Name: combined conditional knockdown and adapted sphere formation assay to study a stemness-associated gene of patient-derived gastric cancer stem cells
Uploaded: 2020-05-09T12:30:00.000+00:00
Duration: 9 min 24 s
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 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>
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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 single cells seeded. We adapted the original method to calculate the mean sizes of all cells/spheres at several time points to improve the results of this assay and to facilitate its reproducibility for other types of cancer stem cells. We certified that the result in this assay is highly dependent on the number of the initial seeded cells. This is a critical point of this assay to maintain initial cell isolation and make an accurate measurement of the number of the spherical colonies (excluding cellular aggregations). Additionally, it is important to optimize the counting time to clearly distinguish the spheres from cellular aggregations and single cells.
Tet-inducible systems are helpful to study the function of genes that are crucial for cell survival in vitro, just like clusterin in this protocol. They are also useful for functional exploration of genes in vivo; this can be done by adding doxycycline into the drinking water of animals. However, leakiness in the uninduced state is an often reported problem of the Tet-inducible systems12. In the presented experiment, a low level of leakiness is also observed with shClu2, as shown in Figure 3. In this case, we can carefully compare the changes of target protein in KD or scrambled control detected in the presence and absence of doxycycline to assess this effect. Another important point is the amount of doxycycline applied in the culture. As the amount of Dox may vary between cell lines, each cell line should be tested with different Dox dosages to decide the effective concentrations for KD and for toxicity on the cells.
The protocol presented here provides an efficient technique for deciphering the stemness-related genes of CSCs and studying CSCs&#39; biology. The protocol can be easily adapted to study the functions of other critical genes in cancer stem cells, such as stemness and survival. Additionally, the conditional knockdown of gene expression in CSCs are feasible to study the biological functions of target genes not only in vitro but also in vivo. However, just some CSCs may not form solid, typical tumorspheres, this protocol should be adapted by using other methods to examine cancer stem cell self-renewal potential in vitro, for example, examining the expression of stemness-related markers.

