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

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 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 self-renewal. This methodology is also applicable to other types of cancer stem cells.

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

1. Gastric cancer stem cell culture

1. Preparation of GCSCs complete culture medium
   1. Prepare GCSCs complete culture medium by adding fresh DME/F12 medium with the following essential ingredients: 20 ng/mL EGF, 10 ng/mL bFGF, 1% Insulin/Transferrin/Sodium selenite, 0.2% glucose, 0.5% B27, 1% Glutamax, 1% Non-essential amino acid, 10 µM 2-mercaptoethanol, 0.75 mg/mL NaHCO₃, 10 µM thioglycerol, 100 IU/mL penicillin and 100 µg/mL streptomycin. Filter and sterilize using a 0.22 µm filter.
      NOTE: GCSCs complete culture medium is recommended stored preferably no more than two weeks at 4 °C.
2. 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 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.
   1. Pre-warm GCSCs complete culture medium at 37 °C for no more than 30 min.
   2. Defrost GCSCs from liquid nitrogen storage and rapidly thaw cryovials in a 37 °C water bath. Keep swirling the vials until the entire content melts totally.
      NOTE: Thaw frozen cells rapidly (<1 min) in a 37 °C water bath.
   3. Transfer the entire contents of the cryovials into a 15 mL centrifuge tube containing 10 mL of GCSCs complete culture medium. Centrifuge at 800 x g for 5 min at RT.
   4. Aspirate the supernatant carefully and suspend the cell pellet in 10 mL of fresh GCSCs complete medium. Plate the cell suspension in a 100 mm Petri dish. Incubate the plate at 37 °C in a 5% CO₂ incubator and add 5 mL of fresh complete medium on the third day.
3. Subculture of GCSCs tumorspheres
   NOTE: GCSCs of the tumorspheres center only have sufficient nutrients before the spheres size growing up to 80-100 µm in diameter. Once dark and low refractivity spheres appear (about 6 days of culture), it is necessary to subculture the tumorspheres.
   1. Shake the dish gently and transfer the GCSCs 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.
   2. Centrifuge at 600 x g for 5 min and carefully dispose of the supernatant. After centrifugation, an off-white pellet will be visible.
   3. Add 2 mL of cell dissociation solution to resuspend the pellet for mechanical and enzymatic dissociation at 37 °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.
   4. Add 10 mL of fresh pre-warmed GCSCs 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.
   5. Discard the supernatant and resuspend the cells with 1 mL of fresh pre-warmed GCSCs complete culture medium. Seed an appropriate number of cells into a new 100 mm Petri dish with 10 mL of fresh pre-warmed GCSCs complete culture medium and incubate at 37 °C, 5% CO₂.
   6. 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 µm in diameter.
4. Cryopreservation of GCSCs
   NOTE: Do not cryopreserve GCSCs cells by adding medium to tumorspheres directly. GCSCs 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.
   1. Harvest GCSCs tumorspheres. Centrifuge at 600 x g for 5 min.
   2. Discard the supernatant and add 2 mL of cell dissociation solution to dissociate GCSCs tumorspheres at 37 °C. Terminate the digestion procedure by adding 10 mL of GCSCs complete culture medium.
   3. Centrifuge at 800 x g for 5 min and collect single GCSCs.
   4. Gently suspend GCSCs with serum-free cryopreservative medium. The recommended final concentration is 5 x 10⁵ - 5 x 10⁶ cells/mL.
   5. Dispense the cell suspension in 1 mL aliquots into marked cryogenic vials.
   6. Immediately place the cryovials containing the cells in an isopropanol chamber and store them at -80 °C. Transfer the vials to liquid nitrogen the following day for long-term storage.

