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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

Abstract

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

Introduction

Gastric cancer is the fifth-most common cancer worldwide and the third leading cause of cancer death. In 2018, over 1,000,000 new cases of gastric cancer were diagnosed globally, and an estimated 783,000 people were killed by this disease1. The incidence and mortality of gastric cancer remain very high in northeastern Asian countries2,3. Despite significant progress in the field of cancer therapeutics, the prognosis of patients with advanced gastric cancer remains poor, with a five-year survival rate of approximately 25%4,5....

Protocol

This human study was approved by the Institutional Ethics Review Board of Sun Yat-sen University Cancer Center (SYSUCC, Guangzhou, China). The animal study was approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University. Note: all experiments were performed in compliance with the relevant laws and institutional guidelines, including the Guideline for occupational exposure protection against blood-borne pathogens.

1. Sample preparation

  1. Obtain gastric c.......

Representative Results

Here, tumor tissues from an operation were preserved in stock solution until the next step. Within 4 hours, tumor tissues were cut into small pieces and implanted into the dorsal flanks of NSG mice that had been anesthetized using isoflurane-soaked cotton. Tumors larger than 1 cm3 could be resected for implantation into new mice (Figure 1) or sliced carefully and preserved in liquid nitrogen following the protocol. In this study, the first-generation tumors grew more slowly than t.......

Discussion

Gastric cancer is an aggressive disease with limited therapeutic options; thus, models of gastric cancer have become a critical resource to enable functional research studies with direct translation to the clinic4,8,17. Here, we have described the methods and protocol of establishing gastric cancer PDX models and primary cell lines. Importantly, both morphological and biological characteristics of gastric cancer specimens were m.......

Acknowledgements

This work was supported by the National Natural Science Foundation of China (81572392); the National Key Research and Development Program of China (2016YFC1201704); Tip-top Scientific and Technical Innovative Youth Talents of Guangdong Special Support Program (2016TQ03R614).

We specifically thank Guangzhou Sagene Biotech Co., Ltd. for aid in the preparation of the figures.

....

Materials

NameCompanyCatalog NumberComments
40 μm Cell StrainerBiologix, Shandong, China15-1040
Biological MicroscopeOLYMPUS, Tokyo, JapanOLYMPUS CKX41
CentrifugeEppendorf, Mittelsachsen, Germany.5427R
CO2 IncubatorThermo Fisher Scientific, Carlsbad, California, USAHERACELL 150i
DPBSBasalmedia Technology, Shanghai, ChinaL40601
Electro-Thermostatic Water CabinetYiheng, Shanghai, ChinaDK-8AXX
Fetal bovine serumWisent Biotechnology, Vancouver, Canada86150040
IsofluraneBaxter, ChinaCN2L9100
Live Tissue Kit Cryo KitCelliver Biotechnology, Shanghai, ChinaLT2601
Live Tissue Thaw KitCelliver Biotechnology, Shanghai, ChinaLT2602
NSGBiocytogen, Beijing, ChinaB-CM-002-4-5W
Penicilin&streptomycinThermo Fisher Scientific, Carlsbad, California, USA15140122
Red blood cell lysis bufferSolarbio, Beijing, ChinaR1010
RPMI-1640 mediumThermo Fisher Scientific, Carlsbad, California, USA8118367
Surgical Suture Needles with ThreadLingQiao, Ningbo, China3/8 arc 4×10
Tissue-processed molds and auxiliary bladesCelliver Biotechnology, Shanghai, ChinaLT2603
Trypsin-EDTAThermo Fisher Scientific, Carlsbad, California, USA2003779
Type 1 collagenaseThermo Fisher Scientific, Carlsbad, California, USA17100017

References

  1. Bray, F., et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. Ca-a Cancer Journal for Clinicians. 68 (6), 394-424 (2018).
  2. Sugano, K.

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Patient derived Xenograft ModelsPrimary Cell LinesTumor Biology ResearchDrug DevelopmentCancer Cell LinesNSG MiceTumor MicroenvironmentTumor DissectionTumor Tissue ProcessingTumor Cryopreservation

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