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><tbody><tr><td>0.22 &amp;mu;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 &amp;micro;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-GV307-RFP-Puro vector following the protocol described above. GCSCs stably transfected with a tetracycline-regulated shRNA-clusterin expression vector were generated and then treated with doxycycline for 48 h (Figure 2) (and shRNA scrambled as a control). The expression level of clusterin was verified by western blot and subsequently quantified by densitometry (Figure 3).
The sphere formation assay was used to test the self-renewal potential of GCSCs. We hypothesized that clusterin promotes the self-renewal potential of GCSCs, and therefore fewer spheres should be observed when clusterin is downregulated by the addition of doxycycline. We demonstrated that the presence of doxycycline and knockdown of clusterin in GCSCs inhibited tumorsphere formation (Figure 4). The cell/sphere sizes of GCSCs were not increasing when clusterin was reduced in GCSCs (Figure 5). No inhibition of tumorsphere formation was observed with GCSCs transduced with the scrambled shRNA controls, indicating that doxycycline had no inhibitory effect on the tumorsphere formation (Figure 5). These results suggested that after clusterin silencing, GCSCs grow slowly and cannot form tumorspheres. Based on the in vitro data, clusterin plays a critical role in promoting the self-renewal activity of GCSCs, indicating that clusterin could be a promising drug target in suppressing CSCs in GC patients.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60799/60799fig1.jpg" /> 
Figure 1: Cell cultures of gastric cancer stem cells. Single cell cultures of gastric cancer stem cells were cultured for 6 days. Phase-contrast microscopic images of these cells/spheres were taken at day 0 (A) and day 6 (B). Original magnification: 10x. Bar size: 20 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60799/60799fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60799/60799fig2.jpg" /> 
Figure 2: Conditional KD of clusterin expression in gastric cancer stem cells. GCSCs&#160;lines were established by infecting lentiviral inducible shRNA control (shCtrl) or inducible shRNA targeting clusterin (shClu1, shClu2). These cell lines were treated with (Dox+) or without Dox (2.5 &#181;g/mL) (Dox-) for 48 h as noted. Phase contrast observation of these cells were shown in top panel. Immunofluorescent observation (red) of these cells were shown in bottom panel. Original magnification: 10x. Bar size: 20 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60799/60799fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60799/60799fig3.jpg" /> 
Figure 3: Expression of Clusterin in inducible knockdown GCSCs&#160;lines with or without Dox treatment. Western blotting analysis of clusterin expression in cell lines stably transfected with tetracycline-regulated shRNA-clusterin (shClu1, shClu2) and scrambled (shCtrl) expression vector after 2 days of doxycycline treatment. The relative expression level of clusterin was quantified by densitometry and normalized against &#946;-actin, then was indicated below the lanes of the Western blots. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60799/60799fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60799/60799fig4.jpg" /> 
Figure 4: Phase-contrast microscopic images of inducible knockdown GCSCs cells/spheres. Single cell of inducible knockdown GCSCs lines were incubated and treated without Dox (top panel) or with Dox (bottom panel) for 6 days. Phase-contrast microscopic images of these cells/spheres were taken at day 6 as indicated. Original magnification: 10x. Scale bar, 20 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60799/60799fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60799/60799fig5.jpg" /> 
Figure 5: Inducible knockdown of Clusterin inhibits GCSC self-renewal capacity. Sphere formation assays were performed in the inducible knockdown GCSCs lines. Phase-contrast microscopic images of these cells/spheres were taken at the indicated day, and the cell/sphere sizes of GCSCs were measured. n&#62;30, &#177; standard error of mean (SEM). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60799/60799fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Name of Gene</td>
			<td>Species</td>
			<td>Gene ID</td>
			<td>Targeting Sequence</td>
		</tr>
		<tr>
			<td>CLU-1</td>
			<td>Human</td>
			<td>1191</td>
			<td>TGAAACAGACCTGCATGAA</td>
		</tr>
		<tr>
			<td>CLU-2</td>
			<td>Human</td>
			<td>1191</td>
			<td>GGGAAGTAAGTACGTCAAT</td>
		</tr>
	</tbody>
</table>
Table 1: Two shRNA targeting sequences against clusterin.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Volume</td>
		</tr>
		<tr>
			<td>Tube A</td>
			<td></td>
		</tr>
		<tr>
			<td>Reduced Serum Medium</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Transfection Reagent</td>
			<td>41 &#956;L</td>
		</tr>
		<tr>
			<td>Tube B</td>
			<td></td>
		</tr>
		<tr>
			<td>Reduced Serum Medium</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>P3000 Enhancer Reagent</td>
			<td>35 &#956;L</td>
		</tr>
		<tr>
			<td>pHelper 1.0 (gag/pol component)</td>
			<td>9 &#956;g</td>
		</tr>
		<tr>
			<td>pHelper 2.0 (VSVG component)</td>
			<td>6 &#956;g</td>
		</tr>
		<tr>
			<td>shCLU 1/2 or control plasmid</td>
			<td>12 &#956;g</td>
		</tr>
	</tbody>
</table>
Table 2: Scale of viral production using transfection.

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 ...

combined-conditional-knockdown-adapted-sphere-formation-assay-to

Nanyang Technological University

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

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains poor when metastasis has occurred. The Cancer Stem Cell (CSC) theory holds that there is a subset of tumor cells within the tumor that have stem-like characteristics, including the capacity to maintain the tumor and to resist multidrug chemotherapy. Therefore, a better understanding of OS biology and pathogenesis is needed in order to advance the development of targeted therapies to eradicate this particular subset and to reduce morbidity and mortality among patients. Isolating CSCs, establishing cell cultures of CSCs, and studying their biology are important steps to improving our understanding of OS biology and pathogenesis. The establishment of human-derived OS-CSCs from biopsies of OS has been made possible using several methods, including the capacity to create 3-dimensional stem cell cultures under nonadherent conditions. Under these conditions, CSCs are able to create spherical floating colonies formed by daughter stem cells; these colonies are termed &#34;cellular spheres&#34;. Here, we describe a method to establish CSC cultures from primary cell cultures of conventional OS obtained from OS biopsies. We clearly describe the several passages required to isolate and characterize CSCs.

