CN110495424B - Construction method of synovial sarcoma xenograft mouse model - Google Patents
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- A—HUMAN NECESSITIES
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Abstract
The invention provides a construction method of a synovial sarcoma xenograft mouse model, which comprises the steps of recovering a synovial sarcoma patient tumor tissue block stored in a liquid nitrogen environment, transplanting the recovered synovial sarcoma tumor tissue block into an immunodeficiency mouse in a xenograft in situ under an aseptic condition, wherein the xenograft in situ comprises selecting an immunodeficiency mouse with the age of 2-6 weeks, keeping the sex of the mouse consistent with that of the patient, soaking the recovered synovial sarcoma tissue block in a sterile incubation liquid containing fetal calf serum, fixing the mouse after anesthesia, implanting the tumor tissue block of the patient into a joint capsule of the mouse, suturing an incision, and breeding the mouse to enable the tumor in the joint capsule to grow. Compared with subcutaneous modeling, the in-situ modeling condition is more real, the screened drug scheme can be better used on the patient, and the in-situ modeling is theoretically easier to generate tumor metastasis than subcutaneous modeling.
Description
Technical Field
The invention relates to the field of establishment of tumor transplantation mouse models, in particular to a construction method of a synovial sarcoma xenograft mouse model.
Background
Synovial sarcoma (Synovial sarcoma, SS) is a highly malignant soft tissue sarcoma, accounting for about 7-10% of soft tissue sarcomas. The origin of SS tissues is complex, no clear risk factors exist, the pathological forms are variable, and the benign and malignant demarcation is difficult, so the diagnosis, differential diagnosis and classification become the clinical problems. SS is common in men of 20-30 years old (about 1.2: 1 for men/women), mainly occurs near the joints of the extremities, but occurs almost anywhere throughout the body, and SS hardly occurs in the joints and has no relation to normal synovial tissue. Approximately 80% of primary SS occurs in the extremities, and around 20% occurs in non-extremities such as the ribs, pericardium, lungs, and abdominal wall. The vast majority of SS can migrate to the lung, and less usually to the abdominal cavity. The previous research considers that the extensive excision of tumor bodies and surrounding excisable tissues is the first choice method for treating SS, but the recurrence rate is high, so that comprehensive treatment becomes an important means for improving the SS prognosis. This brings great mental injury to the patient and his family, and also brings heavy economic burden to the society and even the country, bringing greater social problems. Because of the existence of the heterogeneity of synovial sarcoma tissues, the basic research results based on the synovial sarcoma cell line in the past are difficult to reproduce in clinical synovial sarcoma patients, and the results largely lead to the delay of the clinical synovial sarcoma treatment level.
Tumor models are important vehicles for tumor research. Researches show that the cells are continuously adapted to in-vitro environment in the passage process, the biological characteristics are not in accordance with those of in-vivo tumor cells, the cell line xenograft model has greater consistency with the tumor of a patient, the existing tumor animal model lacks an immune system, and the failure rate of clinical tests of the antitumor drugs caused by the defects of the model is high.
Therefore, the model of human-derived xenotransplantation (PDX) which is from a patient and has better homogeneity with the primary tumor of the patient has far-reaching significance, the pathogenesis of synovial sarcoma can be deeply discussed, the result of research and development based on PDX can be well repeated in the body of the patient, limited synovial sarcoma samples in the body of a clinical patient can be greatly amplified, and a xenograft mouse is used for replacing the clinical patient to carry out different combined drug screening, so that the most appropriate drug is selected, the effectiveness is greatly improved, and the side effect of the drug is reduced.
A human derived tumor xenograft (PDX) model is an in-vivo model, and a large amount of tumor tissues of a patient can be amplified by taking a mouse as a carrier by directly transplanting fresh tumor tissues of the patient to an immunodeficiency mouse after being treated and depending on the environment growth and gradual passage provided by the mouse. The PDX model has the following advantages: firstly, the model is equivalent to a model corresponding to a patient, can well reflect the difference of tumors among different individuals, and can better reflect the actual condition of clinical patients compared with the traditional model; secondly, the PDX model can be passaged, and the passaged tumor tissue can keep high consistency with the initial tumor tissue in gene copy number, gene mutation, expression mode and the like, which is equivalent to the amplification of a patient tumor sample, thereby providing good conditions for drug screening and the study of a patient disease mechanism; and thirdly, the tumor blocks cultured by the PDX can be frozen for resuscitation, and a new PDX model can still be constructed, which is equivalent to the construction of a tumor living body library of different patients.
