 2020. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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