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

Summary

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.

Abstract

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.

Introduction

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 tumor^1,2,3,4 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....

Protocol

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.......

Discussion

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.......

Materials

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