| BACKGROUNDThe mortality of ovarian cancer is always the highest in gynecological malignancy. The main cause lies in the broadcasting of cancer cells in peritoneal cavity. Now, investigations about the mechanism of adhesion between ovarian cancer cell and peritoneal tissue are less. The main reason is that we have no appropriate metastatic cancer cell line and appropriate model of cancer cells adhesion to peritoneal tissue. Metastatic cancer cell line is such cell line that possesses authentic metastatic potential. Although commonly established tumor cell line has some metastatic potential, but the degree of this potential and the metastatic tendency are both ambiguous. As early as 1977, Fidler and Kripke have brought forward experimental testimony and theory about the heterogeneity of tumor cell colony. According to this theory, we can isolate cancer cell subclones from their mother line,which have completely identical heredity background. These cancer cell subclones with least heterogeneity can be valuable for further study on the molecular mechanisms of cancer metastasis and cloning of cancer metastasis related genes. The investigation made by Marding pointed out that although human serous ovarian cancer cell line SKOV3 was the lower metastatic cell line, but it had some subclones with higher metastatic potential. According to this characteristic, we isolated some subclones with different metastatic potential from the mother line SKOV3, and identified their invasiveness and migratory capacity. Concerning the model of cancer cells adhesion to peritoneal tissue, foreigners use outside cultured mesothelium cells as adhesion model, but this model loses natural configuration such as basement membrane and extra-cellular matrix under mesothelium cells. In our country, manual basement membrane (metrigel), laminin, fibronectin etc are often used, but the most important mesothelium cells are scarce. Peritoneal tissue of mice or rat is also used in these peritoneum models, but it's no doubt that human peritoneal tissue is optimal. Now, the model of endometrial fragments adhesion on peritoneum has been established, but the model of ovarian cancer cells adhesion on peritoneum has not been founded.OBJECTIVE1. To isolate cancer cell subclones with different metastatic potential from human ovarian cancer cell line.2. To identify the invasiveness and migratory capacity of these subclones with different metastatic potential using outside and inside experiments.3. To establish an in vitro model of cancer cells adhesion to peritoneal tissue and testify the roles of adhesive molecules such as betal-integrin in this process.METHODS AND RESULTS1. To isolate cancer cell subclones with different metastatic potential:Chose SKOV3 cells in logarithmic growth, made into single cells insuspension, counted the live cell numbers, finitely diluted into ten cells per milliliter, then planted 100μl per hole this liquid into culture board of 96 holes, added culture medium to 200μl per hole, incubated in 37℃ and 5% CO2. Cultured for two or three weeks, chose the hole where subclone grew perfectly, gradually transferred into culture board of 24 holes and 6 holes, finally into culture bottle. After these processes, we got 14 cell subclones, named S1-S14.Chose these cells of the subclones mentioned above which were in logarithmic growth, suspended in l%sucrose solution, blew into single cells in suspension, counted the live cell numbers, adjusted cell's density to 1 × 106 per milliliter, added in cell electrophoretic apparatus, selected 12 or 16 cells per individual subclone, recorded the time they needed during swimming 250 μm in electric field obversely and adversely. According to the formula calculated cell's electrophoretic velocity. According to the result, we can divided these 14 cell subclones into three groups, the first group with high metastatic potential including S1-S3, the second group with mean metastatic potential including S4~S8, and the last group with low metastatic potential including S12-S14.Chose cells of the three groups mentioned above which were in logarithmic growth, made into single cells in suspension, adjusted cell's density to 1×105 per milliliter, tested cell growth cycle by flow cytometer five times apiece, selected the subclones, which rate of G2 or M period in cell cycle are discrepant widely. Finally, we could get three subclones with different metastatic potential, including S1 with highest metastatic potential, S6 with mean metastatic potential, and S14 with lowest metastatic potential.2. To identify the invasiveness and migratory capacity of these three subclones:These three subclones were made into single cells in suspension, joined them respectively into 24 holes of culture plates, there were about 1×104cells in every hole. Next day, chose three holes from every culture plate, digested by enzyme, counted the live cell numbers, then recorded these figures. This course continued for seven days. According to these figures recorded we can draw a chart in vito growth, at the same time by the formula, we can count multiple incremental time of cells. S1 was 20.55 hours, S6 was 28.99 hours, and S14 was 38.48 hours. S1 subclone grew significantly faster than the other two subclones.In the proportion of 1:1, mixed the 1.2% gelose liquor and 2×DMEM (Dulbecco's Modified Eagle Medium) including 20% calf serum, and joined 3 ml this mixed solution into flat vitreous