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Cancer Research

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Published: May 26th, 2023

DOI:

10.3791/65229

1Molecular Epidemiology and Predictive Tumor Markers Group, Area 3, Ramón y Cajal Health Research Institute (IRYCIS), 2The Biomedical Research Network in Cancer (CIBERONC), 3Biobank and Biomodels Platform (PT20/0045), ISCIII research and development platforms in biomedicine and health sciences, BioBank Hospital Ramón y Cajal-IRYCIS, Spanish National Biobanks Network (ISCIII Biobank Register No. B.0000678), Ramón y Cajal Health Research Institute (IRYCIS), 4Faculty of Medicine, University of Alcalá de Henares, 5Institute of Tissue Medicine and Pathology, University of Bern, 6Department of Molecular Oncology, Cancer Research Institute, Biomedical Research Center of the Slovak Academy of Sciences, 7Experimental Oncology, Molecular Oncology Program, Centro Nacional de Investigaciones Oncológicas (CNIO), 8Department of Surgical Oncology, National Cancer Institute, Slovak Medical University, 9Pancreatic and Biliopancreatic Surgery Unit, Hospital Universitario Ramón y Cajal, 10Department of Pathology, Hospital Universitario Ramón y Cajal, 11Department of Radiation Oncology, Hospital Universitario Ramón y Cajal, 12Department of Cancer, Instituto de Investigaciones Biomédicas “Alberto Sols” (IIBM), 13Cancer Stem Cell and Fibroinflammatory Group, Chronic Diseases and Cancer, Area 3, IRYCIS
* These authors contributed equally

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

Tumor organoids are ex vivo three-dimensional (3D) organized cultures that are derived from fresh tumor tissue and provide cancer models. Tumor organoids recapitulate the biological key features of the original primary tumor1,2,3,4 and can be expanded for up to several months and cryopreserved, similar to conventional immortalized cell lines. Tumor organoids provide a biobank of patient-derived tumor models for translational/personalized medicine5 and represent an important advance in cancer cell biology syst....

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All procedures were performed in compliance with the institutional guidelines for the welfare of experimental animals approved by the Universidad Autónoma de Madrid Ethics Committee (CEI 103-1958-A337) and La Comunidad de Madrid (PROEX 294/19) and in accordance with the guidelines for Ethical Conduct in the Care and Use of Animals as stated in The International Guiding Principles for Biomedical Research Involving Animals, developed by the Council for International Organizations of Medical Sciences (CIOMS). The proto.......

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It is important to document how the tumor organoid culture progresses over time, particularly in the first few weeks, in order to estimate how the culture will behave in downstream assays. Figure 2 shows an example of optimal tumor cell isolation and tumor organoid establishment from fresh tissue over a 15 day period. Sometimes, there is a large volume of cell debris in the sample, and it is difficult to see the developing tumor organoids, as shown in Figure 3. .......

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Major advances in pharmacological cancer therapies are challenging, as the likelihood of the approval of drugs in phase I oncology clinical trials is 5.1%, which is the lowest of all disease types23. The main reason is that cancer is very heterogenous, and therefore, patient cohorts do not uniformly respond as expected to the given treatment, which highlights that a more personalized approach is needed. Two-dimensional (2D) cultures have been used in translational cancer research for many years bu.......

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This study was supported by funding from the Plataforma biobancos y biomodelos - Unidades de las Plataformas ISCIII de apoyo ala I+D+i en Biomedicina y Ciencias de la Salud (PT20/00045), The European Union's Horizon 2020 Research and Innovation Programme under grant agreement No. 857381, project VISION (Strategies to strengthen scientific excellence and innovation capacity for early diagnosis of gastrointestinal cancers), Intramural call for new research projects for clinical researchers and emerging research groups IRYCIS (2021/0446), Patient Derived Organoids 2.0 Project (CIBERONC) and the TRANSCAN II project JTC 2017 call "Establishing an algorithm for....

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Name Company Catalog Number Comments
6 well Costar Ultra-low Attachment plates Biofil TCP011006
70 μm pore strainer VWR 732-2758
Ammonium Chloride Potassium (ACK) Lysis Buffer Gibco A10492-01
Amphotericin B Gibco 15290018
Cell culture incubator (21% O2, 5% CO2 and 37 ºC) Nuaire NU-4750E
Cell recovery solution Corning  354253
Collagenase IV Gibco 17104019
DMEM/F-12 (1:1)(1X) with L-Glutamine and HEPES Gibco 31330-038
DNase Roche 10104159001
Fetal Bovine Serum (FBS) Corning 35-079-CV
Freezing container, Nalgene Merck C1562
gentleMACS Octo Dissociator Milteny Biotec 130-096-427
HEPES Gibco 15630056
Human Placenta Growth Factor (PlGF) enQuireBio QP6485-EC-100UG
Immunocompromised female 6-week-old NU-Foxn1nu nude mice Janvier, France 
Insulin-like growth factor-1 (IGF-1) Invitrogen RP10931
L-Glutamine Corning 354235
Matrigel Basement Membrane Matrix  Corning 356234
Normocin InvivoGen ant-nr-2
Pasteur pipettes Deltalab 200007
Penicillin Streptomycin Solution (100x) Corning 30-002-CI
Phosphate-Buffered Saline (PBS) Corning 21-040-CV
Recombinant Human Basic Fibroblast Growth Factor (bFGF) Gibco PHG0026
Recombinant Human Epidermal Growth Factor (EGF) Gibco PHG0311
ROCK Inhibitor Y-27632 (Dihydrochloride) STEMCELL 72304
StemPro Accutase Cell Dissociation Reagent Gibco A1110501
Surgical Blades Nahita FMB018
Trypsin Gibco 25300054

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