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GASTRIC Publication-derived

Locally Advanced Rectal Cancer Tumouroids for Drug Screening

Full SOP with Academic or a plan

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

Authors: 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 Duration: 45 days 8 phases Cell source: Patient-Derived iPSC (Locally Advanced Rectal Cancer)

Abstract

This protocol describes the generation of patient-derived rectal cancer organoids (RCOs) from treatment-naive locally advanced rectal cancer (LARC) patients and their use to predict individual chemoradiation responses. RCOs recapitulate histological and genetic features of original tumors and can predict patient responses to neoadjuvant chemoradiation with 84.43% accuracy, making them a potential companion diagnostic tool for rectal cancer treatment.

Cell source
Patient-Derived iPSC (Locally Advanced Rectal Cancer)
Application
Disease modeling and chemoradiation response prediction

Protocol overview

36 steps across 8 phases

Phase 1 · Biopsy Collection and Tissue Processing Day 0
  1. a Obtain rectal cancer biopsy tissue
  2. b Sort and preserve tissue fragments
  3. c Wash tissue in sterile PBS
Phase 2 · Tumor Cell Isolation and Initial Culture Day 0-1
  1. a Mince tissue into small fragments
  2. b Enzymatic digestion of tissue
  3. c Collect isolated tumor cells
  4. d Seed cells in Matrigel
  5. e Overlay with organoid culture medium
Phase 3 · Organoid Expansion and Maintenance Day 1-14
  1. a Observe and photograph organoids
  2. b Refresh culture medium every three days
  3. c Passage organoids when they reach 100 µm diameter
  4. d Clear Matrigel from organoid fragments
  5. e Re-seed organoid fragments in fresh Matrigel
Phase 4 · Organoid Preparation for Treatment Assays Day 14-21
  1. a Select healthy organoid cultures
  2. b Harvest and passage organoids into 48-well plates
  3. c Allow organoid maturation and stabilization
Phase 5 · Irradiation Response Assay Day 21-45
  1. a Expose organoids to 8 Gy irradiation
  2. b Replace culture medium post-irradiation
  3. c Measure organoid size every three days for 24 days
  4. d Assess organoid viability using CellTiter-Glo 3D assay
  5. e Calculate organoid size recovery ratio
Phase 6 · Chemotherapy Response Assay—5-Fluorouracil (5-Fu) Day 21-45
  1. a Prepare 5-Fu treatment medium
  2. b Replace culture medium with 5-Fu treatment medium
  3. c Refresh drug-containing medium after three days
  4. d Photograph and measure organoid size every three days for 24 days
  5. e Assess cell viability using CellTiter-Glo 3D assay
  6. f Classify 5-Fu response
Phase 7 · Chemotherapy Response Assay—Irinotecan (CPT-11) Day 21-45
  1. a Prepare CPT-11 treatment medium
  2. b Replace culture medium with CPT-11 treatment medium
  3. c Refresh drug-containing medium after three days
  4. d Photograph and measure organoid size every three days for 24 days
  5. e Assess cell viability using CellTiter-Glo 3D assay
  6. f Classify CPT-11 response
Phase 8 · Organoid Cryopreservation Day 14 or ongoing
  1. a Prepare organoids for cryopreservation
  2. b Store cryopreserved organoids
  3. c Resuscitate cryopreserved organoids

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Attribution

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

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