utensil which diameter was 3.5cm, then cooled to concreting. In the same way, mixed the 0.7% gelose liquor and 2 × DMEM in the proportion of 1:1 and joined 0.2 ml cells' suspension including 600 cells into this mixture, then intermixed sufficiently. Joined 3ml this intermixed liquor into the flat utensil where the 1.2% gelose mixture had concreted. Afterwards cultivated in 37℃ and 5%CO2 for 10-14 days, took count of the numbers of the cloning community. The number of S1 was 33.80 ±3.96, S6 was 18.80 ± 5.26, and S14 was 8.00 ± 1.58. There were significantly differences among these three subclones by T test testifying.Refer to Matrigel invasion assay made by Albini etc, in the lower chamber of Boyden chamber, joined 600μl chemotactic liquid. Between the lower and the upper chamber there was a polycarbonate filter, polyvinylpropylene (PVP) free, containing pores of 8μm size. Matrigel glue was diluted by 4℃ RPMI1640, used 50 μg to spread the filter in every Boyden chamber. Joined 400 μl cells' suspension including 2×105 cells into the upper chamber, then cultivated in 37℃ and 5%CO2 for 24 hours. We fixed the filter membrane in formaldehyde for 30 minute, HE-dyed conventionally and counted the penetrated cell under the light microscope of 200 diameters. The penetrated cell number of S1 was 133.80±15.80, S6 was 75.80 ± 27.15, S14 was 31.20±13.77. The invasiveness and penetration ability of S1 washigher than S6 and S14.The method of moving test was similar to invasion assay. The difference was that there was no matrigel glue on the polycarbonate filter. Joined the same cells' suspension in the upper chamber, then cultivated for 12 hours, HE-dyed conventionally and counted ditto. The penetrated cell number of S1 was 450.80±27.99, S6 was 252.20 ± 22.24, S14 was 134.00 ± 14.47. The outer moving ability of S1 was higher than S6 and S14.We used 30 female BALB/c nu/nu nude mice, which were divided into 3 groups. Every group had 10 mice. Subcutaneously injected 0.3ml (including 2.5 × 106 cells) into the back of nude mice apiece. After 6 weeks, executed and anatomized all nude mice. The tumor forming rate of S1 and S6 was 100%, however S14 was only 20%. The tumor's volume of S1 and S6 was 6.42 ± 2.04cm3 and 2.51 ± 1.03cm3 separately, whereas S14 was only 0.52 ± 0.13cm3. The tumor of S1 was multi-knots nodular, its peripheral membrane was incomplete and the tumor connected closely to the peripheral tissue, uneasy to separate. The tumor of S14 was small, lubricous and oval, its peripheral membrane was complete and easy to separate. The group inoculated with S14 subclone cells had no lung metastasis, however the lung metastasis rate of S1 was 70%, S6 was 30%. S1 has higher metastatic ability; however S14 has no metastatic ability.S1 and S14 subclones propagated in sequence for thirty generations, and then we carried out these experiments mentioned above respectively. In vito growth, the multiple incremental time of S1 was 34.00 ± 6.46, S14 was 9.27 ± 3.79, S1 grew significantly faster than S14. In clone forming test using soft agar culture medium, the forming clone number of S1 was 34.00 ± 6.46, S14 was 9.27 ± 3.79, There were significantly differences between them by T test testifying. In matrigel invasion assay, the penetrated cell number of S1 was 141.68 ±7.36, S14 was 35.86±6.57, in moving test, the penetrated cell number of S1 was 456.07±7.05, S14 was 135.86 ± 8.72. In conclusion, theoutside invasiveness and moving ability of S1 was distinctly higher than S14. In spontaneous metastasis assay in nude mice, the tumor's volume of S1 was 7.09 ± 0.78cm3, S14 was 0.36 ± 0.92cm3, the lung metastasis rate of S1 was 60%, however S14 had no lung metastasis. These results above all approved that cell subclones were still stabile in property after propagating in sequence for thirty generations.3. Mechanism of adhesion between ovarian cancer cells and peritoneal tissueTook normal peritoneum during abdomen surgery, sheared into some bits with 1-1.5 cm diameter, conserved in culture medium of 4℃. In the lower chamber of homemade Boyden chamber, joined 0.6ml culture medium, placed peritoneum bit between the lower and the upper chambers. Joined 0.4ml cells' suspension of S1 or S14 including 2× 105 cells into the upper chamber, then cultivated in 37℃ and 5%CO2 for 24 hours. Took peritoneum bits out, fixed them in glutaraldehyde, and then observed under scanning electron microscope conventionally. Mesothelial cells of peritoneum arranged like cobblestone besetting one by one, cancer cells adhered tightly to mesothelial cells and held out many pseudopods. The adhesive cells to the peritoneum of S1 was 36.40 ± 4.35, S14 was 15.20 ± 4.39, this experiment showed that the adhesion ability of S1 was higher than S14 in evidence.Chose cells of S1 and S14 subclones in logarithmic growth, digested and made into cells' suspension, adjusted cell's density to 5×106per milliliter, joined 100μl cells' suspension into sample pipe where were beforehand placed 2μg monoclonal antibodies against the betal-integrin, acted for 40 minutes, negative pipe for comparison had no antibody, centrifugated and washed by PBS, in every sample pipe, joined Sheep(or Goat)anti-Rabbit IgG-FITC which was diluted in 1:100 rates, incubated in dark for 45 minutes, centrifugated and washed by PBS, 1ml PBS liquid suspended cells again, analyzed the results by flow cytometer. The expression rate of betal-integrin on the