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

Cancer stem cells are implicated in cancer relapsed metastasis on drug resistance. This protocol provides an efficient method for investigating the functions of key genes in cancer stem cells. Using a conditional knockdown system and adapted sphere formation assay, this method is easily adapted to study in vitro and in vivo functions on stemness-associated genes in different types of cancer stem cells.

Demonstrating the procedure, will be Yuting Li and Xiangyu Tan, research assistants from my laboratory. To generate lentivirus particles, seed four million 293T lentiviral packaging cells into a 100 millimeter Petri dish with 10 milliliters of DMEM supplemented with 10%FBS. Incubate the cells overnight at 37 degrees Celsius and 5%carbon dioxide, making sure that the cells are 50 to 80%confluent on the day of transfection.

Bring reduced serum medium to room temperature and prepare tubes A and B according to manuscript directions. Add the contents of tube A to tube B.Mix well and incubate the tube for 15 minutes at room temperature to prepare lipid-DNA complexes. Remove five milliliters of medium from the cell culture dish.

Then add five milliliters of the lipid-DNA complex into the cell culture dish drop wise and gently swirl the dish. Incubate the culture dish for 24 hours at 37 degrees Celsius and 5%carbon dioxide. After the incubation, carefully remove the transfection medium and replace it with 10 milliliters of pre-warmed DMEM supplemented with 10%FBS.

Incubate the cells for another 24 hours. Approximately 48 hours post transfection, harvest 10 milliliters of lentivirus containing supernatants and filter them with a 0.45 micrometer pour filter to remove cellular debris. All the cell culture vessels, tips, filters and syringes may contain lentivirus and should be treated with 10%bleach prior to disposal.

Transfer a clarified supernatant to a sterile container. Add Lenti-X concentrator and mix with gentle inversion. Incubate the mixture at four degrees Celsius overnight.

On the next day, centrifuge the sample at 1500 times G for 45 minutes at four degrees Celsius. Carefully remove the supernatant, taking care not to disturb the pellet at the bottom. Gently re-suspend the pellet in one milliliter of DMEM supplemented with 10%FBS and store it at minus 80 degrees Celsius.

Seed 6 million gastric cancer stem cells or GCSC's into a 100 millimeter Petri dish with 10 milliliters of DMEM supplemented with 10%FBS. Culture the cells for 24 hours at 37 degrees Celsius and 5%carbon dioxide. When the cells reach 70 to 80%confluency, aspirate the medium from the dish and add the concentrated lentiviral particles diluted with four millimeters of complete DMEM medium, containing Polybrene reagent.

Return the cells to the incubator for 18 hours. Polybrene should be tested in different conditions to determine the effective concentration, which is usually in the range of two to 10 milligram per milliliter. After 18 hours, replace the medium with 10 milliliters of DMEM with 10%FBS and return the cells to the incubator for 24 hours.

Then, aspirate the supernatant with the cell debris and add fresh DMEM supplemented with 10%FBS and 2.5 micrograms per milliliter of Puromycin. Incubate the cells for another 24 hours. Then, change the medium again to fresh DMEM supplemented with 10%FBS and five micrograms per milliliter Puromycin.

After another 24 hour incubation, rinse the adherent GCSCs twice with five milliliters of DPBS without calcium and magnesium. Dissociate the cells with one milliliter of pre-warmed cell dissociation solution and incubate them for two to three minutes at 37 degrees Celsius. Then, add five milliliters of fresh pre-warmed GCSC complete culture medium to the cell suspension.

Dispense three milliliters of this suspension into a 15 milliliter centrifuge tube for cryopreservation, and the other three milliliters into another tube for induction with doxycycline. Centrifuge both tubes at 800 times G for five minutes. Then, aspirate the supernatant from tube B and re-suspend the cells in one milliliter of fresh pre-warmed GCSC complete culture medium.

Seed an appropriate number of cells into a new 100 millimeter Petri dish with 10 milliliters of fresh pre-warmed GCSC complete culture medium and 2.5 micrograms per milliliter doxycycline. Then, incubate the dish for 48 hours. Use an automated cell counter to determine the density of a 10 microliter sample of cells.

Then, adjust the volume with pre-warmed GCSC complete culture medium to obtain a concentration of 20, 000 viable cells per milliliter. Dispense 100 microliters of the cell suspension into each well of a new, ultra low attachment 96 well culture plate. Incubate the cells at 37 degrees Celsius with 5%carbon dioxide.

Sphere formation should occur within three to 10 days. Image the tumor sphere formation every two days. Gastric cancer stem cells from primary human gastric adenocarcinoma were cultured in serum free culture medium.

After six days, cells expanded from the single-cell like phenotype to large spheres. To assess the function of clusterin in GCSCs, short hairpin RNA sequences against clusterin were cloned into the Tet-GV307-RFP-Puro vector. Cells transfected with the expression vector were treated with doxycycline for 48 hours.

Then, clusterin expression was verified by Western blot and quantified by densitometry. The presence of doxycycline and knockdown of clusterin in GCSCs inhibited tumor sphere formation. While no inhibition of tumor sphere formation was observed in cells transduced with the scrambled shRNA controls, indicating that doxycycline alone did not inhibit tumor sphere formation.

These results suggest that after clusterin silencing, GCSCs grow slowly and cannot form tumor spheres. Based on this data, clusterin plays a critical role in promoting the self-renewal activity of GCSCs, indicating that it could be a promising drug target for suppressing cancer stem cells in gastric cancer patients. The tumors spheres should be solid round structures on individual or aggregated cells, should not be considered tumor spheres.

However, some cancer stem cells may not form typical tumor sphere structures. This procedure can be followed with other methods to examine cancer stem cell for renewable potential in vitro. For example, the expression of stemness-related markers can be studied.

The protocol can be extended to study the functions of the critical genes in cancer stem cells such as stemness on survival.