The Impact Of Blys Antibody On KM3 Tumor Xenografted In Nude Mice

Objective:Multiple myeloma (multiple myeloma, MM) is a malignancy whose plasma cells are unlimitedly proliferative, and it belongs to terminal stages of B cell differentiation malignant tumor. At present, the incidence rate of multiple myeloma in our country is about one hundred thousandth to two hundred thousandths which is the second highest in all hematologic malignancies, more than acute leukemia. In recent years, there are some methods of treating multiple myeloma,for example, stem cell transplantation, combination therapy of velcade and bortezomib, the traditional combination therapy programs,however,multiple myeloma is still a incurable malignant blood disease.With its increasing relapse, the treatment is more difficult, to obtain the mitigation is more difficult, and the time of mitigation is shorter. Therefore,it is so important to look for more therapeutic targets.B-cell malignancy is caused by uncontrolled lymphocyte proliferation and can occur at any time of B cell's development. B lymphocyte stimulator ( BlyS) is a new member of the tumor necrosis factor (TNF) superfamily foundde in 1999, it through three receptors plays an important role in the humoral immune response: B cell maturation protein (BCMA), transmembrane protein activator (TACI) and cell-activating factor receptor (BAFF-R) with the specific binding of B lymphocytes and induce proliferation, differentiation and secretion of IgG, IgA and IgM and other immunoglobulins.Blys is the key B-cell growth factor and plays an important role in the pathogenesis of tumor. Our preliminary study showed: BLyS was highly expressed in the peripheral blood of patients with multiple myeloma ,and then decreased gradually with the improvement of their condition, which suggested that the pathogenesis of multiple myeloma was related to BLyS. Therefore, inhibiting Blys may be used as anti-malignant B-cell tumors. In this study, we select KM3 cell from MM cell line as target cell to study the impact of Blys antibody on KM3 tumor xenografted in nude mice in vitro and in vivo,and provide a theoretical basis and some experimental data for the clinical treatment of B cell tumor.Method:1 experiment in vitro1.1 Cell culture and passage KM3 cells from Human MM cell line were cultured in RPMI1640 medium with 10% fetal bovine serum, 100U/ml penicillin and 100U/ml streptomyc at 37℃, 5% CO2, 95% humidity incubator,and passaged every 2 ~ 3 days . cells in logarithmic growth phase would be selected for the experiment.1.2 MTT colorimetric was used to test the impact of different concentrations Blys antibody on KM3 cells'viabilitywe cultured KM3 cells from Human MM cell line in vitro and tested the impact of Blys antibody in different concentrations (25μg/ml, 30μg/ml, 35μg/ml)on the KM3 cell proliferation at different time(24h,48h,72h)by tetrazolium blue (tetrzolium-based colorimetric assay, MTT) colorimetric assay (The dose of each group was determined according to specifications of Anti-human BLyS Antibody freeze-dried powder).1.3 Flow cytometry was used to test the impact of Blys antibody in different concentrations on apoptosis of KM3 cells.We added Blys antibody in different concentrations (25μg/ml, 30μg/ml, 35μg/ml) to KM3 cells and tested its impact on apoptosis of KM3 cells by flow cytometry 48h later.2. Experiment in vivo2.1 KM3 tumor-bearing nude mice modelKM3 cells in the logarithmic phase were washed two times with serum-free RPMI1640 medium, and re-suspended in mixed liquor of serum-free RPMI1640 medium and Matrigel at the ratio of 1:1,then cell density was adjusted to 1×108/ml. Each of 12 nude mices was inoculated subcutaneously 0.2ml(1×107/ml) cells into the right forelimb lateral. 2.2 The rate of tumor formation in nude micesWe checked each group 15 days after inoculation of tumor cells if there were visible tumors in the outer skin of right forelimbs of nude mices. the rate of tumor formation= the number of nude mices with tumor formation / the number of all nude mices×100%.2.3 Experimental GroupExperimental animals were randomly divided into 3 groups in 16 days after inoculation of tumor cells (n = 3): a high-dose group: intraperitoneal injection of Blys antibody dilution, 0.2ml (including Blys antibody 35μg), 1 / day, d1-5. b. low-dose group: intraperitoneal injection of Blys antibody dilution, 0.2ml (including Blys antibody 25μg), 1 / day, d1-5. c. Control group: intraperitoneal injection of saline, 0.2ml, 1 / day, d1-5. (The