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 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.
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
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.
3. Sphere formation assay
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.
Name of Gene | Species | Gene ID | Targeting Sequence |
CLU-1 | Human | 1191 | TGAAACAGACCTGCATGAA |
CLU-2 | Human | 1191 | GGGAAGTAAGTACGTCAAT |
Table 1: Two shRNA targeting sequences against clusterin.
Component | Volume |
Tube A | |
Reduced Serum Medium | 1.5 mL |
Transfection Reagent | 41 μL |
Tube B | |
Reduced Serum Medium | 1.5 mL |
P3000 Enhancer Reagent | 35 μL |
pHelper 1.0 (gag/pol component) | 9 μg |
pHelper 2.0 (VSVG component) | 6 μg |
shCLU 1/2 or control plasmid | 12 μg |
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.
The authors have nothing to disclose.
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).
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 |