Name: combined conditional knockdown and adapted sphere formation assay to study a stemness-associated gene of patient-derived gastric cancer stem cells
Uploaded: 2020-05-09T12:30:00
Description: Watch this Scientific Journal Video about Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells at JoVE.com

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).

All experimentation using patient-derived gastric cancer stem cells described herein was approved by the local ethical committee7.
1. Gastric cancer stem cell culture
<ol>
	<li>Preparation of GCSCs complete culture medium
	<ol>
		<li>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...

<ol>
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GC is the third leading cause of cancer-related death worldwide. GCSCs are&#160;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 ...

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 capabilities of self-renewal and seeding new tumors, as well as their resistance to traditional chemo- and radiotherapies4. Therefore, targeting CSCs and elimination of CSCs provide an exciting potential to improve the treatment and reduce mortality of cancer patients.
CSCs have been isolated from many types of solid tumors5. In 2009, gastric cancer stem cells (GCSCs) isolated from human gastric cancer cell lines were originally described by Takaishi et al.6. Chen and colleagues firstly identified and purified GCSCs from human gastric adenocarcinoma (GAC) tumor tissues7. These findings not only provide an opportunity to study GCSCs&#160;biology but also provide great clinical importance.
A particular characteristic of CSCs is their capacity to form a sphere8. Single cells are plated in nonadherent conditions at low density, and only the cells possessed with self-renewal can grow into a solid, spherical cluster called a sphere. Thus, the sphere formation assay has been regarded as the gold standard assay and widely used to evaluate stem cell self-renewal potential in vitro.
RNA interference (RNAi) is a powerful research tool to study gene function by the knockdown of a specific gene9. However, long term stable gene knockdown technologies have certain limitations, such as the challenge of exploring the function of a gene that is essential for cell survival. Conditional RNAi systems can be useful for the downregulation of desired genes in a temporal and/or special controlled manner by the administration of an inducing agent. The tetracycline (Tet)-inducible systems are one of the most widely used conditional RNAi systems10. The Tet-inducible systems can induce target gene silencing by controlling the expression of shRNA upon addition of an exogenous inducer (preferentially doxycycline, Dox). The Tet-inducible systems can be divided into two types: Tet-On or Tet-Off systems. The expression of shRNA can be turned on (Tet-On) or turned off (Tet-Off) in the presence of the inducer. In the Tet-ON system without an inducer, the constitutively expressed Tet repressor (TetR) binds to the Tet-responsive element (TRE) sequence containing a Tet-responsive Pol III-dependent promoter for shRNA expression, thus repressing the expression of the shRNA. While upon addition of Dox, the TetR is sequestered away from the Tet-responsive Pol III-dependent promoter. This facilitates the expression of the shRNA and leads to gene knockdown.
The protocol described here employs a functional tetracycline-inducible shRNA system and an adapted sphere formation assay to study the function of clusterin in patient-derived GCSCs. Clusterin has been identified as a novel key molecule for maintaining the stemness and survival of GCSCs in a previous study11. We use the described protocol to study the effects of clusterin in GCSCs&#160;self-renewal. This methodology is also applicable to other types of cancer stem cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#956;m filter</td>
			<td>Millipore</td>
			<td>SLGP033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Thioglycerol</td>
			<td>Sigma-Aldrich</td>
			<td>M6145</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>2068586</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal-Free Recombinant Human EGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X), serum free</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar Ultra-Low Attachment Multiple Well Plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3474</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber Slides</td>
			<td>Invitrogen</td>
			<td>C10228</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II Automated Cell Counter</td>
			<td>Invitrogen</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G6152</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, HEPES</td>
			<td>Gibco</td>
			<td>11330032</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, High Glucose, GlutaMAX, Pyruvate</td>
			<td>Gibco</td>
			<td>10569044</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxycycline hyclate</td>
			<td>Sigma-Aldrich</td>
			<td>D9891</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Gibco</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, Australia</td>
			<td>Gibco</td>
			<td>10099141</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin, Transferrin, Selenium Solution (ITS -G), 100X</td>
			<td>Gibco</td>
			<td>41400045</td>
			<td></td>
		</tr>
		<tr>
			<td>lentiviral vector</td>
			<td>GeneChem</td>
			<td>GV307</td>
			<td></td>
		</tr>
		<tr>
			<td>Lenti-X Concentrator</td>
			<td>Takara</td>
			<td>631232</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 3000 Transfection Reagent</td>
			<td>Invitrogen</td>
			<td>L3000015</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution, 100X</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-HV Syringe Filter Unit, 0.45 &#181;m, PVDF, 33 mm, gamma sterilized</td>
			<td>Millipore</td>
			<td>SLHV033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene General Long-Term Storage Cryogenic Tubes</td>
			<td>Thermo Scientific</td>
			<td>5000-1020</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Cell Culture/Petri Dishes</td>
			<td>Thermo Scientific</td>
			<td>171099</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Gibco</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin, Liquid</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>pHelper 1.0 (gag/pol component)</td>
			<td>GeneChem</td>
			<td>pHelper 1.0</td>
			<td></td>
		</tr>
		<tr>
			<td>pHelper 2.0 (VSVG component)</td>
			<td>GeneChem</td>
			<td>pHelper 2.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Sigma-Aldrich</td>
			<td>H9268</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF-basic</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>STEM-CELLBANKER Cryopreservation Medium</td>
			<td>ZENOAQ</td>
			<td>11890</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Accutase Cell Dissociation Solution</td>
			<td>Gibco</td>
			<td>A1110501</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure 1 M Tris-HCI Buffer, pH 7.5</td>
			<td>Invitrogen</td>
			<td>15567027</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEISS Inverted Microscope</td>
			<td>ZEISS</td>
			<td>Axio Vert.A1</td>
			<td></td>
		</tr>
	</tbody>
</table>

combined conditional knockdown and adapted sphere formation assay to study a stemness-associated gene of patient-derived gastric cancer stem cells

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&#160;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.

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...

Watch this Scientific Journal Video about Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells at JoVE.com

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

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.

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many ...

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