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

Summary

In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

Abstract

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many studies in the last decades. Therefore, it is important to develop methods to investigate the role of key genes involved in cancer cell stemness. Gastric cancer (GC) is one of the most common and mortal types of cancers. Gastric cancer stem cells (GCSCs) are thought to be the root of gastric cancer relapse, metastasis and drug resistance. Understanding GCSCs biology is needed to advance the development of targeted therapies and eventually to reduce mortality among patients. In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

Introduction

Gastric cancer (GC) is one of the most common and mortal types of cancers¹. Despite advances in combined surgery, chemotherapy and radiotherapy in GC therapy, prognosis remains poor and the five-year survival rate is still very low². Recurrence and metastasis are the main reasons cause the post-treatment deaths.

Cancer stem cells (CSCs) are a subset of cancer cells that possess the ability to self-renew and generate the different cell lineages that reconstitute the tumor³. CSCs are believed to be responsible for cancer relapse and metastasis because of their 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.

Protocol

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

1. Gastric cancer stem cell culture

1. Preparation of GCSCs complete culture medium
   1. Prepare GCSCs complete culture medium by adding fresh DME/F12 medium with the following essential ingredients: 20 ng/mL EGF, 10 ng/mL bFGF, 1% Insulin/Transferrin/Sodium selenite, 0.2% glucose, 0.5% B27, 1% Glutamax, 1% Non-essential 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.

Results

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...

Discussion

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

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

Materials

List of materials used in this article

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