人胃癌裸鼠转移模型 z-bo 2020-12-28 303

　　Background: Gastric cancer is the fourth most common malignant tumor in the world and the second leading cause of cancer death after lung cancer. Although the current diagnosis and treatment methods have significantly prolonged the prognosis of patients with early gastric cancer, the 5-year survival rate of patients with gastric cancer in each stage is less than 50% after diagnosis. Metastasis is part of the reason for the high mortality rate of gastric cancer. The proportion of gastric cancer patients who die of peritoneal metastasis is about 50%. Therefore, metastasis has become a hot spot in many gastric cancer research. Transfer is a very complex process involving multiple consecutive steps. Genes related to cell adhesion, movement, proliferation, survival, metabolism, and signal transduction play an important role in tumor metastasis. How these proteins work together to promote metastasis is still poorly understood. The mouse model of metastatic human gastric cancer reported so far has multiple challenges. Orthotopic transplantation of nude mice requires surgery, and the transplanted tumor tissue is derived from human gastric cancer cell lines rather than patients. As a result, the surgical procedure is lengthy, which may cause massive bleeding and death in mice. In addition, although the in situ tumor formation rate is close to 80-100%, the metastasis rate is not high, the liver tumor metastasis rate is 45-60%, and the peritoneal metastasis rate is only 40%. Therefore, the establishment of these mouse models can benefit from improved methods, which will make transplantation easier and lead to more powerful metastases. In this report, we describe a mouse model of metastatic human gastric cancer that solves the problems in previous mouse models. We established a mouse model of metastatic gastric cancer by subcutaneous transplantation of tumor tissue directly derived from gastric cancer patients. Compared with other mouse models previously described, this mouse model forms tumors at a higher rate and, more importantly, shows robust metastasis.

　　 Method: BALB/C nude mice aged 4-6 weeks, male and female weighing 16-18g, fresh tumor tissues were removed and implanted immediately. Experimental study of subcutaneous implantation of fresh tumor tissue into nude mice: The fresh tumor tissue removed from a gastric cancer patient was cut into 1 cubic millimeter, diluted with DMEM diluent, and implanted subcutaneously into the left and right armpits and groin of nude mice with a 16G needle under sterile conditions. Each sample was implanted into four mice, with 5-6 pieces (0.8 ml) per mouse. Once the tumor on the nude mouse grew to 1 cubic centimeter, the mouse was killed by cervical dislocation, and the tumor tissue was examined and removed under sterile conditions. The tumor tissue of each mouse was divided into three parts, one part was used for another round of transplanted tumors in nude mice, and the other part was fixed in 10% formaldehyde for pathological examination and immunohistochemistry (IHC) staining to detect CK8/18, E-cadherin, VCAM-1 and ICAM-1, the third part is stored in liquid nitrogen -80 ℃, real-time PCR analysis of CK8/18, E-cadherin, VCAM-1 and ICAM-1. Dissect and examine the mouse's peritoneum, abdominal cavity, liver, spleen, stomach, intestine, kidney, lung and brain for tumor metastasis. The collected tissue was fixed with 10% formaldehyde for pathological examination. "Establishment and identification of a mouse model of metastatic gastric cancer: Under aseptic conditions, 5-6 pieces (0.8 mL) of the excised tumor tissues were subcutaneously transplanted into the left and right groins of 5 nude mice under aseptic conditions. As mentioned above, the cutting and dilution of tumor tissue, the inspection and excision of tumor growth in nude mice, and the inspection and treatment of implanted tumor tissue and metastatic tumor tissue.

　　 Effect of planting site on metastasis rate: As described above, under aseptic conditions, human gastric cancer tissues transplanted from nude mice were subcutaneously transplanted into three groups of nude mice at different sites. On average, 5-6 pieces were implanted per mouse. One group received tissue on the right and left groin, the other group received tissue on the right and left axilla, and the third group received tissue on two parts of the back. As described above, tumor growth inspection and excision were performed in nude mice. Further analysis includes: detection of tumor growth in different parts, peritoneal and abdominal cavity metastasis. Nude mouse transplantation and metastasis of cryopreserved and passaged human gastric cancer tissues: The human gastric cancer tissues that will be transplanted into nude mice from the fourth and eighth generations are stored in liquid nitrogen and transplanted subcutaneously into nude mice as described above. The rat's right and left groin. Further analysis included tumor growth and peritoneal and abdominal cavity metastasis at different sites.

　　 Pathological observation of nude mice transplantation and metastasis of gastric cancer tissue: fixed with 10% formaldehyde, paraffin embedding, sectioning, hematoxylin-eosin staining (HE) to observe the transplantation and metastasis of human gastric cancer in nude mice.