The prior art is mainly to perform subcutaneous modeling after fresh synovial sarcoma tissues are subjected to a mincing treatment or a cell homogenization treatment. For example, CN201910462582.8 of the applicant's prior application provides a synovial sarcoma cell line hSS-005R and its progeny cell line. The method comprises the steps of taking hSS-005R cells in a synovial sarcoma cell line to carry out subcutaneous unilateral inoculation on a mouse, wherein after 1-3 weeks of inoculation, tumors begin to form and grow, and the mouse 30-50 days after inoculation is a mouse model for medical research.
Disclosure of Invention
However, the homogenization treatment is considered to destroy the characteristics of the microenvironment of the tumor tissue, and the consistency of the tumor, the surrounding stroma and the patient cannot be well maintained. In addition, synovial sarcoma develops well near the joints, and subcutaneous modeling is not considered to be a good model of the tumorigenic environment. The invention successfully establishes a tissue block transplanting mode in the knee joint of the mouse, better maintains the stable relation between the tumor and the peripheral interstitium, successfully establishes an in-situ modeling model of the synovial sarcoma joint, and maintains the consistency of the growth environment of the model tumor and the tumor of a patient.
Therefore, the invention provides a construction method of a synovial sarcoma xenograft mouse model, which comprises the steps of collecting a synovial sarcoma patient tumor tissue block, and then transplanting a fresh synovial sarcoma tumor tissue block into an immunodeficient mouse in a xenograft in situ under aseptic conditions, wherein the xenograft in situ comprises selecting an immunodeficient mouse with the age of 2-6 weeks, the sex of the mouse is consistent with the sex of the patient, soaking the collected fresh synovial tumor tissue block of the patient in a sterile incubation liquid containing fetal calf serum, fixing the mouse after anesthesia, implanting the tumor tissue block of the patient into a joint capsule of the mouse, suturing an incision, and breeding the mouse to enable the tumor in the joint capsule to grow.
In one specific embodiment, the skin of the mouse knee joint is first cut longitudinally with a sterile ophthalmic scissors, and then the joint capsule is opened along the medial longitudinal incision of the patellar ligament with a microscissor or a microslade, followed by implantation of a synovial sarcoma tissue mass in the joint capsule.
In one embodiment, after implantation of the synovial sarcoma tissue mass in the joint capsule, the joint capsule is sutured first, followed by suturing the skin incision; the joint capsule is a knee joint capsule.
In a specific embodiment, the synovial sarcoma patient tumor tissue mass has a unilateral length of less than or equal to 3mm, preferably less than or equal to 1 mm.
In a specific embodiment, the weight of the mouse in orthotopic transplantation is 8-20 g, preferably 12-15g, the sterile incubation liquid further comprises an antibiotic, preferably the sterile incubation liquid comprises an antibiotic and a DMEM medium or an RPMI-1640 medium, and more preferably the antibiotic comprises penicillin and streptomycin.
In a specific embodiment, the immunodeficient mouse is a NOD/SCID mouse.
In a specific embodiment, the method further comprises observing the synovial sarcoma size in the joint capsule of the mouse using a small animal live imager, preferably also observing whether the tumor has metastasized to the viscera of the mouse using a small animal live imager.
In a specific embodiment, the method further comprises breeding the mouse such that the mouse is dissected after the tumor growth in the joint capsule, and the tumor is detached to obtain P0 tumor generation tumor body, and the tumor generation P0 tumor generation tumor body is xenografted in situ into the immunodeficient mouse to obtain P1 tumor generation tumor body, and optionally the tumor generation P1 tumor generation tumor body is xenografted in situ into the immunodeficient mouse to obtain P2 tumor generation tumor body, and so on for passage; preferably the method further comprises the step of programming the P0 generation tumor mass to be cooled and stored in liquid nitrogen, and optionally the steps of programming the P1 and P2 generation tumor mass to be cooled and stored in liquid nitrogen.