The presence of cancer stem cells (CSCs) in bone sarcomas has recently been linked to their pathogenesis. In this article, we present the isolation of CSCs from primary cell cultures obtained from human biopsies of conventional osteosarcoma (OS) using the ability of CSCs to grow under nonadherent conditions.

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains ...

Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median survival following diagnosis is less than 15 months with maximal surgical resection, radiation, and temozolomide chemotherapy. The challenges inherent in developing more effective GBM treatments have become increasingly clear, and include its unyielding invasiveness, its resistance to standard treatments, its genetic complexity and molecular adaptability, and subpopulations of GBM cells with phenotypic similarities to normal stem cells, herein referred to as glioblastoma stem cells (GSCs). Because GSCs are required for tumor growth and progression, differentiation-based therapy represents a viable treatment modality for these incurable neoplasms. 
The following protocol describes a collection of procedures to establish a high throughput screening platform aimed at the identification of small molecules that promote GSC astroglial differentiation. At the core of the system is a glial fibrillary acidic protein (GFAP) differentiation reporter-construct. The protocol contains the following general procedures: (1) establishing GSC differentiation reporter lines; (2) testing/validating the relevance of the reporter to GSC self-renewal/clonogenic capacity; and (3) high-capacity flow-cytometry based drug screening. 
The screening platform provides a straightforward and inexpensive approach to identify small molecules that promote GSCs differentiation. Furthermore, utilization of libraries of FDA-approved drugs holds the potential for the identification of agents that can be repurposed more rapidly. Also, therapies that promote cancer stem cell differentiation are expected to work synergistically with current "standard of care" therapies that have been shown to target and eliminate primarily more differentiated cancer cells.

An efficient screening protocol is presented for the identification of small molecules that promote astroglial differentiation in glioblastoma stem cells (GSCs). The assay is based on a stem cell differentiation reporter whereby the expression of the enhanced GFP (eGFP) is driven by the human GFAP promoter.

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median ...

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 ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 &mu;m in diameter can be observed and are ready for further passaging and expansion.

This video protocol demonstrates the isolation and expansion of stem like cells from surgically resected human glioblastoma mutliforme (GBM) tumor tissue using the neurosphere assay culture method.

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

Neuroscience

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

Discovery of Driver Genes in Colorectal HT29-derived Cancer Stem-Like Tumorspheres

Cancer stem cells play a vital role against clinical therapies, contributing to tumor relapse. There are many oncogenes involved in tumorigenesis and the initiation of cancer stemness properties. Since gene expression in the formation of colorectal cancer-derived tumorspheres is unclear, it takes time to discover the mechanisms working on one gene at a time. This study demonstrates a method to quickly discover the driver genes involved in the survival of the colorectal cancer stem-like cells in vitro. Colorectal HT29 cancer cells that express the LGR5 when cultured as spheroids and accompany an increase CD133 stemness markers were selected and used in this study. The protocol presented is used to perform RNAseq with available bioinformatics to quickly uncover the overexpressed driver genes in the formation of colorectal HT29-derived stem-like tumorspheres. The methodology can quickly screen and discover potential driver genes in other disease models.

Presented here is a protocol to discover the overexpressed driver genes maintaining the established cancer stem-like cells derived from colorectal HT29 cells. RNAseq with available bioinformatics was performed to investigate and screen gene expression networks for elucidating a potential mechanism involved in the survival of targeted tumor cells.

Cancer stem cells play a vital role against clinical therapies, contributing to tumor relapse. There are many oncogenes involved in tumorigenesis and the ...

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

Medicine

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.