Source and preparation of cell lines
The second generation CD133+ spheroid cells (named HUSSLCs) of SK-UT-1 cell line were from previous experiments and were stored in the cell center of Xiangya medical school. SP-2/0 myeloma cells were donated by Professor Liu Rushi of Hunan Normal University 9-11. Balb/c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animal experiments were approved by the Experimental Animal Ethics Committee of the Medical College of Hunan Normal University.
Determination of serum titer of HUSSLCs-immunized Balb/c mice
Mouse immunization experiment and preparation of cell suspension
Two female 6-weeks old BALB/c mice were intraperitoneally injected with HUSSLCs 1×107/0.5 mL saline; the same immunization was performed every 2 weeks after each immunization, totaling three times. Two weeks after the third immunization, each mouse received intrasplenic injection of HUSSLCs 5×105/0.1 mL normal saline for impact immunization. After 3 days, spleen cell suspension was prepared.
Detection of serum antibody titer by ELISA
Two weeks after each immunization, orbital blood of mice was taken for detection of serum titer, and the serum was diluted with a phosphate buffer at a concentration gradient of 1:10, 1:100, 1:1000, 1:10000, 1:100,000, and 1:1000000 respectively for ELISA. The absorbance value was measured at a wavelength of 450 nm using enzyme-labelled spectrophotometer.
Cell fusion and positive clone screening
One unimmunized BALB/c mouse was put to death and intraperitoneally injected with 5 ml of serum-free medium. The peritoneal fluid derived from softly rubbing the peritoneal cavity for 1 to 2 minutes was transferred to 20% FBS-HAT-1640 medium (400 ml) for culture.
One mouse immunized with HUSSLCs was selected and put to death. After that, its spleen was isolated and grinded for culture and collection of cell suspension, which was then treated to pellet-free particles.
SP-2/0 cells selected in logarithmic phase and the spleen cells after treatment were mixed and cultured. The cell mixture of 1000g was centrifuged for 2 minutes to remove the supernatant; 1ml of 37 °C PEG1500 was added to the precipitate and the cells were blown and beaten for even mixture. 30ml serum-free medium was added after 1 minute, and the cell fusion was stopped; 1000g cell mixture after fusion was centrifuged for 2 minutes to remove the supernatant; the precipitate was resuspended using the above-prepared feeder cell-containing medium. The fused cells were inoculated into a 96-well culture plate at 200 μL/well, placed in an incubator, and the culture medium was changed once every 2 days. After 7 days of culture, HT containing (HT is the intermixture of hypoxanthine and thymidine) medium was used instead. When the cell colony covered 1/3-1/2 of the culture well, the next experimental operation was carried out.
50 μl/well of hybridoma cell culture supernatant was added to the ELISA plate. Two wells with no cell growth were chosen for each plate, which was set as a negative control, and another 2 wells with no cell growth added with positive serum was taken as a positive control; The serum of non-immune mice was taken as the negative control while in detection of antibody valence. The ELISA plate was incubated for half an hour at 37 °C. 100 ul of enzyme-labeled secondary antibody was added to each well and incubated at 37 ° C for half an hour. 200 ml of substrate was added to each well and developed at 37 °C for half an hour. The absorbance value (OD value) was measured at a wavelength of 450 nm using an enzyme-labelled spectrophotometer, which was compared with the OD value of the negative control well, with P/N greater than 2.1 serving as a critical point. After successful cell fusion and selective culture, the cell wells detected as positive via ELISA were prepared as cell suspension; the above cell suspensions were diluted at ratios of 1:2, 1:4, 1:8, 1:16, and 1:32 and inoculated into 96-well plates respectively. Each cell concentration was repeated through 8 wells to observe the growth of cells, with suspensions changed every two days; when the cell fusion degree reached 70%, the supernatant was taken for positive detection by ELISA; The positive single colony was selected to repeat the process until the positive rate achieved 100% (clone culture 4 times); the positive clone cells were cultured in a common 24-well plate and a common 6-well plate respectively, which were eventually transferred to a 25 cm2 culture flask for culture and amplification.