The invention also provides a construction method of the synovial sarcoma xenograft mouse model, which comprises the steps of xenogeneically transplanting P0 generation synovial sarcoma tumor tissue, P1 generation synovial sarcoma tumor tissue or P2 generation synovial sarcoma tumor tissue in a fresh mouse model into an immunodeficient mouse under aseptic condition, wherein the xenogeneic transplantation comprises the steps of selecting immunodeficient mice with the age of 2-6 weeks, keeping the sex of the mice consistent with the sex of patients, soaking the synovial sarcoma tumor tissue blocks in sterile incubation liquid containing fetal calf serum, fixing the mice after anesthesia, implanting the synovial sarcoma tumor tissue blocks into joint capsules of the mice, suturing incisions, and breeding the mice to enable tumors in the joint capsules to grow.
The invention also provides application of the mouse model obtained by the method in screening drugs for preventing or treating synovial sarcoma.
The invention also provides a construction method of the synovial sarcoma xenograft mouse model, which is characterized by comprising the steps of recovering the synovial sarcoma patient tumor tissue block stored in a liquid nitrogen environment, then transplanting the recovered synovial sarcoma patient tumor tissue block into an immunodeficiency mouse in a xenograft in situ under an aseptic condition, wherein the xenograft in situ comprises selecting an immunodeficiency mouse with the age of 2-6 weeks, keeping the sex of the mouse consistent with the sex of the patient, soaking the recovered synovial sarcoma patient tumor tissue block in a sterile incubation liquid containing fetal calf serum, fixing the mouse after anesthesia, transplanting the patient tumor tissue block into a joint capsule of the mouse, suturing an incision, and breeding the mouse to enable the tumor in the joint capsule to grow.
In a specific embodiment, the method further comprises the steps of performing programmed cooling and liquid nitrogen preservation and rapid heating resuscitation on the collected synovial sarcoma patient tumor tissue block.
In a specific embodiment, the synovial sarcoma patient tumor tissue block is preserved in DMEM containing DMSO, FBS and NEAA, the temperature is reduced to the liquid nitrogen temperature by more than three sections of procedures, and the synovial sarcoma patient tumor tissue block is preserved in the liquid nitrogen temperature, and the rapid heating resuscitation refers to resuscitating frozen tissues in a constant-temperature water bath at 36-38 ℃.
The invention also provides a construction method of the synovial sarcoma xenograft mouse model, which comprises the steps of heterogeneously and in situ transplanting P0 generation synovial sarcoma tumor tissue, P1 generation synovial sarcoma tumor tissue or P2 generation synovial sarcoma tumor tissue in a mouse model which is frozen in a liquid nitrogen environment and revived under an aseptic condition into an immunodeficient mouse, wherein the heterogeneously and in situ transplanting comprises selecting an immunodeficient mouse with the age of 2-6 weeks, keeping the sex of the mouse consistent with the sex of a patient, soaking the synovial sarcoma tumor tissue block in a sterile incubation liquid containing fetal calf serum, fixing the mouse after anesthesia, transplanting the synovial sarcoma tumor tissue block into a joint capsule of the mouse, suturing an incision, and breeding the mouse to enable the tumor in the joint capsule to grow.
The invention has at least the following beneficial effects:
the invention successfully establishes an in-situ tissue block transplanting mode, better maintains the stable relation between the tumor and the peripheral interstitium, particularly the in-situ modeling of the joint capsule, and maintains the consistency of the growth environment of the model tumor and the tumor of a patient. Compared with subcutaneous modeling, the in-situ modeling condition is more real, the screened drug scheme can be better used on the patient, and the in-situ modeling is theoretically easier to generate tumor metastasis than the subcutaneous modeling. Generally, the establishment and application of the synovial sarcoma in situ model are expected to improve the effectiveness of clinical synovial sarcoma treatment and the prognosis of patients, prevent patients from causing disability and even death to bring great medical social burden, and reduce treatment cost and government financial expenditure.
Drawings
FIG. 1 is a photograph of synovial sarcoma patient and a photograph of synovial sarcoma PDX mouse subcutaneous modeling.
FIG. 2 is the synovial sarcoma PDX mouse knee joint capsule in situ modeling photograph and Micro-CT detection mouse in situ tumor formation.
FIG. 3 is a graph showing the HE comparison of synovial sarcoma of patients and synovial sarcoma of PDX mice.