dose of each group was determined according to vitro experiment).2.4 Treatment Effects(1) We observed the general condition of experimental animals: including mental state, skin, activity, diet, drinking , and weight;(2) We measured long and short diameter of the tumors every 2d,and calculated their volume and tumor growth inhibition rate (IR) according to these formulas: V = ab2 / 2 (a: long diameter, b:short diameter), IR = (V0-Vn) / V0×100% (V0 : tumor volume of the non-treatment group, Vn : tumor volume of the treatment group);Results:1. In vitro cell proliferation assay (MTT) showed that: Blys antibody inhibited multiple myeloma cell line KM3 proliferation in a time-dose-dependent manner in a certain concentration range. When Blys antibody concentration were 25μg/ml, 30μg/ml, 35μg/ml, OD value of 24-hour was 1.36±0.09,0.85±0.03,0.42±0.05; OD value of 48-hour was 0.86±0.02,0.69±0.01,0.29±0.01; OD value of 72-hour was 0.55±0.02,0.41±0.02,0.21±0.01. OD values of 24,48,72-hour in control group were: 2.15±0.16,1.09±0.05,0.7±0.04. The results showed that compared with the control group, the inhibiting effect of Blys antibody on the proliferation of multiple myeloma cell line KM3 gradually increased with the increase of concentration of Blys antibody and action time, and the difference was significant (P <0.05 ). Groups of the same concentration of Blys antibody were compared at different time ,and the difference was significant, with statistical significance (P <0.05). Groups of different concentration of Blys antibody were compared at the same time ,and the difference was also significant, with statistical significance (P <0.05). Those indicated that Blys antibody could inhibit the proliferation of human multiple myeloma cell line KM3 cell in a dose - dependent and time - dependent effect.2. Flow cytometry showed that: Flow cytometry showed that: KM3 cells were added Blys antibody and the cells in S phase reduced gradually with the increase of the concentration of Blys antibody 48 hours later. When the concentration of Blys antibody was 0μg/ml, 25μg/ml, 30μg/ml, 35μg/ml, the percentage of S-phase cells was 61.70±1.99%, 55.23±0.58%, 52.83±11.71%, 43.93±2.07%. The difference was significant, with statistical significance (P <0.05).KM3 cells were added Blys antibody and the typical sub-diploid apoptotic peak appeared 48 hours later. Apoptosis rate of KM3 cells gradually increased with the antibody concentration increased. When the concentration of Blys antibody was 0μg/ml, 25μg/ml, 30μg/ml, 35μg/ml, the apoptosis rate of KM3 cells was3.86±0.04%;8.10±0.11%;8.62±0.08%;9.46±0.13%,the difference was significant, with statistical significance (P <0.05).The data indicated that Blys antibody promoted apoptosis of KM3 cells, and the higher the concentration of Blys antibody was, the stronger the effect of promoting apoptosis was.3. The formation rate of Xenograft tumor: after an average of 10 (7-15) days, there were measurable tumor in outside of the right forelimb of nine nude mices, and the formation rate of Xenograft tumor is 75%.4. The general condition of experimental animals: Group a :mental condition, activity, skin, diet ,water intake and body weight of the nude mices were not anomal; Group b : mental condition of the nude mices was normal ,but the activity compared with group a was slightly worse, the amount of water intake slightly reduced; Group c: these nude mices had apathetic spirit, dull skin, lost weight, and the amount of diet and water intake significantly reduced.5. Inhibition of Blys antibody on tumor: The size of tumor in group a had no significant increase in or even shrink. The size of tumor in group b had slightly increased with time .The size of tumor in group c had increased significantly. After being treated for 4 weeks , the inhibition rate of tumor growth in group a and b was 98.88%, 69.31%,and there was statistically significant difference between the treatment group and control group (P <0.05). The tumor proliferation and the inhibition rate of tumor growth showed that Anti-human BLyS Antibody maked multiple myeloma KM3 tumor xenografts in nude mice minificated.Conclusion:1. In a certain range of concentration, Anti-human BLyS Antibody inhibited the proliferation of multiple myeloma cells, KM3, in a time-dependent and concentration-dependent manner.2. Anti-human BLyS Antibody promoted apoptosis of multiple myeloma cell KM3.3. Anti-human BLyS Antibody maked multiple myeloma KM3 tumor xenografts in nude mice minificated.