Subtype identification of monoclonal antibodies
A 96-well cell culture plate (flat bottom) was added with 50 μL 10 μg/mL of L-poly-L-Lysine per well, placed at room temperature for 30 minutes, and then washed twice with PBS. 50 μL of HUSSLCs cell suspension (2.5 x 106 cells/mL) was added to each well, placed overnight at 4 ° C and washed once the next day with PBS. 50 μL of 0.5% glutaraldehyde was added to each well, fixed at 4 ° C for 15 minutes, and washed twice with PBS. 400 μL of 0.1 mol/L glycine solution was added to each well, placed at 4 ° C for 30 minutes, and washed 3 times with PBS. Incomplete DMEM medium was added each time and stored at -20 ° C for later use. At the time of detection, the cell culture plate was taken out from the -20 ° C refrigerator, and after the liquid in the well was melted, it was washed twice with PBS. 100 μL of the hybridoma cell supernatant to be tested was added to each well, placed at room temperature for 2 hours and washed 4 times with PBS. (1 hour incubation at 37 ° C) 100 μL of enzyme-labeled secondary antibody (goat anti-mouse IgM, goat anti-mouse IgG, goat anti-mouse IgG1, goat anti-mouse IgG2a, goat anti-mouse IgG2b and goat anti-mouse IgG3) were added to each well at room temperature for 2 hours and washed 6 times with phosphate buffer (incubation at 37 ° C for 30 min). 200 μL of freshly prepared substrate (TMB) was added to each well, placed at 37 ° C for 30 minutes. The absorbance value was measured at a wavelength of 450 nm using an enzyme-labeled spectrophotometer.
Identification of monoclonal antibodies
HUSSLCs were taken and inoculated into a common 6-well plate; after cell fusion reached 70%, it was rinsed twice with PBS and fixed by 4% paraformaldehyde at room temperature for 10 minutes; rinsed 3 times for 3 minutes each time, and hybridoma cell culture supernatant was incubated at 37 °C for 1 hour; rinsed with PBS 3 times, 3 minutes each time, with goat anti-mouse-cy7-fluorescent secondary antibody (1:500 dilution) incubated at 37 °C for 1 hour; rinsed with PBS 3 times, 3 minutes each time, with DAPI (1:100, diluted with PBS) incubated and kept out of light at room temperature for 10 minutes, rinsed 3 times with PBS, 3 minutes each time; observed under microscope.
Effect of monoclonal antibodies secreted by positive hybridoma cell lines on spheroid formation of HUSSLCs
SP-2/0 cell supernatant and positive hybridoma cell supernatants with varied dilution ratios (1:1000, 1:500, 1:100) were used to act on HUSSLCs for 48 hours. The spheroid formation was performed as the method mentioned in the literature . After 6 days of culture, non-adherent, clonal-growth tumor spheres were obtained, with a diameter ≥ 50.0 μm defined as tumor spheres. The number of tumor spheres in each well was counted. Spheroid formation rate = average number of tumor spheres per well / total number of viable cells inoculated × 100%.
Effect of monoclonal antibodies secreted by positive hybridoma cell strains on the formation of HUSSLCs agar colonies
SP-2/0 cell supernatant and positive hybridoma cell supernatants with varied dilution ratios (1:1000, 1:500, 1:100) were respectively chosen to act on HUSSLCs for 48 hours. DMEM medium with 20% FBS was proportionally mixed with 1.2% low melting point agarose fluid. The mixture was transferred to a 24-well pate, 0.5ml per well, and was used as bottom agar by the time the mixture was solidified. DMEM medium with 20% FBS was proportionally mixed with 0.6% low melting point agarose fluid. The mixture was transferred to a 24-well pate, and 1000 HUSSLCs cells were added to each well for thorough mixture, which was then used as the top agar. After the top agarose fluid was solidified, it was transferred to an incubator for 14 days, and 500μl of DMEM medium with 20% FBS was added every 5 days. The number of cells ≥ 20 was defined as a colony, and the number of colonies was counted using a fluorescent inverted microscope. Colony formation rate = average number of colonies per well / total number of viable cells inoculated × 100%.