FIG. 4 is a photograph showing the immunohistochemistry comparison of primary synovial sarcoma of patients and PDX mouse synovial sarcoma tumors.
FIG. 5 is a photograph showing the immunohistochemistry comparison of synovial sarcoma relapsed from patients with PDX mouse synovial sarcoma tumors.
Detailed Description
Approved by the ethical committee of hospitals, patients sign informed consent before sample collection, collected tissues are subjected to histopathological examination, and tumor tissue blocks of synovial sarcoma patients, which are pathologically diagnosed, are stored in sterile physiological saline and placed on ice. Dividing the tissue block into 3 parts, and directly treating (see below for details) the tissue block under aseptic conditions in part 1 to perform xenografting into immunodeficient mice; part 2 was placed in DMEM (a medium containing various amino acids and glucose) supplemented with 10% DMSO (dimethyl sulfoxide), 90% FBS (fetal bovine serum) and 1% NEAA (non-essential amino acids), gradient-frozen to-80 ℃, and placed in liquid nitrogen for storage; and the part 3 is put into liquid nitrogen for quick freezing and then is put into the liquid nitrogen for preservation. Wherein, part 1 is used for modeling, part two is used for resuscitation and inoculation, and part three is used for gene detection for submission.
Comparative example and example
The comparative example is subcutaneous inoculation of mice, and the example is in situ implantation in the joint cavity of mice.
Subcutaneous inoculation: selecting 10 NOD/SCID mice (or balb/c nude mice) with 4 weeks of age, namely non-obese diabetic/severe combined immunodeficiency mice, keeping the sex consistent with the sex of a patient (so as not to be interfered by some unknown hormones), weighing about 12-15g, soaking fresh tumor tissue (or synovial sarcoma patient tumor tissue frozen and recovered by liquid nitrogen) in sterile incubation solution of pure FBS + penicillin and streptomycin (the final concentration of the penicillin is 100U/ml, and the final concentration of the streptomycin is 100U/ml), and cutting into 1 x 1mm small blocks by using sterile scissors. Mice were anesthetized and fixed, skin incisions were made near the axilla on the right side, tumor tissue blocks were implanted subcutaneously (P0), and surgical incisions were sutured. Mice were observed daily for tumor growth. Tumors grow to lengths exceeding 5 mm. Tumor volume was calculated by measuring tumor volume 2 times per week with a vernier caliper and recording tumor length (a) and width (b) (V ═ a × b) 2 /2). Meanwhile, a small animal living body imaging instrument is adopted to observe whether the tumor is transferred to the viscera of the mouse or not, and the volume V of the tumor body is increased to 1500mm 3 When the time is about 42 days later, the animal is dissected, the tumor body is stripped and weighed, part of tumor mass is inoculated and passed, and the rest is placed into liquid nitrogen for preservation.
In-situ implantation in the joint cavity:
1. 10 NOD/SCID mice with 4 weeks of age are selected, the sex of the mice is consistent with that of patients, and the weight of the mice is about 12-15 g.
2. Fresh sterile tumor tissue (or tumor tissue after liquid nitrogen preservation and resuscitation) was cut into 1 x 1mm pieces with sterile scissors and stored in pure FBS.
3. After mice were anesthetized, the mouse hairs around the knee joints were shaved and disinfected with 75% ethanol.
4. Fixing the experimental mouse on an operating table, cutting the skin of the knee joint of the mouse by a sterilized ophthalmic scissors longitudinally by about 1cm, and opening the joint capsule along the longitudinal incision on the inner side of the patellar ligament by using a pair of microscissors or a microslip. The minced tumor tissue mass is implanted into the joint capsule.
5. The joint capsule was closed with 8-0 surgical sutures and the skin incision was closed with 6-0 surgical sutures.
6. The post-operative mice were placed on a 37 ℃ hotbed until resuscitated, and penicillin was routinely administered intraperitoneally to prevent post-operative infection.
7. The mice after operation are placed in a cage for breeding, the activity of the mice is not limited, and the mice can be fed with food and water at will. The synovial sarcoma tumorigenesis condition is observed every week, the synovial sarcoma size and the visceral metastasis condition (whether the tumor is transferred to the viscera of the mouse) are observed by using a small animal living body imager every 3 weeks, and the tumor volume is increased to 1500mm 3 Dissecting knee joint at the time or 45-60d later, stripping tumor body, weighing, inoculating partial tumor mass, subculturing, and preserving in liquid nitrogen. The process of preserving tissue mass or whole tumor mass in liquid nitrogen includes programmed cooling, specifically several temperature gradients of 4 ℃, -20 ℃, -80 ℃ (special freezing refrigerator) and liquid nitrogen.