cellmembrane surface of S1 and S14 was both 100%, but their expression intensity was different, S1 was 25.9±1.61, however S14 was 13.42 ± 2.54. In conclusion, the expression intensity of betal-integrin in SI was stronger than in S14 significantly.Joined 400μl cells' suspension of S1 into sample pipe where were beforehand placed 2μg monoclonal antibodies against the betal-integrin, acted for 45 minutes, negative pipe for comparison had no antibody, and joined cells' suspension mentioned above in the upper chamber of Boyden chamber which had been placed peritoneum bit, then cultivated in 37℃ and 5%CO2 for 36 hours. Took peritoneum bits out, fixed them in glutaraldehyde, and then observed under scanning electron microscope conventionally. The cancer cells held out more pseudopods, like wool spherical shape, some connected with the sugar overclothes on the surface of mesothelial cells. In the experimental group that contained antibody the adhesive cells to the peritoneum were lower than cells in the comparative group that had no antibody. The adhesive cells in experimental group were 16.00 ± 4.64, however in comparative group were 37.80 ± 4.59. In conclusion, the antibody against betal-integrin could partially inhibit the adhesion process of ovarian cancer cells to peritoneum.CONCLUSIONS1. The establishment of cancer cell subclones is significant to the study onthe molecular mechanisms of cancer metastasis.As early as1977, Fidler and Kripke had brought forward experimental testimony and theory about the heterogeneity of tumor cell colony. According to this theory, using single cell clone technique we can isolate cancer cell subclones from their mother line, which have homogeneous character and different metastatic potential. Gaojin has pointed out that there is a positive connection between the electrophoretic velocity and metastatic capacity in various tumor cell lines. Subsequently many people make cell'selectrophoretic velocity as the preliminary filtrating criterion, establish cell subclones that originate from the same mother line and have differential metastatic potential. Our experiments use finite diluting method to isolate human ovarian cancer cell line-SK0V3 into single cell colony, and utilize the relationship of cells' electrophoretic velocity and tumor metastatic potential to filtrate these cell subclones primarily, then check up the ratio of G2 or M period in cell growth cycle, finally get three subclones with differential metastatic potential from the same mother line. These cancer cell subclones have completely identical heredity background and least heterogeneity can be valuable for further study on the molecular mechanisms of cancer metastasis and cloning of cancer metastasis related genes.2. The research about the invasiveness and migratory capacity of these subclones validates that S1 subclone has the highest metastatic potential.To study the invasiveness of tumor cells, we need establish an appropriate model and use corresponding method. Basement membrane is the first barrier to avoid the invasiveness and metastasis of tumor cells. Studying the process of tumor cells drilling through basement membrane is an important aspect in exploration the mechanism of tumor invasiveness and metastasis. Boyden chamber is often used outside invasive model in metastatic research. The key of this test is the matrigel glue rich of laminin, collagen IV, fibronectin, et al. Matrigel glue coats the filter with 8 microns size pores to rebuild the human basement membrane. This test ideally simulates tumor cells' invasive process. At the same time we establish a metastatic model inside nude mice by subcutaneously transplanting cancer cells into the back of nude mice that lack immunity system. Via the establishment of the both invasive models, plus vitro growth reflecting cell's basic growth rules and soft agar cloning test reflecting dependent growth ability of tumor cells, we have validated S1 subclone has higher metastatic ability, however S14 subclone has no metastatic ability.3. The study on mechanism of adhesion between ovarian cancer cells and peritoneum approves that S1 subclone has higher adhesion ability than S14 subclone.We need appropriate outside peritoneum model to research the mechanism of adhesion between ovarian cancer cells and peritoneum. Some authors use outside cultivated mesothelium cells as adhesion model, but this model loses natural configuration such as basement membrane and extra-cellular matrix under mesothelium cells, moreover such culture's technique is hardly mastered. Others use culture board coated with rat peritoneum as adhesion model. These models cannot preferably simulate the inside condition and is disadvantageous to observe the treating factors. However in our experiment the outside peritoneum adhesion model we established has following excellences: one is that human peritoneum has normal configuration, this model preferably simulates the adhesion process of tumor cells in body. The other is that this adhesion model is open, we can directly observe the influencing factor. Through these tests we find that the adhesion capacity of S1 subclone is higher than S14 subclone. Antibody against betal-integrin can partly inhibit the adhesion between cancer cells to peritoneum. In conclution, betal-integrin must operate during the course of ovarian cancer cells broadcasting in abdomen.
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