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 amino acid, 10 &#181;M 2-mercaptoethanol, 0.75 mg/mL NaHCO3, 10 &#181;M thioglycerol, 100 IU/mL penicillin and 100 &#181;g/mL streptomycin. Filter and sterilize using a 0.22 &#181;m filter. 
		NOTE: GCSCs&#160;complete culture medium is recommended stored preferably no more than two weeks at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Recovery of GCSCs and culture 
	NOTE: GCSCs were obtained as follows: Tumor samples were subjected to mechanical and enzymatic dissociation. Single cell suspensions were obtained by filtering with nylon net from well-scattered suspension. The resulting cancer cells were cultured in GCSCs&#160;Complete Culture Medium, and some cells grew to form spheres. These spheres were then subjected to enzymatic dissociation, and GCSCs can be obtained by cytofluorometric sorting of the cell population stained with CD44/CD54 markers. The detailed protocol and functional assays of the GCSCs have been reported7.
	<ol>
		<li>Pre-warm GCSCs&#160;complete culture medium at 37 &#176;C for no more than 30 min.</li>
		<li>Defrost GCSCs from liquid nitrogen storage and rapidly thaw cryovials in a 37 &#176;C water bath. Keep swirling the vials until the entire content melts totally. 
		NOTE: Thaw frozen cells rapidly (&#60;1 min) in a 37 &#176;C water bath.</li>
		<li>Transfer the entire contents of the cryovials into a 15 mL centrifuge tube containing 10 mL of GCSCs&#160;complete culture medium. Centrifuge at 800 x g for 5 min at RT.</li>
		<li>Aspirate the supernatant carefully and suspend the cell pellet in 10 mL of fresh GCSCs&#160;complete medium. Plate the cell suspension in a 100 mm Petri dish. Incubate the plate at 37 &#176;C in a 5% CO2 incubator and add 5 mL of fresh complete medium on the third day.</li>
	</ol>
	</li>
	<li>Subculture of GCSCs&#160;tumorspheres 
	NOTE: GCSCs&#160;of the tumorspheres center only have sufficient nutrients before the spheres size growing up to 80-100 &#181;m in diameter. Once dark and low refractivity spheres appear (about 6 days of culture), it is necessary to subculture the tumorspheres.
	<ol>
		<li>Shake the dish gently and transfer the GCSCs&#160;tumorsphere culture medium (the medium and the non-adherent tumorspheres) into a sterile 15 mL centrifuge tube. For larger medium volumes, larger centrifuge tubes may be needed.</li>
		<li>Centrifuge at 600 x g for 5 min and carefully dispose of the supernatant. After centrifugation, an off-white pellet will be visible.</li>
		<li>Add 2 mL of cell dissociation solution to resuspend the pellet for mechanical and enzymatic dissociation at 37 &#176;C. Gently pipet up and down 10 times every 2-3 min in the digestion procedure to break the spheres apart until the tumorspheres are dispersed into single cell suspension. This total dissociation process is recommended to be less than 15 min. 
		NOTE: Perform a visual check under the microscope to confirm that no large spheres or cell aggregates remain.</li>
		<li>Add 10 mL of fresh pre-warmed GCSCs&#160;complete culture medium (5x the volume of the cell detachment solution) to terminate digestion procedure and centrifuge at 800 x g at RT for 5 min.</li>
		<li>Discard the supernatant and resuspend the cells with 1 mL of fresh pre-warmed GCSCs&#160;complete culture medium. Seed an appropriate number of cells into a new 100 mm Petri dish with 10 mL of fresh pre-warmed GCSCs&#160;complete culture medium and incubate at 37 &#176;C, 5% CO2.</li>
		<li>Refeed tumorspheres cultures after 3 days by adding 5 mL of fresh pre-warmed complete medium. After 6 days, passage cells when tumorspheres grow up to 80-100 &#181;m in diameter.</li>
	</ol>
	</li>
	<li>Cryopreservation of GCSCs 
