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OTHER Publication-derived

Head and Neck Squamous Cell Carcinoma Tumouroids

Full SOP with Academic or a plan

Source Driehuis et al., 2019 · Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and University Medical Center Utrecht · 10.3390/jcm8111880

Authors: Else Driehuis, Sacha Spelier, Irati Beltrán Hernández, Remco de Bree, Stefan M. Willems, Hans Clevers, Sabrina Oliveira 7 phases Cell source: Patient-Derived HNSCC and Adjacent Normal Tissue

Abstract

Patient-derived head and neck squamous cell carcinoma (HNSCC) organoids were established and characterized for EGFR expression levels, which recapitulated expression in primary tissue. These 3D organoid models were used to evaluate EGFR-targeted photodynamic therapy using antibody- and nanobody-photosensitizer conjugates, demonstrating selective killing of tumor cells over normal tissue equivalents.

Cell source
Patient-Derived HNSCC and Adjacent Normal Tissue
Application
Disease modeling; EGFR-targeted photodynamic therapy (PDT) drug screening

Protocol overview

42 steps across 7 phases

Phase 1 · Tissue Processing and Organoid Derivation Days 0-14
  1. a Patient Material Collection
  2. b Tissue Preparation
  3. c Trypsin Digestion
  4. d Cell Suspension Filtering and Centrifugation
  5. e BME Resuspension and Plating
  6. f BME Solidification
  7. g Organoid Medium Addition and ROCK Inhibitor Treatment
Phase 2 · Organoid Culture and Maintenance Days 7-ongoing
  1. a Organoid Splitting and Passaging Preparation
  2. b TrypLE Digestion
  3. c Cell Resuspension and Replating
  4. d Post-Splitting ROCK Inhibitor Addition
  5. e Medium Changes and Routine Culture
Phase 3 · RNA Collection and qPCR Analysis for EGFR Expression Days 1-15 (from experimental start)
  1. a Organoid Culture and Single Cell Preparation
  2. b Transfer to Physiological EGF Medium
  3. c RNA Extraction
  4. d cDNA Synthesis
  5. e Quantitative PCR Setup
  6. f qPCR Cycling and Analysis
Phase 4 · Flow Cytometry for EGFR Protein Expression Day 7 post-split (in physiological EGF medium)
  1. a Organoid Culture in Physiological EGF
  2. b Single Cell Preparation
  3. c Antibody Staining
  4. d DAPI Staining and Flow Cytometry
Phase 5 · Immunohistochemistry for EGFR Staining Days 0-14 (2 weeks culture + processing)
  1. a Organoid Culture Preparation
  2. b Organoid Collection and Fixation
  3. c Paraffin Embedding and Section Preparation
  4. d H&E and EGFR Immunostaining
Phase 6 · In Vitro Photodynamic Therapy (PDT) Assay on HNSCC Organoids Days 0-2 (preparation); Day 2 onwards (PDT treatment)
  1. a Organoid Dissociation to Single Cells
  2. b Recovery Culture in Physiological EGF Medium
  3. c Dispase Digestion of BME
  4. d Organoid Harvest and Filtering
  5. e Organoid Plating in 384-Well Format
  6. f Photosensitizer-Conjugate Addition
  7. g Light Activation
  8. h Cell Viability Assessment
  9. i Data Analysis and Kill Curve Generation
Phase 7 · EGFR Overexpression and Doxycycline-Inducible System Variable (virus production ~3 days; organoid infection ~2-3 days; selection ~14 days)
  1. a EGFR Plasmid Construction
  2. b Lentivirus Production in HEK293T Cells
  3. c Viral Supernatant Harvest and Concentration
  4. d Organoid Infection with Lentivirus
  5. e Antibiotic Selection
  6. f Doxycycline Induction Validation
  7. g EGFR Expression Induction and PDT Treatment

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Attribution

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

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