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GASTRIC Publication-derived

Rectal Adenocarcinoma Tumouroids

Full SOP with Academic or a plan

Source Ganesh et al., 2019 · Memorial Sloan Kettering Cancer Center · 10.1038/s41591-019-0584-2

Authors: Karuna Ganesh, Chao Wu, Kevin P. O'Rourke, Bryan C. Szeglin, Youyun Zheng, Charles-Etienne Gabriel Sauvé, Mohammad Adileh, Isaac Wasserman, Michael R. Marco, Amanda S. Kim, Maha Shady, Francisco Sanchez-Vega, Wouter R. Karthaus, Helen H. Won, Seo-Hyun Choi, Raphael Pelossof, Afsar Barlas, Peter Ntiamoah, Emmanouil Pappou, Arthur Elghouayel, James S. Strong, Chin-Tung Chen, Jennifer W. Harris, Martin R. Weiser, Garrett M. Nash, Jose G. Guillem, Iris H. Wei, Richard N. Kolesnick, Harini Veeraraghavan, Eduardo J. Ortiz, Iva Petkovska, Andrea Cercek, Katia O. Manova-Todorova, Leonard B. Saltz, Jessica A. Lavery, Ronald P. DeMatteo, Joan Massagué, Philip B. Paty, Rona Yaeger, Xi Chen, Sujata Patil, Hans Clevers, Michael F. Berger, Scott W. Lowe, Jinru Shia, Paul B. Romesser, Lukas E. Dow, Julio Garcia-Aguilar, Charles L. Sawyers, J. Joshua Smith Duration: 112 days 9 phases Cell source: Patient-Derived Cancer Tissue (Rectal Adenocarcinoma)

Abstract

This protocol establishes a biorepository of patient-derived rectal cancer organoids (tumoroids) from primary, metastatic, and recurrent disease. RC tumoroids retain molecular and histopathologic features of primary tumors and show ex vivo responses to chemotherapy and radiation that correlate with clinical outcomes. An endoluminal in vivo model in NSG mice demonstrates tumor initiation, invasion, metastasis, and chemotherapy response.

Cell source
Patient-Derived Cancer Tissue (Rectal Adenocarcinoma)
Application
Disease modeling and drug screening for personalized treatment response prediction

Protocol overview

53 steps across 9 phases

Phase 1 · Tissue Processing and Crypt Isolation Day 0-1
  1. a Prepare PBS-Abs buffer for tissue washing
  2. b Wash and fragment tissue
  3. c Enzymatic digestion of tissue
  4. d Cell isolation and enrichment
Phase 2 · 3D Culture Initiation and Maintenance Day 1-28+
  1. a Prepare basal culture medium
  2. b Add niche factors to culture medium
  3. c Culture tumoroids in Matrigel suspension
  4. d Passage tumoroids at expansion
  5. e Freeze tumoroids for long-term storage
  6. f Assess tumoroid success criteria
Phase 3 · Ex Vivo Chemotherapy Sensitivity Testing Day 7-13
  1. a Prepare tumoroids for drug testing
  2. b Prepare 5-FU drug dilutions
  3. c Treat tumoroids with 5-FU
  4. d Assess cell viability by CellTiter-Glo
  5. e Generate dose-response curves and calculate AUC
  6. f Perform FOLFOX combination testing
Phase 4 · Ex Vivo Radiation Sensitivity Testing Day 7-20
  1. a Prepare tumoroids for radiation
  2. b Deliver ionizing radiation
  3. c Allow post-radiation recovery
  4. d Assess post-radiation cell viability
  5. e Generate radiation dose-response curves and calculate AUC
Phase 5 · Endoluminal Tumor Implantation in Mice Day 0-112+ (16 weeks)
  1. a Prepare DSS pretreatment
  2. b Allow post-DSS recovery
  3. c Prepare tumoroids for injection
  4. d Anesthetize mice and perform rectal injection
  5. e Monitor tumor engraftment by endoscopy
  6. f Assess metastatic disease
Phase 6 · In Vivo Chemotherapy Treatment and Assessment Week 12-20
  1. a Confirm tumor engraftment and randomize mice
  2. b Prepare 5-FU for intraperitoneal injection
  3. c Administer 5-FU or vehicle control
  4. d Prepare FOLFOX chemotherapy regimen
  5. e Administer FOLFOX treatment
  6. f Monitor tumor response by serial endoscopy
  7. g Assess endpoint and harvest tissues
  8. h Statistical analysis of in vivo response
Phase 7 · Histopathology and Immunohistochemistry Day 14-42 (after tissue fixation)
  1. a Prepare tumoroids and tissues for embedding
  2. b Process tissues and prepare paraffin sections
  3. c Perform hematoxylin and eosin (H&E) staining
  4. d Perform immunohistochemistry for epithelial markers
  5. e Perform immunohistochemistry for mismatch repair proteins
  6. f Perform immunohistochemistry for colorectal markers
  7. g Image and analyze stained sections
  8. h Perform inter-observer concordance analysis
Phase 8 · Genomic Analysis and Molecular Characterization Day 28+ (after organoid establishment)
  1. a Extract genomic DNA from tumoroids and normal organoids
  2. b Perform targeted genomic sequencing using MSK-IMPACT
  3. c Call and annotate somatic variants
  4. d Determine clonal vs. subclonal status of mutations
  5. e Compare tumoroid and primary tumor mutations
  6. f Compare tumoroid mutations to rectal cancer cohort
Phase 9 · Quantitative PCR for Human Cell Detection in Metastases Day 56+ (after tissue harvest)
  1. a Prepare genomic DNA from metastatic tissues and controls
  2. b Design and validate human Alu and mouse SINE primers
  3. c Perform quantitative PCR for Alu and SINE
  4. d Normalize Alu quantification to human DNA standard

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Attribution

This SOP was written by Organthis based on the published method in Ganesh et al., 2019. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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