	NOTE: Do not cryopreserve GCSCs&#160;cells by adding medium to tumorspheres directly. GCSCs&#160;tumorspheres should be digested into single cells so that cell protective agent could enter every cell to ensure the long-term stable storage of cells. Make sure the cells are in healthy situation and without contamination.
	<ol>
		<li>Harvest GCSCs&#160;tumorspheres. Centrifuge at 600 x g for 5 min.</li>
		<li>Discard the supernatant and add 2 mL of cell dissociation solution to dissociate GCSCs&#160;tumorspheres at 37 &#176;C. Terminate the digestion procedure by adding 10 mL of GCSCs&#160;complete culture medium.</li>
		<li>Centrifuge at 800 x g for 5 min and collect single GCSCs.</li>
		<li>Gently suspend GCSCs&#160;with serum-free cryopreservative medium. The recommended final concentration is 5 x 105 - 5 x 106 cells/mL.</li>
		<li>Dispense the cell suspension in 1 mL aliquots into marked cryogenic vials.</li>
		<li>Immediately place the cryovials containing the cells in an isopropanol chamber and store them at -80 &#176;C. Transfer the vials to liquid nitrogen the following day for long-term storage.</li>
	</ol>
	</li>
</ol>
2. Generation of inducible knockdown GCSCs&#160;lines
CAUTION: Recombinant lentiviruses have been designated as Level 2 organisms by the National Institute of Health and Center for Disease Control. Work involving lentivirus requires the maintenance of a Biosafety Level 2 facility, considering that the viral supernatants produced by these lentiviral systems could contain potentially hazardous recombinant virus.
<ol>
	<li>Generation of lentivirus particles
	<ol>
		<li>Synthesize 2 lentiviral vectors carrying inducible shRNA targeting human clusterin and a non-targeting control lentiviral vector (GV307) from GeneChem based on the design of Table 1 (GV307 vector contains: TetIIP-TurboRFP-MCS(MIR30)-Ubi-TetR-IRES-Puromycin).</li>
		<li>Seed 4 x 106 293T lenti-viral packaging cells into a 100 mm Petri dish with 10 mL of DMEM supplemented with 10% fetal bovine serum.</li>
		<li>Incubate 293T cells overnight at 37 &#176;C, 5% CO2. Make sure that 293T cell density is about 50-80% confluent the day of transfection.</li>
		<li>Bring the reduced serum medium to room temperature and prepare Tube A and Tube B as described in Table 2.</li>
		<li>Transfer Tube A into Tube B, mix well, and incubate the complexes for 20 min at room temperature to prepare lipid-DNA complexes.</li>
		<li>Remove 5 mL of medium, before adding lipid-DNA complex, leaving a total of 5 mL.</li>
		<li>Add 5 mL of lipid-DNA complex into the culture dish dropwise and gently swirl the dish to distribute the complex. 
		NOTE: Carefully dispense liquid against the dish wall to avoid disturbing 293T cells.</li>
		<li>Incubate culture dish for 24 h at 37 &#176;C, 5% CO2.</li>
		<li>After 24 hours post-transfection, carefully remove the transfection medium and gently replace with 10 mL of pre-warmed DMEM supplemented with 10% FBS. Incubate for 24 h at 37 &#176;C, 5% CO2. 
		NOTE: All the supernatant and tips should be treated with 10% bleach prior to disposal.</li>
		<li>Approximately after 48 hours post-transfection, harvest 10 mL of lentivirus-containing supernatants. 
		NOTE: All the cell culture vessels and tips should be treated with 10% bleach prior to disposal.</li>
		<li>Filter the lentiviral supernatant using a 0.45 &#181;m pore filter to remove cellular debris. 
		NOTE: All filters and syringes should be treated with 10% bleach prior to disposal.</li>
		<li>Transfer clarified supernatant to a sterile container, add Lenti-X Concentrator (1/3 volume of clarified supernatant) to mix by gentle inversion.</li>
		<li>Incubate mixture at 4 &#176;C overnight.</li>
		<li>Centrifuge samples at 1,500 x g for 45 min at 4 &#176;C. After centrifugation, and off-white pellet will be visible. Carefully remove supernatant, taking care not to disturb the pellet. 
		NOTE: All the supernatant and tips should be treated with 10% bleach prior to disposal.</li>