Resuscitating and inoculating primary and passage tumor tissues of synovial sarcoma: p0, P1 and P2 frozen in liquid nitrogen are taken to replace synovial sarcoma tumor tissues, quickly re-warmed to normal temperature, and respectively inoculated in the armpit and the joint cavity of 10 NOD/SCID mice with the same sex, and the method is the same as the method.
Because synovial sarcoma tissue of a patient is a valuable material for study, the tumor tissue of tumor-bearing mice needs to be passaged to generate more tumor-bearing mice for experiments. The reason why the tumor tissue is frozen and thawed is that too large tumor masses cannot be used in each modeling experiment, and the tumor masses left for seed storage, namely frozen and thawed, can be used in the next experiment. Tumor tissues are passaged through P0, P1, P2 and the like, and the tumor tissues are not changed theoretically, so that the experimental effects of the respective generations can be regarded as the same. And when in-situ modeling or subcutaneous modeling is carried out, as long as the generation of the tumor P0 is successful, the subsequent generation and recovery of P1, P2 and the like are simple and easy and can be completely successful.
FIG. 1 is a photograph of a synovial sarcoma patient and a synovial sarcoma PDX mouse (specifically, a muddy and hairless Balb/c nude mouse) subcutaneous modeling photograph. In which panel a is a pre-operative image of synovial sarcoma from patient 005-PA (where PA represents patient, 005 is patient number) showing swelling of the right knee joint as seen by the + sign. Fig. B, C and D are subcutaneous modeling processes. As can be seen from the graphs C and D, the image of a nude mouse implanted with the patient relapsed synovial sarcoma 005R-P4 (wherein R is recurrence, which means relapse; P4 represents the fourth generation of the PDX model; so 005R-P4 is the fourth generation of the 005 relapsed tumor PDX model of the patient) shows that the maximum diameter of the tumor of the mouse is about 1cm, the tumor body shows swelling growth, the tumor body has a coating and has a regular shape, the subcutaneous tumor of the mouse is dissected, the boundary between the subcutaneous tumor and the surrounding tissues is found to be obvious, the blood circulation is rich, other organs are examined, and the metastasis of lung, bone marrow, liver and the like is not found; the time for the 005R-P4 mouse to touch subcutaneous tumor is about 21 days after being planted.
FIG. 2 is photograph of synovial sarcoma PDX mice (in particular, muddy white-hair NOD/scid mice) knee joint capsule in situ modeling (panels E and F) and Micro-CT detection of in situ tumor formation in mice (panel G).
FIG. 3 is a graph showing the HE comparison of synovial sarcoma of patients and synovial sarcoma of PDX mice. Fig. 3a and 3b are HE staining maps of primary and recurrent synovial sarcoma of patients, respectively, showing that spindle cells are interlaced with fibroblasts. FIG. 3c and FIG. 3d are the HE staining patterns of PDX model established for primary and recurrent synovial sarcoma tissues of patients, respectively, showing the arrangement of spindle cells and fiber-interlaced cells, and the histological features are consistent with the pathology of patients.
FIG. 4 is a photograph showing the immunohistochemistry comparison of primary synovial sarcoma of patients and PDX mouse synovial sarcoma tumors. From a comparison of the patient's primary tumor with PDX mouse immunohistochemistry in FIG. 4, the patient stained essentially identically to PDX synovial sarcoma at Bcl-2, CD34, CD99, CK, EMA, Ki-67, s-100, SMA, Vim, Tle-1.
FIG. 5 is a graph showing the immunohistochemistry of patients with relapsed synovial sarcoma versus PDX mouse synovial sarcoma tumor. As can be seen from fig. 5, the tumors inoculated in the mice were substantially identical to the immunohistochemistry of the synovial sarcoma recurrent in the patient from which they were derived, and the tumor tissues were identical to the recurrent tumor in the patient.
