Experiments on the establishment of an in situ transplantation model of human lung cancer in nude mice

Summary

The establishment of an in situ transplantation model of human lung cancer in nude mice can be used to (1) establish a good platform for animal experimental research on lung cancer; (2) monitor the growth and metastasis of primary tumors in real time; and (3) visualize physiological and pathological processes qualitatively or quantitatively in vivo at the cellular and molecular levels.

Operation method

Surgical in situ transplantation

Principle

The enhanced green fluorescent protein gene was introduced into the human lung cancer macrophage cell line NCI-H460 using retroviral transfection, and the lung cancer in situ transplantation model was established by surgical in situ transplantation method. Tumor growth was regularly observed by a small animal in vivo fluorescence imaging system, and correlation tests were used to analyze the correlation between fluorescence area and tumor volume, and to observe the survival and tumor metastasis of nude mice after in situ transplantation.

Materials and Instruments

BALB cnu nu nude mice Human non-small cell lung cancer NCI-H460
EGFP expression vector Pellet feed for mice Fetal bovine serum Physiological saline Penicillin Streptomycin RPM1-1640 medium
Acetate Filter Membrane C Incubator 96-well plate Needle Syringe Tweezers Cotton swabs Purification bench Scissors

Move

I. Preparation of experimental materials

BALB/c (nu/nu) nude mice, male and female, 4 weeks old-6 weeks old, 22 mice, weighing 18 g-22 g. All nude mice were housed in an SPF-grade barrier system and subjected to experiments (. The relative humidity of the rearing room was ( 55±10 )%, the temperature was ( 22±2 )°C, the light exposure was 12 h with alternating light and dark, and the feed used for nude mice was cobalt 60 radiation sterilized pellet feed for mice (Jiangsu Synergy Pharmaceutical and Bioengineering Co., Ltd.).Establishment of H460 lung cancer cell line expressing green fluorescent protein
1. Cultivate PT67 packaging cells in RPM1-1640 supernatant containing 10% fetal bovine serum, penicillin and streptomycin.

2. Collect the culture medium when the cells reach 80%-90% fusion concentration, and filter it through acetate filter membrane with a pore size of 0.45 um and prepare it for use.

3. 24 h before transfection, update NCI-H460 fresh medium, add the medium containing retroviral solution for 4 h after transfection, and then place it in the incubator at 37℃ for 36 h. Observe the fluorescence under the fluorescence microscope.

4. Collect the transfected cells and culture the cells in G418 selective medium containing 200 ug/mL.

5. Gradually increase the concentration of G418 by 200 ug/mL-400 ug/mL until the resistant clone is formed.

6. Isolate the highly GFP-expressing clones by 96-well plate single cell isolation method, mix the isolated clones, and amplify them using conventional methods.

7. Cultivate the clones for 3 months by in vitro successive passaging, and detect the expression intensity of fluorescence.III. Establishment of in situ transplantation model
1. Suspend H460-GFP cells in PBS solution and adjust the cell concentration to ^5x106/mL. 2.

2. Disinfect the left axillary skin of nude mice, inoculate 0.2 mL of cell suspension into the subcutaneous of 2 nude mice with 1 mL of syringe with 27G1/2 needle to form a dermal mound, and dry cotton swabs for compression.

3. When the subcutaneous grafted tumor grew to about 10 mm in diameter, the grafted tumor was peeled off. Nude mice with well-grown tumors and unbroken tumor nodes were selected, and the tumor nodes were completely peeled off under aseptic manipulation on a purification bench, and the blood stains were washed away with physiological saline.

4. Cut open the tumor nodes, remove the necrotic tissue in the center, and cut them into small rice-like tumor pieces with a diameter of about 1 mm.

5. 20 nude mice were subjected to surgical in situ transplantation.

6. The nude mice were anesthetized with isoflurane inhalation and placed in the right lateral recumbent position with the limbs appropriately immobilized.

7. The skin of the left chest wall was sterilized, and a 0.4 cm-0.5 cm transverse incision was made in the skin near the 4th and 5th intercostal spaces, and the chest wall muscles were sharply separated, exposing the ribs and intercostal muscles to open the chest cavity.

8. The left lung was fixed with forceps, the prepared lump was sutured to the left lung, and the sterile sutures were closed to close the chest cavity.

9. Immediately check the suture and if it leaks, continue until the chest wall is completely closed.

10. Use a 5 mL syringe to make an intrathoracic puncture to extract air and suture the chest wall muscles and skin.

11. Return the nude rat to its original cage and continue rearing.
Monitoring indicators 1. Observe the nude mice every day after surgery for any complications and abnormalities (activity, neurological function, respiration, animal appearance).

2. 2 nude mice were anesthetized and executed every week for 4 weeks after tumor implantation.

3. Sharp separation of the left skin muscle.

4. The thoracic and abdominal cavities were opened under a fluorescent stereomicroscope and photographed under natural light and fluorescence excitation, respectively.

5. Detect the expression of GFP using a small animal in vivo imaging system with an emission wavelength of 520 nm, an excitation wavelength of 480 nm, an exposure time of 1 s, and quantitative analysis of the fluorescence area by IPP software.

