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INTESTINAL Publication-derived

Visualization and Targeting of LGR5+ Human Colon Cancer Stem Cells

Source Shimokawa et al., 2017 · Keio University School of Medicine · 10.1038/nature22081

👤 Mariko Shimokawa, Yuki Ohta, Shingo Nishikori, Mami Matano, Ai Takano, Masayuki Fujii, Shoichi Date, Shinya Sugimoto, Takanori Kanai, Toshiro Sato ⏱ 180 days 📋 10 phases 🧫 Patient-Derived CRC (Colorectal Cancer) Organoids

Abstract

This protocol describes the generation of genetically engineered patient-derived colorectal cancer organoids with fluorescent reporters for LGR5+ and KRT20+ cells, enabling lineage tracing, ablation, and functional characterization of cancer stem cell plasticity in intact tumor tissues. The approach facilitates visualization of self-renewal and differentiation capacity, assessment of CSC targeting efficacy, and evaluation of chemotherapy potentiation.

Cell source
Patient-Derived CRC (Colorectal Cancer) Organoids
Application
Cancer stem cell modeling and drug screening

Protocol overview

57 steps across 10 phases

Generation of LGR5-GFP and KRT20-GFP Reporter Organoids Days 1-14
  1. 1 Design and clone CRISPR–Cas9 targeting plasmids
  2. 2 Construct donor vectors for GFP knock-in
  3. 3 Electroporationof CRISPR constructs into organoids
  4. 4 Select successfully targeted organoids with puromycin
  5. 5 Isolate clonal organoids and validate knock-in
  6. 6 Remove puromycin selection cassette via Cre-loxP recombination
Generation of LGR5-CreER/Rainbow and KRT20-CreER/Rainbow Lineage-Tracing Organoids Days 15-35
  1. 1 Design and construct LGR5-CreER knock-in plasmid
  2. 2 Construct PiggyBac rainbow reporter vector
  3. 3 Co-electroporate LGR5-CreER and rainbow reporter into organoids
  4. 4 Select and expand clonal reporter organoids
  5. 5 Verify CreER inducibility in vitro
  6. 6 Repeat for KRT20-CreER/rainbow
Generation of LGR5-iCaspase9-tdTomato (LGR5-iCT) Ablation Organoids Days 36-56
  1. 1 Design LGR5-iCaspase9-tdTomato knock-in construct
  2. 2 Electroporate LGR5-iCT construct into organoids
  3. 3 Select LGR5-iCT clones with puromycin
  4. 4 Validate dimerizer-inducible apoptosis in vitro
  5. 5 Remove puromycin cassette (optional)
Organoid Culture and Maintenance Ongoing (parallel to other phases)
  1. 1 Embed organoids in Matrigel
  2. 2 Prepare basal culture medium
  3. 3 Add niche factors to basal medium
  4. 4 Change culture medium every 2–3 days
  5. 5 Passage organoids when necessary
Xenotransplantation and In Vivo Lineage Tracing Days 57-120
  1. 1 Prepare organoid cell suspension for xenotransplantation
  2. 2 Prepare NOG mice for xenotransplantation
  3. 3 Perform subcutaneous or subrenal capsule xenotransplantation
  4. 4 Monitor tumor growth
  5. 5 Administer tamoxifen for lineage tracing
  6. 6 Harvest tumors for analysis
LGR5+ Cancer Stem Cell Ablation In Vivo Days 30-90 (post-transplantation)
  1. 1 Establish LGR5-iCT xenografts
  2. 2 Administer dimerizer (AP20187) for short-term ablation
  3. 3 Monitor tumor response to ablation
  4. 4 Harvest tumors at multiple timepoints
Combined Chemotherapy and CSC Ablation Days 30-60 (post-transplantation)
  1. 1 Establish LGR5-iCT xenografts and assess baseline LGR5 expression
  2. 2 Administer cetuximab or oxaliplatin
  3. 3 Combine chemotherapy with dimerizer ablation
  4. 4 Monitor combined treatment response
  5. 5 Harvest and analyze tumors
Histological Analysis, Immunofluorescence, and Imaging Days 121-180
  1. 1 Prepare frozen tissue sections for rainbow imaging
  2. 2 Perform confocal imaging of rainbow reporters
  3. 3 Perform digital light-sheet (DLS) imaging for whole-tumor 3D reconstruction
  4. 4 Perform immunofluorescence for lineage markers
  5. 5 Perform in situ hybridization for LGR5 and KRT20 mRNA
  6. 6 Acquire high-resolution microscopy images
  7. 7 Quantify clonal structures and clone-formation efficiency
Flow Cytometry and Cell Sorting Days 1-180 (as needed for analysis)
  1. 1 Dissociate organoids to single cells
  2. 2 Stain cells with 7-AAD viability dye
  3. 3 Gate singlets and analyze GFP expression
  4. 4 Sort GFP+ and GFP− populations
  5. 5 Culture sorted cells in Matrigel for colony-formation assay
  6. 6 Assess organoid colony formation
Gene Expression Analysis Days 1-180 (as needed for analysis)
  1. 1 Extract RNA from sorted cells
  2. 2 Assess RNA quality
  3. 3 Perform microarray gene expression profiling
  4. 4 Perform gene-set enrichment analysis (GSEA)
  5. 5 Perform quantitative real-time RT-PCR validation
  6. 6 Extract RNA from tumor tissues for in vivo validation
  7. 7 Quantify LGR5 and KRT20 mRNA in tumor tissues

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Shimokawa et al., 2017. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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