Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells

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.......

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 .......

Materials

Name	Company	Catalog Number	Comments
0.22 μm filter	Millipore	SLGP033RB
1-Thioglycerol	Sigma-Aldrich	M6145
2-Mercaptoethanol	Gibco	2068586
Animal-Free Recombinant Human EGF	Peprotech	AF-100-15
B-27 Supplement (50X), serum free	Gibco	17504044
Corning Costar Ultra-Low Attachment Multiple Well Plate	Sigma-Aldrich	CLS3474
Countess Cell Counting Chamber Slides	Invitrogen	C10228
Countess II Automated Cell Counter	Invitrogen	AMQAX1000
D-(+)-Glucose	Sigma-Aldrich	G6152
DMEM/F-12, HEPES	Gibco	11330032
DMEM, High Glucose, GlutaMAX, Pyruvate	Gibco	10569044
Doxycycline hyclate	Sigma-Aldrich	D9891
DPBS, no calcium, no magnesium	Gibco	14190250
Fetal Bovine Serum, qualified, Australia	Gibco	10099141
GlutaMAX Supplement	Gibco	35050061
Insulin, Transferrin, Selenium Solution (ITS -G), 100X	Gibco	41400045
lentiviral vector	GeneChem	GV307
Lenti-X Concentrator	Takara	631232
Lipofectamine 3000 Transfection Reagent	Invitrogen	L3000015
MEM Non-Essential Amino Acids Solution, 100X	Gibco	11140050
Millex-HV Syringe Filter Unit, 0.45 µm, PVDF, 33 mm, gamma sterilized	Millipore	SLHV033RB
Nalgene General Long-Term Storage Cryogenic Tubes	Thermo Scientific	5000-1020
Nunc Cell Culture/Petri Dishes	Thermo Scientific	171099
Opti-MEM I Reduced Serum Medium	Gibco	31985070
Penicillin-Streptomycin, Liquid	Gibco	15140122
pHelper 1.0 (gag/pol component)	GeneChem	pHelper 1.0
pHelper 2.0 (VSVG component)	GeneChem	pHelper 2.0
Polybrene	Sigma-Aldrich	H9268
Recombinant Human FGF-basic	Peprotech	100-18B
Sodium bicarbonate	Sigma-Aldrich	S5761
STEM-CELLBANKER Cryopreservation Medium	ZENOAQ	11890
StemPro Accutase Cell Dissociation Solution	Gibco	A1110501
UltraPure 1 M Tris-HCI Buffer, pH 7.5	Invitrogen	15567027
ZEISS Inverted Microscope	ZEISS	Axio Vert.A1