		<li>Gently resuspend the pellet in 1 mL of DMEM supplemented with 10% FBS as virus stock, store at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Generation of stable transfected cell lines
	<ol>
		<li>Seed 6 x 106 GCSCs&#160;into a 100 mm Petri dish with 10 mL DMEM supplemented with 10% FBS for 24 h at 37 &#176;C, 5% CO2 (70-80% confluence prior to infection).</li>
		<li>Aspirate the medium in the dish, add the concentrated lentiviral particles diluted with 4 mL of complete DMEM medium containing polybrene reagent (5 &#181;g/mL) into the dish. Incubate for 18 h at 37 &#176;C, 5% CO2. 
		NOTE: The optimal concentration of polybrene depends on cell type and may need to be test in different concentrations to decide the effective concentrations. Otherwise, it may be empirically determined, usually in the range of 2-10 &#181;g/mL. All the tubes and tips should be treated with 10% bleach prior to disposal.</li>
		<li>Change the medium, replace with 10 mL of DMEM with 10% FBS medium and incubate for 24 h at 37 &#176;C, 5% CO2. 
		NOTE: All the medium and the tips should be treated with 10% bleach prior to disposal.</li>
		<li>Aspirate the supernatant with cell debris, replace with fresh DMEM supplemented with 10% FBS medium containing puromycin (2.5 &#181;g/mL) and incubate for 24 h at 37 &#176;C, 5% CO2. Then replace fresh DMEM supplemented with 10% FBS medium containing puromycin (5 &#181;g/mL) and incubate at 37 &#176;C, 5% CO2 for additional 24 h.</li>
		<li>Rinse the adherent GCSCs&#160;twice with 5 mL of DPBS without calcium and magnesium.</li>
		<li>Dissociate GCSCs with 1 mL of pre-warmed cell dissociation solution and incubate 2-3 min at 37 &#176;C.</li>
		<li>Add 5 mL of fresh pre-warmed GCSCs complete culture medium to the cell suspension.</li>
		<li>Dispense 3 mL into a 15 mL centrifugal tube (tube A) for cryopreserving the cells, and the other 3 mL into a 15 mL centrifugal tube (tube B) for inducing by doxycycline.</li>
		<li>Centrifuge tube A and tube B at 800 x g for 5 min.</li>
		<li>Resuspend the pellet of tube A with 1 mL of serum-free cryopreservative medium, transfer the vial to -80 &#176;C overnight, and remove it into liquid nitrogen storage.</li>
		<li>Aspirate the supernatant and resuspend the cells of tube B in 1 mL of fresh pre-warmed GCSCs&#160;complete culture medium. Seed an appropriate number of cells into a new 100 mm Petri dish of 10 mL fresh pre-warmed GCSCs&#160;complete culture medium with doxycycline (Dox) (2.5 &#181;g/mL) and incubate for 48 h at 37 &#176;C, 5% CO2. 
		NOTE: The optimal concentration of Dox may vary between cell lines. Each cell line should be tested in different Dox concentrations to decide the effective concentrations for KD and for toxicity on the cells.</li>
		<li>Confirm stable repression of clusterin in GCSCs&#160;by western blotting.</li>
	</ol>
	</li>
</ol>
3. Sphere formation assay
<ol>
	<li>Thaw the frozen inducible knockdown GCSCs&#160;lines (see step 1.2).</li>
	<li>Determine viable cell density of a 10 &#181;L sample using an Automated Cell Counter.</li>
	<li>Adjust the volume with pre-warmed GCSCs&#160;complete culture medium to obtain a concentration of 2 x 104 viable cells/mL.</li>
	<li>Dispense into 3 new 96-well ultra-low-attachment culture plate wells (0.1 mL/well) each group.</li>
	<li>Incubate the cells in an incubator at 37 &#176;C with 5% CO2. Sphere formation should occur within 3-10 days. Monitor and record the visualization of tumorspheres formation every 2 days. 
	NOTE: The medium is not recommended to be changed in case of any disturbance of the tumorspheres formation. These tumorspheres should be easily distinguished from single and aggregated cells.</li>
	<li>Determine tumorsphere formation results by evaluating the sizes of the formed tumorspheres using imaging software.</li>
</ol>