6. Observe the growth of in situ transplanted tumors, and measure the tumor volume by the classical method: use vernier calipers to measure the longest diameter of the tumor (a) and the shortest diameter perpendicular to it (b), and calculate the volume of the tumor according to the formula: Tumor Volume (V) = ab2/2 (a, b in mm).

7. The in situ tumor growth curve was plotted using the number of days of tumor inoculation as the horizontal coordinate and the fluorescence area of the tumor inoculation site as the vertical coordinate.

8. The remaining nude mice were opened weekly with chest flaps, and the nude mice were anesthetized by intraperitoneal injection of sodium pentobarbital 45 mg/kg, or the nude mice were fixed by hand and photographed under natural light source and fluorescence excitation.

9. When the nude mice developed malignant disease, the ruffed mice were dissected, and the tumor growth and the involvement of surrounding organs were observed visually and photographed.V. Statistical processing
SPSS 13.0 software was used for statistical analysis, fluorescence area and tumor volume were expressed in Mean±SD, Pearson correlation test was used to analyze the correlation between in vivo tumor area and tumor volume, and the difference was considered statistically significant at P<0.05.

Caveat

1. Different experimental groups and control groups should be designed, and the number of animals in each group is usually 5-10. 2;2. Generally, after 6-8 weeks of tumor inoculation, when the tumor of mice can grow to 15-20mm in diameter (i.e. the mice will be on the verge of death), blood can be taken from the eyeball, serum can be separated and preserved, the mice can be executed, and tumor tissues can be photographed, and part of the tumor tissues can be used for frozen tissue section (or preserved at -80℃), and immunohistochemical staining accordingly, and some of the tumor tissues can be fixed with 3% neutral formaldehyde, and embedded in paraffin for conventional HE staining. Some tumor tissues were fixed with 3% neutral formaldehyde, embedded in paraffin, and stained with conventional HE staining.

Common Problems

I. Experimental discussion

In vivo transplantation model is the main method for experimental study of tumors.After Rygaard et al. successfully transplanted human colon cancer in nude mice for the first time in 1969, people began to use nude mice for the study of tumor transplantation. Subcutaneous transplantation and transgenic animal models could not well reflect the clinical metastasis and drug sensitivity, and in 1991, Fu et al. created the surgical in situ transplantation method, which used grafts directly from fresh surgical specimens or transplanted human tumor cells or tumor cell lines, and then surgically transplanted the tumors into nude mice to establish a tumor model in situ. Numerous scholars have confirmed that the in situ transplantation model technology is a great progress through the animal experimental models of prostate cancer, pancreatic cancer, breast cancer and other in situ transplanted tumors, and its metastasis rate and metastasis pattern are closer to the clinic.

In 1994, Chalfie et al. expressed GFP in Escherichia coli and nematodes for the first time, which created the precedent of GFP application research. In recent years, green fluorescent protein has become one of the most widely used labeling proteins. green fluorescent protein expressed by GFP gene can spontaneously emit green fluorescence when irradiated by blue light or violet light, the fluorescent signal intensity is high, easy to be captured, and can be directly detected by fluorescence microscope or flow cytometer without the assistance of other substrates or cofactors, which is intuitive and convenient to observe. At the same time, it also has the advantages of good stability, strong tolerance, non-toxicity to tissues or cells, and easy to construct carriers, etc. Therefore, GFP can be used in tumor growth and development. Therefore, GFP has been widely used in tumor growth and therapeutic evaluation.

In vivo optical imaging in living animals directly transfects the GFP gene into tumor cells, which directly reflects the biological characteristics of the studied cells based on the observation and quantitative analysis of fluorescent proteins. The intrinsic chromophore of fluorescent proteins can emit blue excitation light to stimulate fluorescent protein luminescence under the irradiation of simple instruments such as blue light emitting diodes with narrow band filters and excitation filters. The observer is phototransferred through a suitable viewing lamp and a light-conducting fiber at a wavelength of about 490 nm. As researchers delved deeper into fluorescent proteins, it was found that fluorescent proteins with emission spectra in the near-infrared band were more suitable for imaging deep animal tissues. Fluorescent imaging produced by red fluorescent proteins will be more sensitive, with less absorption and scattering from surrounding tissues. The brightest fluorescent protein that has been identified is a red fluorescent protein called Katushka, which emits at a wavelength of more than 620 nm, and has rapid maturation, high pH stability, and photostability, which may make non-invasive whole-body imaging of living animals with lung tumors a reality in the future.

In summary, the in situ model of lung cancer in nude mice expressing green fluorescent protein constructed by the SOI method in this experiment was able to objectively evaluate the growth of NCI-H460-EGFP cells in nude mice by observing the green fluorescent protein, thus discovering tiny metastatic foci that could not be detected by the naked eye, which is valuable in the study of exploring the mechanisms of lung cancer growth and metastasis. Meanwhile, this study also confirms that the tumor volume can be inferred by measuring the fluorescence area in this model, so as to quantitatively analyze the growth of tumor in vivo, and to provide a new experimental tool for preclinical research of drug therapy.

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