Effect of monoclonal antibodies secreted by positive hybridoma cell strains on migration of uterine sarcoma stem cell-like cells
SP-2/0 cell supernatant and positive hybridoma cell supernatants with varied dilution ratios (1:1000, 1:500, 1:100) were respectively chosen to act on HUSSLCs for 48 hours. The treated HUSSLCs cells were inoculated onto a 6-well cell culture plate and diluted to 5 × 105 cells/well with DMEM complete medium containing 10% fetal bovine serum, photographed when the cell fusion reached 90%. The tip head was used to scratch the bottom center of the 6-well plate. PBS was then adopted to rinse and remove the debris and floating cells for 2 times. The cells continued to be cultured after the scratches were made, photographed at the same wound site 24 hours later to count cells in the wound area. The cells treated with SP-2/0 cell supernatant were set as the control group, and the relative cell migration rate was calculated.
Effect of monoclonal antibodies secreted by positive hybridoma cell strains on the expression of HUSSLCs CD133
SP-2/0 cell supernatant and positive hybridoma cell supernatants with varied dilution ratios (1:1000, 1:500, 1:100) were respectively chosen to act on HUSSLCs for 48 hours, which was inoculated onto William's E medium (containing 20% FBS) at 105 cells/ml and incubated for 15-30 minutes at room temperature to block non-specific sites. The cells were rinsed twice with PBS and resuspended in 990 μl of PBS. Then, 10 μl of antibody (including PE-CD133 and isotype control PE-IgG2b) was added to cell suspension. After incubating for 30 minutes at 4 ° C away from the light, the cells were rinsed twice with PBS, fixed with 0.1% formaldehyde and detected by FACS CaliburTM system. All data was analyzed using Flow jo7.6.1 software.
Effects of monoclonal antibodies secreted by positive hybridoma cell strains on the expression of HUSSLCs CD44, ABCG2, Bmi1, Nanog, Oct4 and ALDH1 proteins
SP-2/0 cell supernatant and positive hybridoma cell supernatants with varied dilution ratios (1:1000, 1:500, 1:100) were respectively chosen to act on HUSSLCs for 48 hours. Whole-cell extracts were prepared. Bradford assay was applied to detect the protein content in cell lysates (supernatants). 40 μg of the extracted protein was taken for electrophoretic separation using SDS-polyacrylamide gel, and transferred to polyvinylidene difluoride membrane. The membrane was sealed with 5% bovine serum albumin for 2 hours at room temperature. The membranes were incubated overnight at 4 °C with respective antibodies as primary antibodies. The polyvinylidene difluoride membrane was washed with 1X Tris and incubated along with horse radish peroxidase secondary antibody for 2 hours at room temperature. Polyvinylidene difluoride membrane was washed with 1X TBS, and the protein expression was detected using enhanced chemilum inescence. The ratio between CD44, ABCG2, Bmi1, Nanog, Oct4, ALDH1 and β-actin protein band grayscales was respectively analyzed and calculated by image analysis software. The ratio between CD44, ABCG2, Bmi1, Nanog, Oct4, ALDH1 and β-actin protein band grayscales after HUSSLCs has been treated with SP-2/0 medium supernatant was defined as 1.00, which was standardized to relative density. The above experiment was repeated 3 times, and the data of 3 independent experiments were expressed as mean ± standard deviation (n = 3).
Experimental data were expressed as mean ± standard deviation (Mean ± SD), with all performed using SPSS 18.0 software. LSD was adopted for comparison between homoscedasticity mean values, and Tukey's test for homoscedasticity mean values in multiple sets, with P < 0.05 suggesting statistical difference.