Establishment Of Cloning Cell Strains Of Murine Ascites Hepatocarcinoma With Different Lymphatic Metastatic Potential

Objective: Invasiveness and metastasis are one of the most important basicbiological characteristics of cancer, and also a major cause of death in cancer patients.However, the clear mechanisms of cancer have been unknown. Therefore, scientistshave been focusing on the research of the metastasis of cancer. To establish ideal cellmodels of tumor metastasis is of great significance to clearly study the mechanismsunderlying tumor lymphatic metastasis. Murine ascites hepatocarcinoma cell lineH₂₂ belongs to the mouse liver carcinoma which can be subcutaneously inoculatedinto mice and grow into a spherical nodule form. The tumor cells proliferates insuspension when inoculated into mouse abdominal cavity. To establish experimentalmetastatic cell model of H₂₂ is very important to investigate H₂₂ itself and toscreen medicine to inhibit cancer metastasis. Two cloning cell strains HCa-F andHCa-P with different lymphatic metastatic potentials were separated from a murineascites hepatocarcinoma cell line, HCa-F is with high lymphatic metastatic potentialwith70%～80%lymphatic metastasis rate, and HCa-P is with low lymphaticmetastatic potential with0～20%lymphatic metastasis rate. The two cell strains hadbeen widely used in studying the mechanism underlying tumor lymphatic metastasisand screening drugs. Experimental lymphatic metastatic cell model H22is moreapplicable in accordance with the natural law of tumor metastasis in animal modelsand has been widely applied in studying the mechanism underlying tumor lymphaticmetastasis and screening drugs.To research the mechanism underlying tumor lymphatic metastasis, whether inhistopathologically tissue level, biologically cell level, or molecular level, it isnecessary to develop a good reference system of tumor metastatic modles. Theshortcoming of this model is lack of the control of non-metastatic cell strain, so it is an urgent work to develop a novel series of mouse heptocellular carcinoma cell strainswith increasing lymphatic potential on the base of a mouse ascites heptocellularcarcinoma cell modle with different lymphatic metastatic potenchial.In this paper, murine ascites hepatocarcinoma cell strain with low lymphaticmetastatic potential （HCa-P） was separated by limiting dilution analysis and the rateof lymphatic metastatic potential was detected by mouse foot pad inoculationexperiments. Then, the biological characteristics of cloning cell strains was observedby means of morphology, cell growth curve, population doubling time, secretion ofmatrix metalloproteinase2and9（MMP-2, MMP-9）, cell migration, and invasion. Thepurpose is to establish a novel series of cloning cell strains of murine asciteshepatocarcinoma with different lymphatic metastatic potential and set up a platformfor a novel series of mouse heptocellular carcinoma cell strains with increasinglymphatic potential on the base of a mouse ascites heptocellular carcinoma cell modlewith different lymphatic metastatic potenchial.Methods: Murine ascites hepatocarcinoma cell strain with low lymphaticmetastatic potential （HCa-P） was separated by limiting dilution analysis and a seriesof single cells were selected and marked in96-well plates. Clone cells were culturedin96-well plates and named according to the location of the cells in96well plates,and then were in expanding culture in vitro. When confluencing in96-well plates,clone cells were transferred them24-well plates, and then into culture bottles inexpanding culture. In order to detect the rate of transplanted tumor formation andlymphatic metastasis in animals, each cell clone strain was subcutaneously inoculatedinto the mouse615foot pad. A novel series of clone cell strains of murine asciteshepatocarcinoma with different lymphatic metastatic potential were developed.Detection of biological characteristics of clone cell strains: Detection ofbiological characteristics of clone cell strains and their ancestral cell line HCa-P wereanalyzed by use of morphology, cell growth curve, population doubling time,secretion of matrix metalloproteinase2and9（MMP-2, MMP-9）, cell migration, andinvasion. Cell morphology was observed under an inverted microscope; cellproliferation was observed by making growth curve and population doubling times.The expression level of matrix metalloproteinase2（MMP-2） from each clone cellstrain was determined by western blot; the secretion amount of matrixmetalloproteinase2and9（MMP-2, MMP-9） was observed by the use of gelatinzymography assay. Invasion and migration of cell strains were tested by the use of Boyden Chamber.Results: Four clone cell strains of murine ascites hepatocarcinoma withdifferent lymphatic metastatic potential were separated from HCa-P with lowlymphatic potehtial, and named as G2, H5, B8, and E10. After cells weresubcutaneously inoculated into the mouse615foot pad for28days，the lymph nodemetastasis rate from transplanted tumor of every clone cells respectively wasG2-33.3%, H5-0, B8-13.3%, E10-20%, and Hca-P-26.7%. The metastatic rate ofG2was higher than that of its ancestral cells HCa-P. No lymphatic metastasis wasfound in H5. There was significant difference between H5and G2, as well as HCa-P,in lymphatic metastatic rate （x2test, P <0.05）. The metastatic rates of B8and E10were not different from that of their ancestral cells HCa-P.There are differences in some biological behavior among clone cell strains.Cell morphology analysis showed that the G2cell strain had a bit bigger number ofbig cells significantly than other strains and H5had the smallest mummer of big cells.As cell population doubling time, G2was5.76±0.86h, H5was18.18±1.43h, andG2was significant different from H5（t test, P <0.05）. Western Blot analysis showedthat the expression levels of matrix metalloproteinase2（MMP-2） secretion of G2cells were significantly different from H5cells （t test, P <0.05）. Transwell chambersdetection demonstrated that migration number of cells, breaking through matrixmembrane, of the cell of G2was178.6±3.5in24hours, and that of H5was23.6±1.5, and the difference was statistically significant （t test, P <0.05）. Invasion propertyof cancer cells was detected by Transwell chambers, and the results showed that therewas significant difference of the number of cells break throughing cell stromalmembrane from G2with138.6±7.6invasion cells and H518.6±3.2in24hours.Thedifference was statistically significant （t test, P <0.05）.Conclusion: Four clone cell strains of murine ascites hepatocarcinoma withdifferent lymphatic metastatic potential were separated from HCa-P with lowlymphatic potehtial, and named as G2, H5, B8, and E10. G2, B8, and E10were withlow lymphatic potehtial while H5was non metastatic clone cell strain. Among G2, B8,and E10, the rate of metastasis of G2is the highest at33.3%. The metastatic rates ofB8and E10were not different from that of their ancestral cells HCa-P.There were no significant differences between B8with E10and their ancestralcells HCa-P in some biological behaviors. There were significant differences betweenG2and H5in morphology, cell growth curve, population doubling time, expression and amount of secretion of matrix metalloproteinase2and9（MMP-2, MMP-9）, cellmigration, and invasion.