2. Generation of inducible knockdown GCSCs 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.

1. Generation of lentivirus particles
   1. 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).
   2. 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.
   3. Incubate 293T cells overnight at 37 °C, 5% CO₂. Make sure that 293T cell density is about 50-80% confluent the day of transfection.
   4. Bring the reduced serum medium to room temperature and prepare Tube A and Tube B as described in Table 2.
   5. Transfer Tube A into Tube B, mix well, and incubate the complexes for 20 min at room temperature to prepare lipid-DNA complexes.
   6. Remove 5 mL of medium, before adding lipid-DNA complex, leaving a total of 5 mL.
   7. 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.
   8. Incubate culture dish for 24 h at 37 °C, 5% CO₂.
   9. 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 °C, 5% CO₂.
      NOTE: All the supernatant and tips should be treated with 10% bleach prior to disposal.
   10. 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.
   11. Filter the lentiviral supernatant using a 0.45 µm pore filter to remove cellular debris.
       NOTE: All filters and syringes should be treated with 10% bleach prior to disposal.
   12. Transfer clarified supernatant to a sterile container, add Lenti-X Concentrator (1/3 volume of clarified supernatant) to mix by gentle inversion.
   13. Incubate mixture at 4 °C overnight.
   14. Centrifuge samples at 1,500 x g for 45 min at 4 °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.
   15. Gently resuspend the pellet in 1 mL of DMEM supplemented with 10% FBS as virus stock, store at -80 °C.
2. Generation of stable transfected cell lines
   1. Seed 6 x 10⁶ GCSCs into a 100 mm Petri dish with 10 mL DMEM supplemented with 10% FBS for 24 h at 37 °C, 5% CO₂ (70-80% confluence prior to infection).
   2. Aspirate the medium in the dish, add the concentrated lentiviral particles diluted with 4 mL of complete DMEM medium containing polybrene reagent (5 µg/mL) into the dish. Incubate for 18 h at 37 °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 µg/mL. All the tubes and tips should be treated with 10% bleach prior to disposal.
   3. Change the medium, replace with 10 mL of DMEM with 10% FBS medium and incubate for 24 h at 37 °C, 5% CO₂.
      NOTE: All the medium and the tips should be treated with 10% bleach prior to disposal.
   4. Aspirate the supernatant with cell debris, replace with fresh DMEM supplemented with 10% FBS medium containing puromycin (2.5 µg/mL) and incubate for 24 h at 37 °C, 5% CO₂. Then replace fresh DMEM supplemented with 10% FBS medium containing puromycin (5 µg/mL) and incubate at 37 °C, 5% CO₂ for additional 24 h.
   5. Rinse the adherent GCSCs twice with 5 mL of DPBS without calcium and magnesium.
   6. Dissociate GCSCs with 1 mL of pre-warmed cell dissociation solution and incubate 2-3 min at 37 °C.
   7. Add 5 mL of fresh pre-warmed GCSCs complete culture medium to the cell suspension.
   8. 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.
   9. Centrifuge tube A and tube B at 800 x g for 5 min.
   10. Resuspend the pellet of tube A with 1 mL of serum-free cryopreservative medium, transfer the vial to -80 °C overnight, and remove it into liquid nitrogen storage.
   11. Aspirate the supernatant and resuspend the cells of tube B in 1 mL of fresh pre-warmed GCSCs complete culture medium. Seed an appropriate number of cells into a new 100 mm Petri dish of 10 mL fresh pre-warmed GCSCs complete culture medium with doxycycline (Dox) (2.5 µg/mL) and incubate for 48 h at 37 °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.
   12. Confirm stable repression of clusterin in GCSCs by western blotting.

3. Sphere formation assay

1. Thaw the frozen inducible knockdown GCSCs lines (see step 1.2).
2. Determine viable cell density of a 10 µL sample using an Automated Cell Counter.
3. Adjust the volume with pre-warmed GCSCs complete culture medium to obtain a concentration of 2 x 10⁴ viable cells/mL.
4. Dispense into 3 new 96-well ultra-low-attachment culture plate wells (0.1 mL/well) each group.
5. Incubate the cells in an incubator at 37 °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.
6. Determine tumorsphere formation results by evaluating the sizes of the formed tumorspheres using imaging software.

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.

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 µm. Please click here to view a larger version of this figure.

Figure 2: Conditional KD of clusterin expression in gastric cancer stem cells. GCSCs 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 µg/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 µm. Please click here to view a larger version of this figure.

Figure 3: Expression of Clusterin in inducible knockdown GCSCs 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 β-actin, then was indicated below the lanes of the Western blots. Please click here to view a larger version of this figure.

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 µm. Please click here to view a larger version of this figure.

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>30, ± standard error of mean (SEM). Please click here to view a larger version of this figure.

Table 1: Two shRNA targeting sequences against clusterin.

Table 2: Scale of viral production using transfection.

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

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