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GASTRIC Publication-derived

Patient-Derived Organoids from Locally Advanced Rectal Cancer: Generation, Culture, and Chemoradiation Response Testing

Source Yao et al., 2020 · Fudan University Shanghai Cancer Center, Department of Radiation Oncology · 10.1016/j.stem.2019.10.010

👤 Ye Yao, Xiaoya Xu, Lifeng Yang, Ji Zhu, Juefeng Wan, Lijun Shen, Fan Xia, Guoxiang Fu, Yun Deng, Mengxue Pan, Qiang Guo, Xiaoxue Gao, Yuanchuang Li, Xinxin Rao, Yi Zhou, Liping Liang, Yaqi Wang, Jing Zhang, Hui Zhang, Guichao Li, Lixing Zhang, Junjie Peng, Sanjun Cai, Chen Hu, Jianjun Gao, Hans Clevers, Zhen Zhang, Guoqiang Hua ⏱ 39 days 📋 10 phases 🧫 Patient-Derived Primary Rectal Cancer Tissue

Abstract

This protocol describes the generation of patient-derived organoids (PDOs) from locally advanced rectal cancer (LARC) biopsies, their culture and maintenance, and in vitro assessment of sensitivity to irradiation, 5-fluorouracil (5-Fu), and irinotecan (CPT-11). Organoid responses were validated against patient clinical outcomes from a phase III neoadjuvant chemoradiation trial, demonstrating predictive accuracy of 84.43% for chemoradiation response.

Cell source
Patient-Derived Primary Rectal Cancer Tissue
Application
Disease modeling, Drug screening, Chemoradiation response prediction

Protocol overview

68 steps across 10 phases

Tissue Biopsy Collection and Processing Day 0 (collection); same day processing
  1. 1 Obtain patient rectal cancer biopsy
  2. 2 Transport biopsy tissue to laboratory
  3. 3 Divide biopsy tissue for downstream applications
  4. 4 Prepare tissue for enzymatic digestion
Tumor Cell Isolation and Initial Culture Day 0–1
  1. 1 Perform enzymatic digestion of tumor tissue
  2. 2 Collect and wash isolated tumor cells
  3. 3 Prepare cell-Matrigel suspension for seeding
  4. 4 Overlay with organoid culture medium and incubate
Rectal Cancer Organoid Culture Establishment and Maintenance Day 1–14+ (ongoing expansion)
  1. 1 Prepare RCO culture medium
  2. 2 Monitor organoid growth daily; refresh medium every 3 days
  3. 3 Passage organoids every 1–2 weeks upon confluency
  4. 4 Wash organoid fragments and prepare for re-seeding
  5. 5 Re-seed washed organoid fragments in fresh Matrigel
  6. 6 Cryopreserve organoid stocks as needed
Histopathological and Molecular Characterization Day 7–14 (concurrent with culture expansion)
  1. 1 Fix organoids for H&E and immunohistochemistry staining
  2. 2 Process and embed fixed tissues in paraffin
  3. 3 Perform H&E staining
  4. 4 Perform immunohistochemistry staining for marker expression
  5. 5 Capture and document staining images
Organoid Preparation and Dosimetry Setup for Drug and Radiation Testing Day 10–14 (before testing)
  1. 1 Select healthy organoid lines for drug and radiation testing
  2. 2 Prepare organoids for seeding in 48-well plates
  3. 3 Polymerize Matrigel and overlay with culture medium
  4. 4 Allow organoids to reach target size (~100 μm diameter) before treatment
  5. 5 Photograph organoids at day 0 (baseline before treatment)
Organoid Irradiation Response Assay Day 14–39 (following baseline documentation)
  1. 1 Irradiate organoid cultures with 8 Gy X-rays
  2. 2 Refresh culture medium post-irradiation
  3. 3 Photograph and measure organoid size every 3 days post-irradiation for 24 days
  4. 4 Refresh culture medium every 3 days during the 24-day observation period
  5. 5 Calculate organoid size recovery ratio
  6. 6 Validate organoid survival by CellTiter-Glo 3D cell viability assay
  7. 7 Optional: Perform dose-response irradiation assay in selected organoid lines
Organoid Drug Response Assays (5-Fluorouracil and Irinotecan) Day 14–39 (performed in parallel with irradiation assay or sequentially)
  1. 1 Treat organoids with 5-Fluorouracil (5-Fu) at 10 μM
  2. 2 Refresh 5-Fu-containing medium after 3 days
  3. 3 Continue 5-Fu treatment for 24 days with media refreshes every 3 days
  4. 4 Photograph and measure organoid sizes every 3 days during 5-Fu treatment
  5. 5 Calculate organoid size recovery ratio after 5-Fu treatment
  6. 6 Treat parallel organoid wells with 10 μM Irinotecan (CPT-11)
  7. 7 Refresh CPT-11-containing medium after 3 days
  8. 8 Continue CPT-11 treatment for 24 days with media refreshes every 3 days
  9. 9 Photograph and measure organoid sizes every 3 days during CPT-11 treatment
  10. 10 Calculate organoid size recovery ratio after CPT-11 treatment
  11. 11 Validate drug responses by CellTiter-Glo 3D cell viability assay
Combinatorial Chemoradiation Treatment Assay (Selected Organoid Lines) Day 14–39 (in select organoid lines showing triple-resistance or specific response patterns)
  1. 1 Identify organoid lines requiring combinatorial treatment testing
  2. 2 Prepare combinatorial treatment medium: irradiation + 5-Fu + CPT-11
  3. 3 Irradiate organoid wells with 8 Gy, then add combinatorial drug medium
  4. 4 Refresh combinatorial medium every 3 days for 24 days
  5. 5 Photograph and measure organoid sizes every 3 days during combinatorial treatment
  6. 6 Assess synergy between irradiation and chemotherapy
Molecular Characterization: Whole-Exome Sequencing and Genomic Analysis Day 1–30 (concurrent with organoid expansion; sent for external sequencing)
  1. 1 Extract DNA from organoid lines and paired patient biopsies
  2. 2 Assess DNA quality and quantity
  3. 3 Prepare whole-exome sequencing (WES) libraries
  4. 4 Perform cluster generation and DNA sequencing
  5. 5 Perform read alignment and quality control
  6. 6 Call somatic mutations using MuTect2
  7. 7 Detect copy number variations (CNVs) using Control-FREEC
  8. 8 Analyze mutational signatures and patterns
  9. 9 Calculate mutation concordance between organoids and tumors
  10. 10 Archive sequencing data and deposit in public repository
Clinical Outcome Correlation and Statistical Analysis Ongoing after organoid testing (final analysis when patient outcome data mature)
  1. 1 Obtain patient clinical outcome data
  2. 2 Classify patients into responder/non-responder groups
  3. 3 Classify organoid responses to single treatments
  4. 4 Match organoid responses to patient clinical outcomes
  5. 5 Calculate diagnostic performance metrics
  6. 6 Identify cutoff value for organoid response prediction
  7. 7 Analyze organoid responses by treatment modality
  8. 8 Correlate genomic alterations with organoid drug responses
  9. 9 Perform statistical analysis using appropriate tests
  10. 10 Prepare publication and deposit data in public repository

Full SOP

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Attribution

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

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