<ol>
	<li>Bray, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+cancer+statistics+2018:+GLOBOCAN+estimates+of+incidence+and+mortality+worldwide+for+36+cancers+in+185+countries.">Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.</a> CA: A Cancer Journal for Clinicians. 68 (6), 394-424 (2018).</li><li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics,+2016.">Cancer statistics, 2016.</a> CA: A Cancer Journal for Clinicians. 66 (1), 7-30 (2016).</li><li>Valent, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cell+definitions+and+terminology:+the+devil+is+in+the+details.">Cancer stem cell definitions and terminology: the devil is in the details.</a> Nature Reviews Cancer. 12 (11), 767-775 (2012).</li><li>P&#252;tzer, B. M., Solanki, M., Herchenr&#246;der, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+cancer+stem+cell+targeting:+How+to+strike+the+evil+at+its+root.">Advances in cancer stem cell targeting: How to strike the evil at its root.</a> Advanced Drug Delivery Reviews. 120, 89-107 (2017).</li><li>Saygin, C., Matei, D., Majeti, R., Reizes, O., Lathia, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+Cancer+Stemness+in+the+Clinic:+From+Hype+to+Hope.">Targeting Cancer Stemness in the Clinic: From Hype to Hope.</a> Cell Stem Cell. 24 (1), 25-40 (2019).</li><li>Takaishi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+gastric+cancer+stem+cells+using+the+cell+surface+marker+CD44.">Identification of gastric cancer stem cells using the cell surface marker CD44.</a> Stem Cells. 27 (5), 1006-1020 (2009).</li><li>Chen, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+expansion+of+cancer+stem+cells+in+tumor+tissues+and+peripheral+blood+derived+from+gastric+adenocarcinoma+patients.">Identification and expansion of cancer stem cells in tumor tissues and peripheral blood derived from gastric adenocarcinoma patients.</a> Cell Research. 22 (1), 248-258 (2012).</li><li>Pastrana, E., Silva-Vargas, V., Doetsch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eyes+wide+open:+a+critical+review+of+sphere-formation+as+an+assay+for+stem+cells.">Eyes wide open: a critical review of sphere-formation as an assay for stem cells.</a> Cell Stem Cell. 8 (5), 486-498 (2011).</li><li>Hannon, G. J., Rossi, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unlocking+the+potential+of+the+human+genome+with+RNA+interference.">Unlocking the potential of the human genome with RNA interference.</a> Nature. 431 (7006), 371-378 (2004).</li><li>Seibler, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+gene+knockdown+in+mice+using+a+tight,+inducible+shRNA+expression+system.">Reversible gene knockdown in mice using a tight, inducible shRNA expression system.</a> Nucleic Acids Research. 35 (7), e54 (2007).</li><li>Xiong, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Verteporfin+blocks+Clusterin+which+is+required+for+survival+of+gastric+cancer+stem+cell+by+modulating+HSP90+function.">Verteporfin blocks Clusterin which is required for survival of gastric cancer stem cell by modulating HSP90 function.</a> International Journal of Biological Sciences. 15 (2), 312-324 (2019).</li><li>Ohkawa, J., Taira, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+the+functional+activity+of+an+antisense+RNA+by+a+tetracycline-responsive+derivative+of+the+human+U6+snRNA+promoter.">Control of the functional activity of an antisense RNA by a tetracycline-responsive derivative of the human U6 snRNA promoter.</a> Human Gene Therapy. 11 (4), 577-585 (2000).</li></ol>