　　 Immunohistochemical staining: Immunohistochemical method was used to detect the expression levels of E-cadherin, VCAM-1, ICAM-1 and CK8/18 in transplanted tumors and metastatic tumor tissues of nude mice. The sections for staining were taken from surgical specimens, implanted and metastatic tumor tissues, and tissues containing metastatic tumors. The reagents used for staining include SP-9000 histidine plus kit, 3-3′-diaminobenzidine tetrachloride (DAB) kit, primary mouse monoclonal antibody against E-cadherin (1 : 200 dilution), ICAM-1 (1:500 dilution) and rabbit anti-VCAM-1 primary polyclonal antibody (1:500 dilution). The IHC stained sections were independently evaluated by two pathologists, and it was decided that any discrepancies in the results were resolved by consensus. The staining intensity was rated as negative, weak, moderate or strong.

　　 Total RNA extraction and real-time PCR: Trizol was used to extract total RNA from transplanted and metastatic tumor tissues grown in nude mice and surgical specimens used for transplantation. CDNA is synthesized by reverse transcriptase. SyBR premix probes are used for real-time PCR. The 20μL reaction contained 10μL of SyBr premix EX TaqTM, 1μL of DNA template, 0.4μL of each primer and 8.2μl of sterilized water. The PCR cycle conditions are: 37°C for 5 min, 95°C for 30 s, 95°C, 5s, 40 cycles, 60°C, 30s. Β-actin mRNA was used as an internal control, and the reaction mixture without template DNA was used as a negative control. All samples were tested independently three times, and the quantitative PCR data was analyzed by comparative CT method.

　　 Conclusion: Tumor formation and metastasis: Among the four mice implanted with the first surgical specimen, only one developed a tumor at the implantation site at 76 days. Twenty-five days later, the mice had cervical dislocation and tumor analysis. The average size of tumor tissue is 1 cubic centimeter, showing a complete capsule and hard texture. Visual inspection revealed retroperitoneal metastasis. There was no metastasis in the peritoneum, abdominal cavity, liver, spleen, stomach, intestine, kidney, lung, and brain. Pathological analysis showed that the transplanted tumor and metastatic tumor tissue were composed of poorly differentiated cancer cells, with only a small amount of mesenchyme and blood vessels. In a parallel study involving the implantation of tumor tissue in 4 mice, similar results were obtained; at 26 days after implantation, only one mouse developed tumors. No metastasis was found in the peritoneum, abdominal cavity, liver, spleen, stomach, intestine, kidney, lung, and brain. The other mice implanted with the second and fourth surgical specimens showed no tumor growth.

　　The stability of the implanted tumor after multiple generations: the first surgical specimen was passed down to ten generations. The tumor growth rate is 100%, and the retroperitoneal and visceral metastasis rate is 80-100% (average 94%), regardless of whether the original tissue is fresh or frozen. Metastases were found in peri-esophageal lymph nodes, subgastric mucosa, serosal stomach, spleen, hilar area, central vein and sinusoids, liver parenchyma, liver capsule, kidney hilar, renal parenchyma, adrenal glands, intestinal serosa, pancreas and vas deferens.

　　The rate of metastasis of tumors in different parts: the effect of implants in different sites on the rate of tumor metastasis, not the rate of tumor growth. Inguinal implants caused 94% of retroperitoneal and visceral metastases, and back implants caused 30% of retroperitoneal and 10% of visceral metastases. There was no retroperitoneal metastasis and 20% organ metastasis after axillary implantation. The occurrence time was: 16 days for inguinal tumor implantation, 20 days for back tumor implantation, and 14 days for axillary tumor implantation. Metastatic organs include liver (50%), kidney (44%), intestine (28%), esophagus (12%), pancreas (12%), stomach (6%), spleen (6%) and vas deferens (6%) ). "Characteristics of transplanted and metastatic tumors: IHC and real-time PCR results show that ICAM-1, VCAM-1 and CK8/18 are mainly expressed in primary and first-generation implantable tumors, while E-cadherin is not expressed. Primary and first-generation tumors stained positively for VCAM-1 and CK8/18, but subsequent generations showed weak staining for these proteins: VCAM-1 staining was moderately positive in the first generation (++), Weak signal (+), CK8/18 is dyed as the first generation of weak signal (+). The E-cadherin staining of tumors of each stage was negative, while the E-cadherin, ICAM-1, VCAM-1 and CK8/18 staining of metastatic tumors of each generation were negative.

　　 Conclusion: Tumor metastasis is a complex multi-stage process. Although many genes and factors are related to tumor metastasis, the exact molecular mechanism is still unclear. In this study, we established a mouse model of metastatic human gastric cancer with a strong metastatic phenotype, which will help understand the molecular mechanism of this process.