The human hepatocellular carcinoma nude mouse subcutaneous-hepatic in situ transplantation tumor model can be applied to (1) study the biological properties of malignant tumors and (2) screen anticancer drugs.
Operation method
Indirect in situ transplantation model
Principle
Histologically intact fresh human hepatocellular carcinoma tissue was first inoculated subcutaneously in nude mice to form a subcutaneous grafted tumor, and then this grafted tumor tissue was used to reinoculate into the liver of nude mice to establish a hepatic in situ grafted tumor model (indirect hepatic in situ grafted tumor model), which was compared with the direct hepatic in situ grafted tumor model, the subcutaneous grafted tumor model, and the intraperitoneal grafted tumor model.
Materials and Instruments
NC nude mice Balb c nude mice Move I. Preparation of an indirect liver in situ graft tumor model Common Problems I. Experimental discussion At present, recurrence and metastasis of hepatocellular carcinoma are still the main obstacles to its long-term survival after surgery. In order to study the metastatic mechanism of tumors, it is particularly important to establish an animal model of tumors close to the human body. In the animal transplanted tumor model established in the early stage, the transplanted tumor rarely metastasized although it could maintain the histological structure and biochemical properties of the source tumor, thus affecting its value in studying the metastatic properties of human tumors. Further studies have shown that the ability of human cancer to express invasive metastasis in the host not only requires the availability of a suitable transplantation environment, but also depends on the interactions between tumor cells and between tumor cells and the host. For more product details, please visit Aladdin Scientific website.
Hepatocellular carcinoma surgical resection specimen Hanks' solution Sodium pentobarbital Formaldehyde Paraffin wax
Needles Optical microscope Scalpel Scissors Gauze Sterilizing solution Masks
1. A fresh surgically resected specimen of hepatocellular carcinoma (from Changhai Hospital, the patient was a 47-year-old male, pathological diagnosis: hepatocellular carcinoma of the left lobe of the liver, rough beam type, grade II) was used in Hanks' solution, and cut into 1-2 mm3 small pieces after removing necrotic and non-cancerous tissues.
2. 2 pieces of tumor tissue were taken and implanted subcutaneously in the lumbar back of nude mice with a coarse needle within 40 min of ex vivo, and when the subcutaneous grafted tumors grew to a diameter of about 1 cm, the tumors were excised, and in Hanks' solution, necrotic tissue was removed and then cut into 1～2 mm3 pieces. After the nude mice were anesthetized with sodium pentobarbital intraperitoneally, a transverse incision was made in the left epigastric region, and the livers were exposed.
3. The above 2 pieces of tumor tissue were taken and implanted into the deep parenchyma of the right lobe of the liver of nude mice with a coarse needle within 40 min of ex vivo, and the abdomen was closed at full length.II. Preparation of direct hepatic in situ graft tumor model
1. In the same case of fresh surgical resection specimen of hepatocellular carcinoma, the treatment method was the same as that of subcutaneous transplantation, and the treatment of nude mice was the same as that of indirect hepatic in situ transplantation. 2 pieces of tumor tissues were taken and implanted into the deep parenchyma of the right lobe of the liver of nude mice with a coarse needle within 40 min of ex vivo.2. Preparation of subcutaneous graft tumor model and intraperitoneal graft tumor model.III. Pathological examinations and related indicator tests1. Anatomical and histological examination
(1) After inoculation, all nude mice were kept in separate cages, fed freely, and observed once or twice a day.
(2) When the nude mice were in a state of general exhaustion, they were executed and grossly dissected. The inoculated and metastatic tumors were observed and measured respectively, and the tumor invasion and metastasis were recorded; the important organs (mainly liver and lung) were fixed with 10% neutral formaldehyde, and then routinely paraffinized and examined by light microscope.2. Peripheral blood alpha-fetoprotein (AFP) test
Prior to execution, blood was obtained by removing the eyeballs for blood collection, and the amount of AFP secreted was detected by biochemical methods.
