Establishment of an in situ implantation metastasis model for human non-small cell lung cancer in nude mice: (1) to investigate distant metastasis of lung cancer and block its distant metastasis; (2) to mimic the naturally occurring process of metastasis of advanced non-small cell lung cancer; and (3) to assess the dissemination of tumor cells and the growth of tumors in the intact organs of living animals.
Operation method
In situ implantation model
Principle
Human lung adenocarcinoma cells A549 were transfected with GFP-expressing plasmid pRNAT-U6/Neo, and G418 was screened to obtain stable GFP-expressing cells, and the growth activity and tumorigenicity of the cells were compared before and after transfection. The transfected cells were planted in situ in nude mice with predetermined standards for execution.HE staining and immunohistochemistry were used to detect the location and number of metastatic foci. Tumor dissemination was detected using the KODAK IS2000MM system.
Materials and Instruments
BALB c nu nu nude mouse Adenocarcinoma cell line A549 Move I. Preparation of experimental materials Caveat Strict biosafety standards should be followed when growing in situ to avoid bacterial contamination. Common Problems The in situ implantation method simulates the natural environment where lung cancer occurs, which is the best method to establish the metastatic model of lung cancer. The subcutaneous implantation method, which is the most widely used method in oncology research, establishes a model with only a local mass without metastasis and dissemination to distant organs, and without tumor-related symptoms. Intrathoracic in situ implantation is easy to operate and can reproduce the process of clinical NSCLC metastasis; the transfer of GFP into human lung adenocarcinoma A549 cells has no effect on the growth activity and tumorigenicity, and the results of A549/GFP used in NSCLC metastasis model are reliable; with the help of fluorescence imaging equipment, NSCLC metastasis can be detected in a non-invasive way by using the tracer function of GFP. For more product details, please visit Aladdin Scientific website.
Calf Serum RPMI-1640 Culture Solution pRNAT-U6 Neo Calcium Phosphate Ready-to-Use SP Ultrasensitive Sense Immunohistochemical Staining Kit DAB Kit Color Developing Kit Formaldehyde EDTA Sodium pentobarbital
Fluorescence Microscope Optical Microscope
1. 40 BALB/c nu/nu nude mice, male, 4-6 weeks old, weighing 18-20 g, raised in specific pathogen free (SPF) grade.
2. Lung adenocarcinoma cell line A549, cultured in RPMI 1640 medium containing 10% calf serum (Gibcobrl, USA), 5% CO237°C, and routinely passaged.
3. Plasmids and screening drug plasmid pRNAT-U6/Neo (Genscript, USA), carrying the Neomycin resistance gene for screening, encoded cGFP under the control of the CMV promoter as a transfection marker. g418 (Promega, USA) was screened at 800 μg/ml initially followed by 200 ug/ml maintenance screening.II. Transfection of cells
1. Mammalian Calcium Phosphate Transfection System (Promega, USA), the plasmid pRNAT-U6/Neoo was transfected into A549 cells according to the operation guide, and the transfection efficiency was observed under the fluorescence microscope at 24~48 h after transfection.
2. Stable transfection was obtained by adding G418 into the post-culture medium.
3. GFP-positive cells were obtained by limited dilution after the clone formation rate of cells was >10% as determined by plate clone formation assay.
4. 1 month later, the transfected cells were harvested, routinely cultured, and screened by adding G418 (200 ug/ml) to the culture medium.
5. Determine the proliferation kinetic indexes of A549 cells after transfection, and compare the growth curves of A549 cells before and after transfection, and detect whether the growth activity of the transfected cells is affected by transfection.
6. After a certain amount of cell proliferation, nude mice were injected subcutaneously with cells before and after transfection, the tumor formation time was recorded, and the tumor volume was calculated by using the formula V=1/2xlengthxwidth, and the tumor sizes of the two groups of nude mice before and after transfection were compared to detect whether there was any difference in tumorigenicity.Establishment of human lung adenocarcinoma A549 in situ planting model in nude mice
1. Take the transfected cells, rinse them twice with serum-free RMPI 1640 culture medium and adjust the final concentration of cells to 5x106/0.2 ml.
2. Anesthetize the nude mice with 0.5% sodium pentobarbital (50 mg/kg) (China Pharmaceutical Shanghai Chemical Reagent Company) by intraperitoneal injection, and then use a 26-gauge needle to suck up 0.2 ml of cell suspension and inject it into the left thoracic cavity of the nude mice, and pay attention to controlling the depth of the needle in the process of injection.
3. The whole operation was completed within half an hour after cell harvesting, and the principle of asepsis was strictly followed.
4. Confirm the success of in situ planting under KODAK IS2000MM after injection, and the injection site was the thoracic cavity.
5. After injection, continue to feed, observe once every other day and record the body weight. 6.
6. When the nude mice showed depression, shortness of breath, arching of the back and obvious emaciation, and a 20% loss of body weight compared with the Et injection, the nude mice were executed by cervical dislocation.
7. The chest was opened for observation, and the organs were removed and fixed with 10% formaldehyde for preservation.
IV. Inspection items
1. fluorescence microscope imaging of post-transfected cells Fluorescence microscope observation 24～48 h after transfection to confirm the success of transfection. Observe the growth of cell clones under microscope during monoclonization.
2. In vivo GFP labeling imaging detection Apply KODAK IS2000MM to perform in vivo fluorescence imaging to confirm the injection site is the thoracic cavity, and then intermittently apply it to in vivo to confirm the distant metastasis of tumor cells.
3. Anatomical and histological examination The executed nude mice were all subjected toAfter detailed anatomical examination, the organs were removed, fixed and embedded, and then serially sectioned at a thickness of 4 tun, the sections were strictly numbered, and HE staining was performed on the odd-numbered slices for light microscopic observation, in order to initially determine the metastatic foci.
4. Immunohistochemical staining
Lung, liver and brain were selected as the main organs for study, and on the basis of HE staining to determine the location of metastatic foci, the adjacent even-numbered sections were taken for immunohistochemical detection, CEA mouse anti-human monoclonal antibody (ZM-0062), LRP mouse anti-human monoclonal antibody (ZM.0335) working solution, ready-to-use SP Ultra-sensitive Immunohistochemical Staining Kit (sP-9000), Antigen Repair solution, DAB Kit chromogenic kit (zU I 9032) were purchased from Beijing Zhongsui Jinqiao Biotechnology Co.
V. Statistical processing
The cell growth activity and tumor volume x ± s were expressed, and the comparison of the values was performed by ANOVA. x2 test was used to compare the results of HE assay and in vivo imaging assay. SPSSl3.0 software was used for statistical analysis.