Establishment Of Multifocal Liver Cancer Model In Mice Via Hydrodynamic Injection

Lots of patients suffering liver cancer present multiple foci in the liver when they consult. Compared with single nodule liver cancer, the prominent features of multifocal liver cancer involves rapid progress, strong invasion, poor prognosis, short survival, recurrence after treatment. Presently, there is no effective treatment for it.In order to explore the growth in nature and development discipline of multifocal liver cancer and search for the antitumor drugs and therapy strategies, the animal model of multifocal liver cancer has been critical tool. The existing methods for liver cancer models development have following drawbacks: low success rate, complicate operation, dangerous peritoneal seeding, and few possibility to get tumor mass with similar biological characteristic at the same time. Therefore, it is very essential to develop a novel animal model.Objective:In this study, multifocal liver cancer model in mice was established via hydrodynamic injection. The aim is to offer a new model choice for studying multifocal liver cancer.Methods:1.6 ml or 200μl of saline containing 2×105 H22 cells were injected into the tail vein of mice by hydrodynamic injection (large volume injection) or small volume injection respectively at speed of 200μl/s. The mice were sacrificed 15 min, 30 min, 1 h, 8 h, 24 h, 3 d, 7 d after injection. The rest mice were kept to monitor the survival period. The liver, lungs, spleen, kidneys and heart were taken from all mice. The tumor growth was observed with naked eyes, under light microscope and electron microscope. The biological behaviors of tumor were evaluated by immunohistochemical staining.Results:1. Survival of mice and changes of gross liver after injection of tumor cells: After large volume injection of tumor cells, the mean survival time of mice was 14.38 d. At 15 min~1 h after injection, the ratios of liver weight and original body weight of mice were significantly higher than mice without any injection. The ratio was highest when the mice died naturally. The dense hemorrhage spots appeared on the surface of the liver at 8 h , reduced significantly at 24 h, were not evident at 3 d and almost disappeared at 7 d after injection. No obvious grey-white nodules were found on the surface of liver at 7 d. The grey-white miliary nodules were found in all of lobes of livers when the mice naturally died. In mice receiving small volume injection, there were no significant changes in the ratio of liver weight and body weight, no nodules were seen when the mice died naturally. The mean survival time was 20.17 d.2. Features of intrahepatic tumor nodules under light microscopy after injection: In mice receiving large volume injection, a few typical tumor cells were seen at 15 min~24 h after injection. Tumor formation rates were 100% at 3 d and 7 d group and natural death group. The multifocal tumor nodules mainly distributed near the surface of the liver. As time goes on, the number of lobes with nodules increased, the number of nodules was more and more, the areas of nodules were larger and larger, the compression to the surrounding liver tissue became more severe. In mice receiving small volume injection, very few typical tumor cells were observed at all time points after injection. No any tumor masses formed until the mice died naturally, but there were numerous tumor cells and exudates attaching to hepatic capsule.3. Ultrastructural changes of liver after tumor cells injection: In mice receiving large volume injection, disrupted sinusoidal endothelium, and the enlarged endothelial gap were observed at 15 min~8 h after injection. There were tumor cells and Kupffer cells in sinusoid. Microvillus sheded from the surface of the hepatocyte. There were abundant vesicles in the cytoplasm near to the hepatic sinusoid. Red cells and lymphocytes and tumor cells presented inside the hepatocytes. The mitochondrias matrix of hepatocytes condensed and endoplasmic reticulum expanded obviously. At 24 h after injection, lymphocytes infiltration was observed. On day 3, the enlargement of endothelia gap became no obvious. The hepatocytes looked normal besides the tumor cells between the hepatocytes. On day 7, group of tumor cells appeared and squeezed the peripheral hepatocytes. In mice receiving small volume injection, no significant differences were seen at different time points in the liver, the hepatocytes were with normal appearance.4. Impact of large volume injection of tumor cells on the liver histological structure: In addition to the above changes of tumor cells, hepatic sinusoid expansion and congestion, muotifocal hemorrhage and necrosis in the liver were observed at 15 min~8 h after injection. Hemorrhage and necrosis were catabatic at 24 h and inflammatory cells infiltration appeared on day 3. The liver tissue far away the tumor nodules recovered to normal on day 7 and when the mice died. No hemorrhage and necrosis were seen in the liver of mice receiving small volume injection.5. Immunohistochemical staining of liver tissue: The labeling index of PCNA and MMP-2 in tumor nodules of natural death group was significant higher than that of 7 d group. But there was no obviously difference of c-myc and VEGF expression between these two groups.6. Tumor formation in non-liver tissue after tumor cells injection: In mice receiving large volume injection, tumor nodules were found in the lung 7 days after injection, the tumor formation rate was 100%. When the mice died naturally, the tumor cells didn't form nodules, but grew along the alveolar wall, or aggregated in loose groups. In addition, tumor cells were seen in the kidneys, splenic red pulp, and myocardium interstitial. The splenic corpuscle decreased in size and the germinal center could not be observed obviously. In mice receiving small volume injection, tumor nodules were observed in the lung 3 days after injection, the tumor formation rate was 40% on day 3 and 100% on day 7. When the mice died naturally, the tumor nodules in the lung had clear boundary, some tumor cells attached to the spleen and kidney capsule. The splenic corpuscle decreased in size and the germinal center could not be observed obviously.Conclusions:1. Multifocal liver cancer model in mice was successfully developed by hydrodynamic injection of H22 hepatic cancer cells, the success rate are 100%. The multiple nodules mainly distribute near the surface of the liver and possess strong proliferative and invasive ability.2. The reversible enlargement of endothelial gap, endothelium rupture, membrane injury of hepatocytes and cytoplamic vesicles formation induced by hydrodynamic injection are the essential pathway for the entry of tumor cells into the liver parenchyma.3. Advantages of multifocal liver cancer model establishment method via hydrodynamic injection includes easy handling, safty, short experimental cycle, good reproduction, easy preparation in large quantities and low cost. It would provide a new model option for studying multifocal liver cancer.4. Small volume injection of H22 liver cancer cells mainly induces formation of multifocal tumor in lung. It also induced cancerous pleural effusion and ascites but hepatic tumor nodules.