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LIVER Publication-derived

Effective therapeutic targeting of CTNNB1-mutant hepatoblastoma with WNTinib

Source Balaseviciute et al., 2025 · Mount Sinai Liver Cancer Program, Icahn School of Medicine at Mount Sinai; Institut d'Investigacions Biomediques August Pi i Sunyer (IDIBAPS), Hospital Clínic, Universitat de Barcelona · 10.1002/1878-0261.70168

👤 Ugne Balaseviciute, Julián Huguet-Pradell, Jordi Abril-Fornaguera, Albert Gris-Oliver, Alex Rialdi, Elisa Fernandez-Martínez, Carla Montironi, Vanessa Del Pozo, Peter Houghton, Laura Zanatto, Agavni Mesropian, Ieva Keraite, Swan Thung, Carolina Armengol, Pau Sancho-Bru, Ernesto Guccione, Roser Pinyol, Josep M. Llovet ⏱ 28 days 📋 6 phases 🧫 Patient-Derived xenograft (PDX) HB tumors; PDX-derived cell lines (TT001); HepG2 cell line; Patient-derived organoids from CTNNB1-mutant HB

Abstract

This protocol evaluates the efficacy of WNTinib, a Wnt/CTNNB1 inhibitor, in CTNNB1-mutant hepatoblastoma using patient-derived xenograft (PDX) models, cell line-derived xenografts (TT001 and HepG2), and patient-derived organoids. WNTinib delays tumor growth, extends survival, and demonstrates greater efficacy than cisplatin in organoid models, providing preclinical evidence for targeted therapy in pediatric hepatoblastoma.

Cell source
Patient-Derived xenograft (PDX) HB tumors; PDX-derived cell lines (TT001); HepG2 cell line; Patient-derived organoids from CTNNB1-mutant HB
Application
Disease modeling and drug screening for CTNNB1-mutant hepatoblastoma

Protocol overview

37 steps across 6 phases

Patient-Derived Xenograft (PDX) Model Generation and Treatment Days 0-28
  1. 1 PDX tumor sample collection and implantation
  2. 2 Monitor tumor growth and randomize animals
  3. 3 Administer WNTinib or vehicle control
  4. 4 Monitor animal weight and tumor volume
  5. 5 Sacrifice and collect tumors at endpoint
Establishment of PDX-Derived 2D Cell Lines Days 0–14 (post-sacrifice)
  1. 1 Implant PDX tumor in mice
  2. 2 Monitor tumor growth to target volume
  3. 3 Harvest and mince tumor tissue
  4. 4 Enzymatic digestion of tumor
  5. 5 Strain and wash dissociated cells
  6. 6 Plate cells in primary media on collagen-coated dishes
Subcutaneous Xenograft Models (HepG2 and TT001 Cell Lines) Days 0–21 (HepG2); Days 0–22 (TT001)
  1. 1 Prepare cell suspensions for injection
  2. 2 Subcutaneous implantation in athymic nude mice
  3. 3 Monitor tumor growth pre-randomization
  4. 4 Randomize animals to treatment arms
  5. 5 Administer WNTinib or vehicle via oral gavage
  6. 6 Monitor body weight, tumor growth, and survival
  7. 7 Determine survival endpoint and sacrifice
Histological and Immunohistochemical Analysis Post-sacrifice processing
  1. 1 Fix and embed tumor tissue
  2. 2 Prepare histology sections and stain with H&E
  3. 3 Perform antigen retrieval
  4. 4 Block and incubate with anti-Ki67 antibody
  5. 5 Apply EnVision™+ HRP system and DAB
  6. 6 Counterstain with hematoxylin
  7. 7 Digital image acquisition and Ki67 quantification
  8. 8 Quantify necrotic area
Patient-Derived Organoid (PDO) Generation Days 0–7 (per treatment cycle)
  1. 1 Obtain and document patient consent
  2. 2 Plate tumor tissue in Basement Membrane Extract (BME)
  3. 3 Expand organoids through weekly mechanical and enzymatic disruption
  4. 4 Treat organoids with WNTinib, cisplatin, combination, or vehicle
  5. 5 Assess organoid viability using CellTiter-Glo assay
CTNNB1 Genotyping by Sanger Sequencing Post-experiment (1–2 weeks)
  1. 1 Extract genomic DNA from cell lines
  2. 2 Design and prepare PCR primers
  3. 3 Perform PCR amplification
  4. 4 Purify PCR product
  5. 5 Submit for Sanger sequencing
  6. 6 Analyze sequencing results and call mutations

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Balaseviciute et al., 2025. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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