Murine Colon Carcinoma Metastasis Model And Its Activation Of Hepatic Stellate Cells

[Objective] Liver is the primary distant metastatic organ of colon cancer and the mechanism is remained to be examined in detail. Therefore, to establish a model of colon cancer liver metastasis is helpful for the basic research of this field and the screening of antitumor drugs and the clinical treatment.. We here to establish a model of colon cancer liver metastasis in a new method. We observe the expression of aHSC in liver metastatic foci, extratumoral liver tissue and liver tissue without metastasis via immunohistochemisty and image analysis software and reveal the relationship between the expression of aHSC and colon cancer liver metastasis.[Methods] (1) Model establishing:BALB/C mouse were subcutaneously injected mice colon carcinoma cells(CT26) and a neoplasm formed. Then the neoplasm was excised and cut into small(2mm) size tissue. The orthotopic transplantation of tumor tissue was performed in pouch-making method.(2) Observing the expression of a-SMA:Liver tissue were fixed with 10% formalin neutral buffer solution and embedded in paraffin. The sections were cut at the thickness of 5 mm. Paraffin-embedded sections were deparaffinized and incubated for 5 min with 1% H2O2 to eliminate endogenous peroxidase. Then, for antigen retrieval, Subsequently, endogenous avidin and biotin were blocked and nonspecific protein binding was blocked Thereafter, the sections were incubated with mouse anti-aSMA, the immune complexes were visualized using SABC kit.[Results] (1) The neoplasm grew to about 2cm when 3 weeks after injection. Exploratory laparotomy (n=5) were performed 3 weeks after operation, neoplasm formed in the left lower quadrant without abdominal metastasis and cancerous ascites. When 4 weeks after operation (n=5), the neoplasm in the left lower quadrant were bigger than that in 3 weeks group. Metastatic foci appeared at the adjacent intestine wall in 2 mouse. Metastatic foci appeared in the liver in 3 mouse. The liver metastatic rates were 40% and 60% respectively when 3 weeks and 4 weeks after operation.(2) The a-SMA expressed in all metastatic foci (n=5) and extratumoral tissue (n=5) and 1 nonmetastatic tissue (n=5). The expression of aHSC in the liver metastasis is significantly higher than that in the extratumoral liver tissue and the nonmetastatic liver tissue. The positive cell area are 26594.99±10170.42μm2,6157.03±2164.24μm2,1404.22±2901.31μm2 respectively in metastatic foci (group A),extratumoral tissue (group B) and nonmetastatic tissue (group C), the positive cell density are 0.01274±0.007475/μm2,0.00310±0.001575/μm2,0.00064±0.001327/μm2 respectively in group A,B and C. P<0.05 between the 3 groups.[Conclusions] The pouch-making method orthotopic transplantation of liver metastasis model provides useful tools for the study of mechanism of metastasis. The increasing quantity and intensity of HSC activation cohere with the progression of colon cancer. The alteration of microenvironment evoked by aHSC is a potential factor which may cause liver metastasis of colon cancer.