Study on Functional Monoclonal Antibodies of Anti-Human Uterine Sarcoma Stem Cell-Like CellsStudy on Functional Monoclonal Antibodies of Anti-Human Uterine Sarcoma Stem Cell-Like Cells

Background: To establish a functional monoclonal antibody library using Human Uterine Sarcoma Stem Cell-Like Cells (HUSSLCs) to screen and identify functional monoclonal antibodies that can recognize and inhibit HUSSLCs. Methods: B lymphocytes in proliferative state were prepared by using the second generation CD133+spheroid cells of SK-UT-1 cell line, i.e. HUSSLCs, as antigens; Spheroid formation, agar colony formation, wound healing, flow cytometry, and Western blotting were adopted to detect the effect of monoclonal antibodies with varied dilution ratios on HUSSLCs spheroid formation, agar colony formation, cell migration, CD133 expression, and expression of CD44, ABCG2, Bmi1, Nanog, Oct4 and ALDH1. Results: Myeloma cells of SP2/0 cell line can achieve 85% degrees of fusion and results of 1-2F monoclonal cell supernatants with different dilution ratios reduced HUSSLCs spheroid formation rate, agar colony formation rate, cell migration rate, CD133 positive cell expression and protein expression levels of CD44, ABCG2, Bmi1, Nanog, Oct4, and ALDH1 in concentration-dependent manner (P <0.05). Conclusion: The antibody valence produced by HUSSLCs-immunized mice reached the requirement for preparation of monoclonal antibody. Anti-HUSSLCs monoclonal antibodies feature functions of inhibiting the self-renewal, unrestricted proliferation, migration, invasion and multidrug resistance of HUSSLCs and functions characterized by tumor stem cells. anchorage-independent and tolerance of of agar colony formation rate assay the supernatants of 1-2F monoclonal cells with different dilution ratios reduced the rate of HUSSLCs agar colony formation in concentration-dependent manner, that 1-2F monoclonal antibodies inhibit anchorage-independent growth of HUSSLCs and has effect. vitro migration, 13, 16-21 cells various tissues including sarcoma and is associated with poor in patients with various types of tumors 22-28 . Analysis results of PE-labeled CD133 antibody flow cytometry revealed that the supernatant of 1-2F monoclonal cells with different dilution ratios reduced the percentage of CD133-positive cells of HUSSLCs in concentration-dependent manner, indicating that the supernatant of 1-2F monoclonal cell culture medium can inhibit the expression of CD133 protein of HUSSLCs tumor stem cell marker or can effectively reduce the number of tumor stem cells. Ample studies have shown that 29-34 CD44 overexpression can facilitate and maintain TSC self-renewal, migration and invasion, serving as a marker protein for a variety of TSC; ABCG2 overexpression contributes to and maintains multidrug resistance of tumor stem cells 35-38 ; Abnormal expression of Bmi1 contributes to and maintains the self-renewal, unrestricted proliferation and multidrug resistance properties 39-42 of tumor stem cells; Nanog abnormal expression and functional effect of self-renewal and unrestricted proliferation 43-46 of tumor stem cells; Oct4 abnormal expression is associated with self-renewal and unrestricted proliferative function of tumor stem cells, as well as epithelial-mesenchymal transition phenotype 47-51 ; ALDH1 abnormal expression enhances the cytotoxic effects of tolerant drugs of stem cells and tumor stem cells, as well as other compounds, contributing to and maintaining multidrug resistance of tumor stem cells and cell anoikis resistance 52-55 . Western blot analysis showed that the supernatants of 1-2F monoclonal cells with different dilution ratios reduced the expression levels of CD44, ABCG2, Bmi1, Nanog, Oct4 and ALDH1 proteins of HUSSLCs in concentration-dependent manner (P < 0.05). These results indicate that 1-2F monoclonal antibodies can inhibit the expression of the above various tumor stem cell markers of HUSSLCs, or can effectively inhibit the self-renewal, unrestricted proliferation, migration, invasion, and multidrug

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.

Clone screening
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 6well plate respectively, which were eventually transferred to a 25 cm 2 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 10 6 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 antimouse 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, 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%. The treated HUSSLCs cells were inoculated onto a 6-well cell culture plate and diluted to 5 × 10 5 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 10 5 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 Calibur TM system. All data was analyzed using Flow jo7.6.1 software. 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 βactin 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).

Statistical analysis
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. Figure 1A showed that the antibody level in mice had lower valence after the first immunization, suggesting no significant difference from the negative control; after the second and third impact immunization, the antibody valence increased significantly (P <0.05); after the third immunization, the anti-HUSSLCs antibody valence can reach 1.0 or more in a million-fold diluted immune serum. The myeloma SP2/0 cell line cells grew semiadherently in complete 1640 medium of 10% fetal bovine serum (Fig. 1B). The cells were round, morphological integral and neatly arranged. After 3 days of culture, the degree of fusion could achieve 85%. After 8 days of screening through liquid culture medium, the fused hybridoma cells grew into clusters to form small cell colonies, with the cells mixed and translucent, proliferating in fast pace (see Fig. 1C). Positive hybridoma cells were detected by ELISA, and 6 positive wells were found. The culture well numbers were named 4-2, 9-15, 10-2, 10-4, 10-11 and 10-12, respectively. After 4 times cloning of positive wells by method of limiting dilution, it was found that 6 hybridoma cell strains could stably secrete anti-HUSSLCs monoclonal antibodies, named 1-2F, 1-2G, 1-3F, 1-9B, 1-3G, 3-3I respectively and frozen in conventional liquid nitrogen. As shown in Fig. 1D, 1-2F, 3-3I, 1-2G, 1-3G, 1-9B were IgG2a, and 1-3F was IgG2b. The results of cell immunofluorescence assay indicated that red fluorescence in varying degrees were displayed in HUSSLCs incubated by monoclonal cell supernatants of 1-2F, 1-3F, 1-2G, 1-3G, 3-3I and 1-9B, among which 1-2F monoclonal cell supernatants incubated the highest number of positive cells labeled red fluorescence, followed by 1-3G monoclonal cell supernatants, reaching more than 50% (Fig. 1E).

Discussion
Since the successful isolation of Tumor Stem Cells (TSCs) from human acute myeloid leukemia in 1997 by Bonnet et al. 12 , TSCs were isolated from a variety of malignant tumors such as breast cancer, pancreatic cancer, prostate cancer, and liver cancer.
Through a series of studies, it is found that TSCs are closely related to drug resistance, metastasis and recurrence of malignant tumors. Therefore, TSCs targeted treatment is expected to become a new and effective approach for malignant tumors.
The spheroid formation rate was determined by referring to the methods and procedures of literature 4, 9-11, 13 . The results revealed that the supernatants of 1-2F monoclonal cells with different dilution ratios reduced the rate of HUSSLCs spheroid formation in concentration-dependent manner, indicating that 1-2F monoclonal antibody has the effect of inhibiting self-renewal of HUSSLCs. Agar colony formation is commonly used to detect anchorage-independent growth and anoikis tolerance of tumor stem cells 14

Conclusion
In conclusion, our study demonstrates that antibody valence produced by HUSSLCs-