The Experimental Research Of Metastasis Models Construction Of Human Colorectal Carcinoma In Nude Mice

Colorectal carcinomas are commonly seen in alimentary tract, which have high malignancy, tending to spread via lymphatic metastasis and implantation in the digestive tract. To study the mechanism of tumor metastasis and strategy of prevention and management is very important because the infiltration and metastasis are main causes of relapses and mortality. For a better understanding on the mechanism of colorectal carcinoma metastasis and setup effective preventive and therapeutic strategies, it is necessary to setup a stable, reliable and repeatable animal model, which can act as an experimental platform for further studies. Up to now, there is no satisfactory simple lymphatic metastasis or in situ implantation model for colorectal carcinomas, So it is of great research and clinic value that we construct the nude mice food pad simple lymphatic metastasis and tissue block colon mucous membrane in situ implantation models. PART ONE ESTABLISHMENT OF A HUMAN COLORECTAL CANCER LYMPHATIC METASTASIS MODEL IN NUDE MICEObjective To establish a simple, practicable and stability claw pad inoculation and lymphatic metastasis model of colorectal cancer in nude mice.Methods Human colon cancer cell line HCT-116 cells were cultured,and the subcutaneous inoculation in nude mice claw pad was employed. At the time point of 1st to 6th and 8th week post- inoculation, the planted tumor, the popliteal and groin lymph nodes (1st station), the neighbor to iliac artery lymph nodes (2nd station), the renal hilum lymph nodes (3th station), the liver and the lungs were collected. Then the HE staining and human carcinoma antigen (CEA) immunohistochemistry were performed and the tumor formation rate and growth characteristics and metastasis condition were confirmed.Results The tumor formation rate was 100% at the time point of one week post- inoculation. The growth curve of implanted tumor showed exponential growth three weeks after tumor inoculation. The tumor lymphatic metastasis rate of 1st station, 2nd station and 3th station lymph nodes all were 0% at the first and the second week. And it was 40%, 0%, 0% in 1st station, 2nd station and 3th station lymph nodes at the 3th weeks, respectively. Similarly, it was 60%, 0%, 0% at the 4th week; 60%, 20%, 0% at the 5th week; 100%, 40%, 0% at the 6th week; 100%, 60%, 20% at the 8th week. No metastasis was observed in the liver and the lungs at the time points mentioned above. The morphological characteristics of metastasis in lymph nodes were confirmed by HE staining, and the diffuse cancer cells expressed CEA, which were detected by immunohistochemistry.Conclusion It was a simple lymphatic metastasis model, with high metastasis rate. That could be successfully established by subcutaneous inoculation of human colon cancer cell lines (HCT-116) in nude mice claw pad. The model provided useful tools for the mechanisms studies of human colorectal cancer lymphatic metastasis and the anti-metastasis therapeutics. PART TWO ESTABLISHMENT AN ORTHOTOPIC IMPLANTATION AND METASTASIS MODEL IN MUCOUS MEMBRANE OF RECTUM OF HUMAN INTESTINUM CRASSUM CANCER IN MUDE MICEObjective To establish an orthotopic implantation and metastasis model in mucous membrane of rectum of human intestinum crassum cancer in nude mice.Methods Tumor cell line HCT-116, derivated from of human colon adenocarcinoma ,was inoculated submucous stratum of rectum into nude mice to develop implantation tumor. After 4 weeks, the tumors were obtained and the tumors blocks were prepared, then it was implanted on the surface of rectal mucosa. At the different time points, from the 2nd to 12th weeks post- inoculation, the rectum tumor tissue, the superior rectal artery lymph nodes, the inferior mesenteric artery lymph nodes, the abdominal aorta lymph node, the liver and the lungs were collected. Then the HE staining and human carcinoma antigen (CEA) immunohistochemistry were performed and the tumor formation rate and growth characteristics and metastasis condition were confirmed.Results The tumor formation rate was 91% at the time point of two weeks post- inoculation. The growth curve of implanted tumor showed exponential growth 6-12 weeks after tumor inoculation. The tumor lymphatic metastasis rate of superior rectal artery lymph node, inferior mesenteric artery lymph node and abdominal aorta lymph node all were 0% at the 2nd week ,and it was 60%, 20%, 0% in superior rectal artery lymph node, inferior mesenteric artery lymph node, abdominal aorta lymph node at the 4th weeks, respectively. Similarly, it was 80%, 60%, 40% at the 6th week; 100%, 80%, 80% at the 8th week; 100%, 100%, 100% at the 10th week and 12th week. The distant metastasis in both the liver and the lungs was observed at the 10th week post-implantation, with the same metastasis rate, that was 20%. With the development of tumors, the metastasis rate was up to 40% at 12th week. The metastasis cancer cells in lymph nodes ,liver and lungs were detected by HE staining, and the cancer cells expressed CEA, which were detected by immunohistochemistry.Conclusion This method is simple and reproducible, with high metastasis rate, simulating both the occurring and development process of the human colorectal cancer. That could be successfully established by implantion of tumors block on the surface of rectal mucosa. The model provided useful tools for the metastasis mechanisms studies of human intestinum crassum cancer and the anti-metastasis therapeutics.