Experimental establishment of an in situ tumor model of the prostate gland

Summary

The establishment of in situ prostate tumor models can be (1) used for continuous in vivo monitoring of prostate cancer development and progression, (2) used for studies of in situ prostate tumor growth and treatment, and (3) used to study the biological behavior of prostate cancer.

Operation method

in situ planting

Principle

Ten BALB/C nude mice were injected with 20 μL of red fluorescent protein (RFP)-carrying prostate cancer PC-3 cells (PR7 cells) suspension into the dorsal peritoneum of the prostate, and the in situ prostate tumors and their metastasis were observed by a non-invasive in vivo molecular imaging system in the 2nd, 4th, 6th, and 8th weeks, respectively. Tissues of prostate in situ tumor, lung, liver, kidney, femur, pelvic lymph nodes and other tissues were taken at autopsy, and frozen sections were made, and tumorigenesis and the presence or absence of metastasis were detected by fluorescence microscopic observation and HE staining, respectively.

Materials and Instruments

BALB C nude mice Prostate cancer PR7 cell line
Feed Drinking water Pancreatic enzymes EDTA Hank's balanced salt solution Chloral hydrate Paraformaldehyde Hematoxylin-eosin
Incubator Inverted fluorescence microscope Cryostat tubes Swabs Needles Optical microscope Syringes Non-invasive in vivo molecular imaging system

Move

I. Preparation of experimental materials

1. 10 male BALB/C nude mice, 6 weeks old, provided by Shanghai Slaughter Laboratory Animal Co. They were housed in ultra-clean laminar flow racks under SPF conditions with free intake of feed and water.2. Stable expression of red fluorescent protein (prostate cancer PR7 cell line) was cultured in RPMI-1640 medium (Gibco, USA) containing 5% fetal bovine serum (v/v) (Hangzhou Sijiqing Bioengineering Co., Ltd.) in 37, 5% _CO2 (v/v) incubator, digested and passaged with 0.25% trypsin + 0.02% EDTA mixture (v/v).

II. RFP expression
RFP expression in prostate cancer PC-3 and PR7 cells was observed under inverted fluorescence microscope.III. Establishment of in situ prostate tumor models
1. Logarithmic growth phase PR7 cells were digested with 0.25% trypsin + 0.02% EDTA mixture (v/v), washed and centrifuged in Hank's Balanced Salt Solution (HBSS) and then resuspended to prepare ^2×107/mL cell suspension, which was transferred into sterile cell freezing tubes and brought to the Laboratory Animal Center for spare parts.

2. Nude mice were anesthetized with 10% chloral hydrate (v/v) (4 mL/mg) by intraperitoneal injection and then fixed. After disinfecting the skin with iodine povidone, a 1-cm longitudinal incision was made in the lower median of the abdomen to expose the bladder and the seminal vesicle glands, and a cotton swab assisted in fully exposing the prostate gland.

3. The 29G needle of the disposable 1 mL insulin syringe was carefully inserted into the prostate peritoneum with the bevel upward, and 20 μL of the above cell suspension was slowly injected, and the prostate peritoneum could be seen to be obviously bulging without extravasation, the needle was withdrawn, and the anatomical position of the organs was restored, the muscularis propria layer was closed by interrupted sutures of 7-0 absorbable sutures, and the skin was closed by sutures of 4-0 silk sutures.

4. The nude mice were returned to the cage after awakening.V. Non-invasive in vivo molecular imaging system for monitoring in situ tumors and metastasesIn situ tumor formation, growth, and metastasis were monitored with a non-invasive in vivo molecular imaging system (Xenogen, USA) at weeks 2, 4, 6, and 8 postoperatively.
VI. Pathological examination1. All nude mice developed malaise 6 to 8 weeks after surgery.

2. Cervical dislocation method of execution was used to remove in situ prostate tumors, lungs, livers, kidneys, femurs, and pelvic lymph nodes.

3. 2 frozen sections of 10 μm thickness were made.

4. 1 slice of each tissue was stained with DAPI (4′,6-diamidino-2-phenylindole) for 2 min, and washed 3 times with PBS for 3 min each.

5. Observe the tissues under a fluorescence microscope with green light to stimulate red light and ultraviolet light to stimulate blue light.