No conflicts of interest declared.

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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Research

JoVE Journal

Cancer Research

4.3K Views.  Shenzhen University School of Medicine. 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.

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

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more than 50 histological subtypes of these rare solid tumors have been identified. Thus, due to their extraordinary diversity and low incidence, our understanding of the biology of these tumors is still limited. Patient-derived cultures represent the ideal platform to study STS pathophysiology and pharmacology. We thus developed a human preclinical model of STS starting from tumor specimens harvested from patients undergoing surgical resection. Patient-derived STS cell cultures were obtained from the surgical specimens by collagenase digestion and isolated by filtration. Cells were counted, seeded, and left for 14 days in standard monolayer cultures and then processed by downstream analysis. Before performing molecular or pharmaceutical analyses, the establishment of STS primary cultures was confirmed through the evaluation of cytomorphologic features and, when available, immunohistochemical markers. This method represents a useful tool 1) to study the natural history of these poorly explored malignancies and 2) to test the effects of different drugs in an effort to learn more about their mechanisms of action.

The following protocol focuses on the establishment of a primary culture of patient-derived soft tissue sarcoma (STS). This model could help us to better understand the molecular background and pharmacological profile of these rare malignancies and could represent a starting point for further research aimed at improving STS management.

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in adults. Initially, AML is a disease of hematopoietic stem cells (HSC) characterized by arrest of differentiation, subsequent accumulation of leukemia blast cells, and reduced production of functional hematopoietic elements. Heterogeneity extends to the presence of leukemia stem cells (LSC), with this dynamic cell compartment evolving to overcome various selection pressures imposed upon during leukemia progression and treatment. To further define the LSC population, the addition of CD90 and CD45RA allows the discrimination of normal HSCs and multipotent progenitors within the CD34+CD38- cell compartment. Here, we outline a protocol to detect simultaneous expression of several putative LSC markers (CD34, CD38, CD45RA, CD90) on primary blast cells of human AML by multiparametric flow cytometry. Furthermore, we show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts. This method of detection and quantification of putative LSC may be used for clinical follow-up of chemotherapy response (i.e., minimal residual disease), as residual LSC may cause AML relapse.

We outline a protocol to detect simultaneous expression of leukemia stem cell markers on primary acute myeloid leukemia cells by flow cytometry. We show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts.

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including osteosarcoma--one of the most frequent primary non-hematologic malignancies in the childhood and adolescence. Therefore, LFS provides an ideal model to study this malignancy. Taking advantage of iPSC methodologies, LFS-associated osteosarcoma can be successfully modeled by differentiating LFS patient iPSCs to mesenchymal stem cells (MSCs), and then to osteoblasts--the cells of origin for osteosarcomas. These LFS osteoblasts recapitulate oncogenic properties of osteosarcoma, providing an attractive model system for delineating the pathogenesis of osteosarcoma. This manuscript demonstrates a protocol for the generation of iPSCs from LFS patient fibroblasts, differentiation of iPSCs to MSCs, differentiation of MSCs to osteoblasts, and in vivo tumorigenesis using LFS osteoblasts. This iPSC disease model can be extended to identify potential biomarkers or therapeutic targets for LFS-associated osteosarcoma.

Here, we present a protocol for the generation of induced pluripotent Stem Cells (iPSCs) from Li-Fraumeni Syndrome (LFS) patient derived fibroblasts, differentiation of iPSCs via mesenchymal stem cells (MSCs) to osteoblasts, and modeling in vivo tumorigenesis using LFS patient-derived osteoblasts.

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

A Melanoma Patient-Derived Xenograft Model

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

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.

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.

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...