3. Analysis of tumor cell DNA content
A portion of the transplanted tumor specimen was retained for analysis by flow cytometry DNA content analysis was performed.
In recent years, the in situ transplantation model of human carcinoma established according to the theory of in situ transplantation overcomes this defect, which not only maintains the structure of the original tumor tissue, but also keeps most of the biological properties of human tumors, especially the metastatic properties, and it can display its malignant behaviors in the host in a manner similar to that in the patient. Inspired by this novel model, nude mouse in situ transplantation tumor models of various human cancer tumors have been successively established, providing an ideal tool for humans to better study the metastatic mechanism of tumors.
Sun et al. used histologically intact patient liver cancer specimens to establish a human liver cancer in situ graft tumor model LCI-D20 in nude mice, which had a high spontaneous metastasis rate. Compared with the nude mice subcutaneous graft tumor model, the metastatic ability of the two models was very different, although their histological features were similar, with the spontaneous metastasis rate of the intrahepatic in situ graft tumors reaching 100%, whereas the metastasis of the subcutaneous graft tumors was rare. The only shortcoming of LCL-D20 is the low survival rate of transplanted tumors (1/16).
Yamada et al. inoculated nude mice subcutaneously with a human hepatobiliary cell carcinoma cell line to form subcutaneous grafts and injected through the splenic vasculature to form liver grafts, and found that the metastatic rate of liver grafts was significantly higher than that of subcutaneous grafts (100% vs. 20%).
Similar results were obtained by Kimura et al. and Kuo et al. With reference to the above experimental method, we improved it by establishing a subcutaneous transplantation model of human liver cancer in nude mice, and then inoculating the liver of nude mice to establish an in situ transplantation model. The rationale is to allow liver cancer tissues to be adapted to the in vivo environment of nude mice under the nude mice's skin, and then inoculate them in the liver, thus guaranteeing the survival rate of transplanted tumors. It was shown that this model could fully demonstrate the biological characteristics of human hepatocellular carcinoma as well as the direct hepatic in situ transplantation tumor model, and its invasive and metastatic abilities were superior to those of the nude mice subcutaneous and peritoneal transplantation tumors compared with the latter.
In order to explore the mechanism of the difference in tumor biological behavior between in situ and subcutaneous transplanted tumors, the oncogene expression of p53 and other oncogenes and multiple chemotherapeutic drug treatment could be studied for in situ and subcutaneous transplanted tumors respectively, and it was found that the intensity of p53 expression in in situ transplanted tumors was significantly higher than that in subcutaneous transplanted tumors, and the sensitivity of both of them to a variety of chemotherapeutic drugs was also different. The reason for this difference may lie in the different microenvironments in which the tumors are located, thus affecting the performance of their biological properties.
Fukumura et al. concluded that the effects of the hepatic microenvironment on the vascularization and establishment of microcirculation in colon cancer graft tumors were different from those of the subcutaneous microenvironment in nude mice, and that the effects of the hepatic microenvironment on the graft tumors were different from those of the subcutaneous microenvironment.
Cui et al. pointed out that local invasion of tumors is a prerequisite for the occurrence of metastasis, and only tumors with the ability of local invasion can have the ability to metastasize. Subcutaneous and intraperitoneal implanted tumors tend to form a fibrous envelope around the tumor due to the adverse effect of the microenvironment of the site in which they are located, thus restricting the ability of the tumor to invade the surrounding tissues, lymphatic vessels, and blood vessels, and so the chances of metastasis are greatly reduced, whereas the site of the in situ transplantation is itself the soil for tumor growth, where the tumor receives sufficient nourishment, and the tumor is free of envelope formation, and thus is able to fully demonstrate its biological properties. This suggests that in order to express the complete metastasis pattern, a better implantation environment must be available, and only the in situ transplantation model can fully express the metastatic characteristics of human cancer tumors.