Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

Summary

This protocol describes optimization procedures in a virus-based dual fluorescence-labeled tumor xenograft model using larval zebrafish as hosts. This heterogeneous xenograft model mimics the tissue composition of pancreatic cancer microenvironment in vivo and serves as a more precise tool for assessing drug responses in personalized zPDX (zebrafish patient-derived xenograft) models.

Abstract

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and basic cancer researches. Generations of PDX models in traditional host mice are time-consuming and only working for a small proportion of samples. Recently, zebrafish PDX (zPDX) has emerged as a unique host system, with the characteristics of small-scale and high efficiency. Here, we describe an optimized methodology for generating a dual fluorescence-labeled tumor xenograft model for comparative chemotherapy assessment in zPDX models. Tumor cells and fibroblasts were enriched from freshly-harvested or frozen pancreatic cancer tissue at different culture conditions. Both cell groups were labeled by lentivirus expressing green or red fluorescent proteins, as well as an anti-apoptosis gene BCL2L1. The transfected cells were pre-mixed and co-injected into the 2 dpf larval zebrafish that were then bred in modified E3 medium at 32 °C. The xenograft models were treated by chemotherapy drugs and/or BCL2L1 inhibitor, and the viabilities of both tumor cells and fibroblasts were investigated simultaneously. In summary, this protocol allows researchers to quickly generate a large amount of zPDX models with a heterogeneous tumor microenvironment and provides a longer observation window and a more precise quantitation in assessing the efficiency of drug candidates.

Introduction

Precision oncology aims to find the most beneficial therapeutic strategies for individual patient¹. Currently, numerous preclinical models such as in vitro primary culture, in vitro organoid culture², and patient-derived xenografts (PDX) in mice before or after organoid culture are proposed for diagnosis and to screen/assess the potential therapeutic choices³. PDX model generated by the injection of human primary cancer cells into immune-compromised mice, is one of the most promising tools for personalized drug screening in clinical oncology^3,4<....

Protocol

All animal procedures were approved and followed the guidelines of the Animal Ethics Committee at Fudan University and all pancreatic cancer specimens were obtained from Fudan University Shanghai Cancer Center. Ethical approval was obtained from the FUSCC Ethics Committee, and written informed consent was obtained from each patient.

1. Preparing the Equipment for the Microinjection

1. Preparing the injection plate.
   1. Prepare a 50 mL solution of 1% agarose di.......

Discussion

Both PDX and CDX models are vital platforms in the field of tumor biology²², and the critical step of a successful inter-species transplantation is to improve the survival of the xenograft.  Recently, some studies have shown that transient expression of BCL2L1 (BCL-XL) or BCL2 may significantly improve the viability of human embryonic stem cells in mice hosts without affecting the cell identities and fates^23,24

Materials

Name	Company	Catalog Number	Comments
DMEM	GIBCO	C11995500BT
FBS	Hyclone	sv30087.03
Y-27632	Cliniscience	Y0503	Rho kinase inhibitor
Primocin	invivogen	ant-pm-1	an antibiotic for primary cell cultures
Putrescine dihydrochloride	Sigma	P5780
Nicotinamide	Sigma	N3376
penicillin streptomycin	GIBCO	15140122.00
phosphate buffer (PBS)	GIBCO	C10010500CP
HBSS	GIBCO	14170112.00
collagenase type IV	GIBCO	17104019.00
hyaluronidase	Sigma	H3884
DnaseⅠ	Sigma	D5025
insulin	Sigma	I9278
b-FGF	GIBCO	PHG0264
EGF	GIBCO	PHG0314
pancreatic cancer fibroblasts inhibitor	CHI Scientific	FibrOUT
0.45 μm sterile filter	Millipore	SLHV033RB
concentration column	Millipore	Millipore UFC910008	Concentrate the virus
polybrene	Sigma	H9268
Hyaluronic Acid Sodium Salt	Sigma	H7630
L-glutamine	GIBCO	21051024.00
gemcitabine	Gemzan
methylcellulose	Sigma	M0262
Navitoclax(ABT-263)	Selleck	S1001	Bcl-xL inhibitor
Equipment
Microinjector	NARISHIGE
stereomicroscope	OLYMPUS	MVX10
Confocal Microscope	LEICA	SP8	0.00