6. The in situ tumor was used as a positive control.

7. The other 1 sheet was fixed with 4% paraformaldehyde for 10 min and then stained with hematoxylin-eosin (HE) to look for metastatic foci under ordinary light microscope.

Common Problems

I. Experimental discussion

With the discovery of fluorescent proteins and their wide application in the biomedical field and the development of in vivo fluorescence imaging of animals, it has become possible to dynamically observe the formation, growth and metastasis of primary tumors without executing animals. The fluorescent proteins used to label tumor cells mainly include RFP and green fluorescent protein (green fluorescent protein).Compared with GFP, RFP has longer excitation and emission wavelengths, and its emission peaks are located outside the range of fluorescence background generated by culture medium, culture equipment and cellular components, so it has a high signal-to-noise ratio and sensitivity; and fluorescence conversion within the cell is efficient and easier to detect. Moreover, the fluorescence conversion within the cell is more efficient and easier to detect. Therefore, in this experiment, RFP-labeled prostate cancer PR7 cells were inoculated in situ in the peritoneum of the prostate, and then the fluorescence signals and their intensity changes were observed continuously in vivo with a non-invasive in vivo molecular imaging system to monitor the formation, growth and metastasis of in situ tumors.

Currently, the prostate tumor models with more research applications include subcutaneous inoculation, in situ prostate inoculation and intravascular inoculation models. The method of direct inoculation of human prostate cancer cells into nude mice subcutaneously is simple and easy to carry out, easy to observe, but spontaneous metastasis rarely occurs; intravascular inoculation has two methods of injection into the tail vein and left ventricular inoculation, which is not widely used due to the absence of the formation of the primary foci and the separation of tumor cells from the primary foci and the invasion of blood vessels or lymphatic vessels, and metastasis, etc.; the establishment of the [in-situ inoculation model of prostate tumor] can also be divided into the periprostatic membrane, which can be used as a model of in situ tumor growth and metastasis. The in situ prostate tumor inoculation model can be divided into two types: in situ injection of cell suspension into the peritoneum of the prostate gland and in situ tumor block transplantation. In situ tumor block transplantation requires subcutaneous implantation of tumor cell suspension in nude mice, and then transplanting the tumor block into nude mice prostate by microsurgical technique after tumor formation, which increases the steps of subcutaneous tumor formation, makes it difficult to control the size of tumor blocks transplanted in different nude mice, and requires high surgical techniques. Therefore, we used the method of direct injection of tumor cell suspension into the peritoneum of the prostate to construct an in situ tumor model of the prostate, which is an important method to construct an in situ tumor model of the prostate due to its ability to better simulate the pathophysiological process of prostate tumor development and its simplicity.

In this experiment, the non-invasive in vivo molecular imaging system was used to continuously observe the formation and growth of in situ prostate tumors in the same batch of nude mice in the 2nd, 4th, 6th and 8th weeks after the in situ injection of tumor cell suspension, and it was found that there were already red fluorescent signals representing the tumor cells in the 2nd week after the operation when the tumor could not be touched in vitro, and the fluorescence signals became stronger and stronger with the increasing volume of the tumor. This indicates that the non-invasive in vivo molecular imaging system has high sensitivity and feasibility for monitoring the formation and growth of in situ prostate tumors, and it has successfully established an animal model for in vivo monitoring of in situ prostate tumor development. In addition, the traditional method of pathological confirmation of tumors or metastases is HE staining observation of sections after paraffin embedding.

In this experiment, the prostate cancer PR7 cells used to construct the in situ prostate tumor model could stably express RFP, so we took the specimens for frozen section immediately after the animals were executed, and then observed the in situ tumor and the tissues and organs that might have metastasis by fluorescence microscope, and the results confirmed that the in situ tumor was observed to have red fluorescence, and no metastatic foci were found in the tissues and organs, which is in line with the results of the HE staining pathology. This method is intuitive and simple, and can play an important role in the study of tumor models with labeled fluorescent proteins.

The prostate tumor model established in this experiment did not find obvious spontaneous metastatic foci, which can be used for basic research on the mechanism of prostate cancer and screening of anti-tumor drugs, but limits its application in the study of the metastatic process and mechanism of prostate cancer, which may be related to the short survival time of nude mice and the selection of cell lines. The establishment of in situ prostate tumors with a higher incidence of spontaneous metastasis needs to be further explored.

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