The establishment of in situ prostate tumor model in nude mice can (1) establish an animal model of prostate cancer that is closer to the natural growth process of human tumors in vivo, and (2) be used for the study of the mechanism of prostate cancer invasion and metastasis as well as effective therapeutic measures.
Operation method
In situ graft tumor model
Principle
An incision was made in the lower abdomen of 10 nude mice to reveal the bladder and prostate, and 50 μL of human prostate cancer DU145 cells, totaling 6 × 106 cells, were inoculated under the peritoneum of the right and left ventral lobes of the prostate gland with a 1 mL TB cartridge and a 29G needle to establish an in situ tumor model, and to observe the changes in vital signs of the nude mice. The nude mice were executed under near-death condition, and specimens of in situ tumors, bladder, pelvic lymph nodes, lungs and livers were taken, fixed with 40 g/L formaldehyde, embedded in paraffin and stained with HE, and observed under microscope, and X-ray irradiation was performed to show the bone metastasis.
Materials and Instruments
Male BALB c nude mice Du145 cell line Move I. Experimental preparation Common Problems For more product details, please visit Aladdin Scientific website.
Fetal bovine serum RPM I 1640 culture medium Sodium pentobarbital Trypsin EDTA
CO2 incubator Cotton swab Scalpel Gauze Syringe Needle Formaldehyde Optical microscope
Ten male BALB / c nude mice, 6 wk old, with body mass of about 20 g, were fed and watered ad libitum in a laminar flow rack under SPF conditions. The Du145 cell line was cultured with RPM I 1640 culture medium (Gibco, USA) containing 100 mL /L fetal bovine serum (Hangzhou Four Seasons) in an incubator at 37°C, 50 mL /L CO2, and digested and passaged with a mixture of 1. 25 g/L trypsin and 0. 2 g/L EDTA.
II. Specific operations
1. Nude rats were anesthetized with 10 g/L sodium pentobarbital (75 mg/kg), placed in the supine position, and the limbs were fixed with adhesive tape. A 1-cm transverse incision was made in the middle of the lower abdomen to expose the bladder and seminal vesicle glands, and a cotton swab was used to gently press on the top wall of the bladder, the abdominal wall and to free the bladder to the bladder neck, and the prostate was further exposed by pushing away the muscle and the gonads, and was seen to be about 1.0 mm × 1.5 mm × 2.0 mm in size, with 2 lobes on the right and left. It was pink in color and had obvious glandular tissue characteristics.
2. Use a 1 mL TB syringe and a 29G needle to gently pick up the peritoneum of one side of the prostate, and slowly advance the needle until it lifts up the peritoneum of the opposite side, and then slowly inject 50 μL (6 × 106 cells ) of Du145 cell suspension after the tip of the needle enters into the peritoneum, and the peritoneum will bulge upward, and detach from the surface of the gland as a satisfactory criterion.
3. The anatomical position of the organs was restored, and the muscle and skin layers of the abdominal wall were sutured intermittently with 720 absorbable thread, and the rats were returned to the cage after awakening.
4. The nude mice were executed by cervical dislocation method with the appearance of malignant fluid and in a state of near death as the end point of observation.
5. Necropsy and observation of tumor growth at the tumor site.
6. Immediately after necropsy, X-rays were performed at 100 mA, 40 kV, and 5.23 ms.
7. The distance between the nude mice and the bulb tube was 1.0 m. Then the prostate, bladder, pelvic lymph nodes, liver and lung specimens were taken, fixed in 40 g/L formaldehyde, and paraffin thin sectioned at 4 μm, and the metastatic foci or lack of metastasis were observed under the light microscope after HE staining.
