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In This Article

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

Summary

In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

Abstract

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many studies in the last decades. Therefore, it is important to develop methods to investigate the role of key genes involved in cancer cell stemness. Gastric cancer (GC) is one of the most common and mortal types of cancers. Gastric cancer stem cells (GCSCs) are thought to be the root of gastric cancer relapse, metastasis and drug resistance. Understanding GCSCs biology is needed to advance the development of targeted therapies and eventually to reduce mortality among patients. In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

Introduction

Gastric cancer (GC) is one of the most common and mortal types of cancers1. Despite advances in combined surgery, chemotherapy and radiotherapy in GC therapy, prognosis remains poor and the five-year survival rate is still very low2. Recurrence and metastasis are the main reasons cause the post-treatment deaths.

Cancer stem cells (CSCs) are a subset of cancer cells that possess the ability to self-renew and generate the different cell lineages that reconstitute the tumor3. CSCs are believed to be responsible for cancer relapse and metastasis because of their capabilitie....

Protocol

All experimentation using patient-derived gastric cancer stem cells described herein was approved by the local ethical committee7.

1. Gastric cancer stem cell culture

  1. Preparation of GCSCs complete culture medium
    1. Prepare GCSCs complete culture medium by adding fresh DME/F12 medium with the following essential ingredients: 20 ng/mL EGF, 10 ng/mL bFGF, 1% Insulin/Transferrin/Sodium selenite, 0.2% glucose, 0.5% B27, 1% Glutamax, 1% Non-essential amin.......

Representative Results

Gastric cancer stem cells from primary human gastric adenocarcinoma were cultured in serum-free culture medium. After 6 days, cells expanded from the single cell-like phenotype (Figure 1A) to form large spheres (Figure 1B).

To assess the function of clusterin in GCSCs, shRNA sequences against clusterin and scrambled were cloned into Tet.......

Discussion

GC is the third leading cause of cancer-related death worldwide. GCSCs are critical in gastric cancer relapse, metastasis and drug resistance. Using GCSCs from gastric cancer patients will allow us to explore their weak spot and develop the targeting drugs for the treatment of GC patients.

The sphere formation assay is a useful method to examine cancer stem cell self-renewal potential in vitro. Results can be presented as the percentage of spheres formed divided by the original number of .......

Acknowledgements

This work was supported by the Nature Science Foundation of Guangdong Province (2018A030310586, 2020A1515010989), the Medical Scientific Research Foundation of Guangdong Province (A2019405), the National Natural Science Foundation of China (81772957), the Science and Technology Program of Guangdong Province in China (2017B030301016), and the Industry and Information Technology Foundation of Shenzhen (20180309100135860).

....

Materials

NameCompanyCatalog NumberComments
0.22 μm filterMilliporeSLGP033RB
1-ThioglycerolSigma-AldrichM6145
2-MercaptoethanolGibco2068586
Animal-Free Recombinant Human EGFPeprotechAF-100-15
B-27 Supplement (50X), serum freeGibco17504044
Corning Costar Ultra-Low Attachment Multiple Well PlateSigma-AldrichCLS3474
Countess Cell Counting Chamber SlidesInvitrogenC10228
Countess II Automated Cell CounterInvitrogenAMQAX1000
D-(+)-GlucoseSigma-AldrichG6152
DMEM/F-12, HEPESGibco11330032
DMEM, High Glucose, GlutaMAX, PyruvateGibco10569044
Doxycycline hyclateSigma-AldrichD9891
DPBS, no calcium, no magnesiumGibco14190250
Fetal Bovine Serum, qualified, AustraliaGibco10099141
GlutaMAX SupplementGibco35050061
Insulin, Transferrin, Selenium Solution (ITS -G), 100XGibco41400045
lentiviral vectorGeneChemGV307
Lenti-X ConcentratorTakara631232
Lipofectamine 3000 Transfection ReagentInvitrogenL3000015
MEM Non-Essential Amino Acids Solution, 100XGibco11140050
Millex-HV Syringe Filter Unit, 0.45 µm, PVDF, 33 mm, gamma sterilizedMilliporeSLHV033RB
Nalgene General Long-Term Storage Cryogenic TubesThermo Scientific5000-1020
Nunc Cell Culture/Petri DishesThermo Scientific171099
Opti-MEM I Reduced Serum MediumGibco31985070
Penicillin-Streptomycin, LiquidGibco15140122
pHelper 1.0 (gag/pol component)GeneChempHelper 1.0
pHelper 2.0 (VSVG component)GeneChempHelper 2.0
PolybreneSigma-AldrichH9268
Recombinant Human FGF-basicPeprotech100-18B
Sodium bicarbonateSigma-AldrichS5761
STEM-CELLBANKER Cryopreservation MediumZENOAQ11890
StemPro Accutase Cell Dissociation SolutionGibcoA1110501
UltraPure 1 M Tris-HCI Buffer, pH 7.5Invitrogen15567027
ZEISS Inverted MicroscopeZEISSAxio Vert.A1

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. Siegel, R. L., Miller, K. D., Jemal, A.

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