005-F1 PDX in FIGS. 4 and 5 refers to the first generation PDX model in which patient tumor tissue was implanted, and F1 and P0 (tumor grown after patient tumor tissue was first implanted in mice) represent the same meaning.
The in-situ modeling in PDX has profound significance, the tumor obtained by in-situ modeling has better homogeneity with the primary tumor of a patient, the pathogenesis of synovial sarcoma can be deeply discussed after successful modeling, the research and development results can be better repeated in the body of the patient based on the in-situ modeling, meanwhile, a large number of limited synovial sarcoma samples in the body of a clinical patient can be amplified, and a xenograft mouse is used for replacing the clinical patient to carry out different combined drug screening, so that the most appropriate drug is selected, the effectiveness is greatly improved, and the side effect of the drug is reduced. Therefore, the in-situ modeling and application can hopefully improve the effectiveness of clinical synovial sarcoma treatment and the prognosis of patients, prevent the patients from being disabled and even dying to bring great medical and social burden, reduce the treatment cost and the government financial expenditure, have great practical significance in the aspects and have great economic benefit.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, numerous and varied simplifications or substitutions may be made without departing from the spirit of the invention, which should be construed as falling within the scope of the invention.
Claims (9)
1. A construction method of a synovial sarcoma xenograft mouse model is characterized by comprising the steps of recovering a synovial sarcoma patient tumor tissue block stored in a liquid nitrogen environment, transplanting the recovered synovial sarcoma patient tumor tissue block into an immunodeficient mouse NOD/SCID mouse in a xenograft in situ under an aseptic condition, wherein the unilateral length of the patient tumor tissue block is less than or equal to 1mm, the xenograft in situ comprises selecting an immunodeficient mouse with the age of 2-6 weeks, keeping the sex of the mouse consistent with the sex of the patient, soaking the recovered synovial tumor tissue block in a sterile incubation solution containing fetal calf serum, fixing the mouse after anesthesia, transplanting the patient tumor tissue block into a knee joint capsule of the mouse, specifically, cutting the knee joint skin of the mouse longitudinally by using a sterilized ophthalmic scissors, opening the knee joint capsule along the inner side longitudinal incision of a patellar ligament by using a pair of microscleres or a microsclers, then implanting a synovial sarcoma patient tumor tissue block in the knee joint capsule; suturing the incision, and breeding the mouse to enable the tumor in the knee joint capsule to grow; the method also comprises breeding the mice to ensure that the mice are dissected after the tumor in the knee joint capsule grows, and stripping the tumor body to obtain P0 generation tumor body.
2. The method of claim 1, further comprising programmed cooling and liquid nitrogen preservation of the collected synovial sarcoma patient tumor tissue mass and rapid warming resuscitation.
3. The method according to claim 2, wherein the synovial sarcoma patient tumor tissue block is preserved in DMEM containing DMSO, FBS and NEAA, and the three or more sections are cooled to liquid nitrogen temperature and preserved in the liquid nitrogen temperature, and the rapid warming resuscitation refers to resuscitating the frozen tissue in a constant temperature water bath at 36-38 ℃.
4. The method according to claim 1, wherein the weight of the mouse is 8-20 g when the mouse is transplanted in situ, the sterile incubation solution comprises antibiotics and DMEM medium or RPMI-1640 medium, and the antibiotics comprise penicillin and streptomycin.
5. The method of claim 1, further comprising observing the size of synovial sarcoma in the knee capsule of the mouse using a small animal in vivo imager.
6. The method of claim 1, further comprising xenogeneic transplantation of the tumor mass P0 into the knee joint capsule in immunodeficient mice to give tumor mass P1, xenogeneic transplantation of the tumor mass P1 into the knee joint capsule in immunodeficient mice to give tumor mass P2, and so on.
7. The method of claim 6, further comprising programming and storing the P0 generation tumor mass fraction in liquid nitrogen, and comprising programming and storing the P1 and P2 generation tumor mass fraction in liquid nitrogen.
8. The method of claim 1, wherein the implantation of the synovial sarcoma tumor tissue mass in the knee capsule is followed by suturing the knee capsule and then suturing the skin incision.
9. The method of claim 1, wherein the tumor mass volume in the knee capsule of the mouse is increased to 1500mm 3 The knee joints of the mice are dissected at the same time or 45 days later, and the tumor bodies are stripped to obtain P0 generation tumor bodies.
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