Human prostate cancer animal model is an indispensable experimental tool to study the pathogenesis of prostate cancer, the relationship between tumor and host, the invasion and metastasis process of cancer and the effectiveness of therapeutic measures. Therefore, the establishment of an ideal prostate cancer animal model should be able to simulate the process of human prostate cancer to the greatest extent possible, and have the following characteristics: (1) human tumor, which can secrete prostate-specific antigen (PSA); (2) tumor characteristics similar to human prostate cancer; (3) response to hormone therapy similar to human prostate cancer; and (4) tumor characteristics similar to human prostate cancer. The characteristics of the tumor are similar to those of human prostate cancer; ③ The response to hormone therapy is similar to that of human prostate cancer.
Because of its immunodeficiency, nude mice are the best vehicle for establishing animal models of human tumors. In the past, subcutaneous inoculation was chosen as the tumor loading site for the production of nude mice models of prostate cancer, which was convenient for inoculation and easy to observe. However, according to Paget's "Seed and Soil" theory, tumor growth and metastasis depend on a suitable microenvironment, so in situ planting has higher tumorigenicity and metastasis rates than subcutaneous inoculation. Therefore, in situ implantation has a higher tumorigenicity and metastasis rate than subcutaneous inoculation, and the subcutaneous implantation model does not well simulate the natural growth process of prostate cancer in the human body as well as many biological behaviors, whereas in situ implantation not only makes the tumor cells obtain a microenvironment closer to that of the human body, but also rich in endogenous growth factors, which can improve the success rate of the implantation and form a metastatic pattern similar to that of the clinic.
Sato et al. compared prostate cancer LNCaP cell suspension for SCID mice after subcutaneous and in situ implantation respectively, the results of the subcutaneous group of 100% tumor, none of the lymph nodes and distant metastasis; in situ group 89% tumor, but all occurred in the retroperitoneum and mediastinal lymph node metastasis, 40% were found to have microscopic lung metastasis.
Rembrink et al. and Wang et al. used prostate cancer PC23, PC23212521L, TSU2Pr1 and LNCaP cells as the in situ model for nude mice, which showed high tumorigenicity, lymph node and lung metastasis. Since the prostate gland of nude mice is very small (about 50 mg), tumor cell injection requires particularly delicate manipulation. Moreover, 10-180 min after tumor cell injection, even if the tumor cells are not directly punctured into blood vessels, they are prone to spontaneous lymphatic and bloodstream metastasis, resulting in artificial dissemination.
Several pre-tests were performed, and the requirement of the envelope bulging upward and detaching from the gland surface was finally achieved, with a final tumorigenicity rate of 60%, which was similar to the results of Wang Pengfei et al. in China. It was observed that all mice developed acute severe hepatitis postoperatively, the nature of which may be an autoimmune acute severe hepatitis (A IH).
The pathogenesis of A IH is currently unknown, but some studies have suggested that drugs, the environment, and viral infections may be contributing factors to A IH.... Because mice develop liver failure prematurely. Therefore, lung metastasis, lymph node metastasis and bone metastasis could not be observed in this group of models. In addition, implanting histologically intact tumor blocks resulted in higher tumorigenicity and metastasis rates than tumor cell suspensions but required more microscopic instrumentation and manipulation finesse. Therefore, in situ injection of tumor cells is gradually becoming the current mainstream technique.
In addition, sophisticated modeling techniques can help to "breed" new cell lines with biological properties that are closer to clinical reality. For example, most prostate cancer cell lines do not secrete PSA, which is not conducive to the monitoring of tumor models, and LNCaP, although it can secrete PSA, belongs to an androgen-dependent cell line, which restricts its application in the study of androgen-independent prostate cancer.
Thalmann et al. used LNCaP to establish different immunodeficient animal models, and cultivated a series of subcell lines, which retained the secretory properties of PSA, but were also androgen-independent and more malignant. These subcell lines were inoculated subcutaneously and in situ in the prostate of nude mice, and both of them established animal models with higher tumorigenicity and bone metastatic potential than LNCaP cells.
Harper et al. also successfully developed two new cell sublines, TEN12F and TEN12C, after modeling with TEN12 under different conditions, which created the conditions for explaining the mechanism of the transition from androgen-dependent to non-androgen-dependent human prostate cancer at the molecular level.