Construction method and application of osteosarcoma PDX model

Abstract

This invention relates to the field of tumor animal model technology, and provides a method for constructing an osteosarcoma PDX model and its application. The method for constructing the PDX model includes the following steps: S1 pretreatment of tumor samples from osteosarcoma patients, and S2 subcutaneous inoculation in mice. S1 specifically includes: first washing the tumor samples with physiological saline, then storing and transporting the tumor samples in a low-temperature culture medium at 0-4°C, and then washing the tumor samples with room-temperature PBS solution; and mechanically cutting the tumor samples into 2-3 mm pieces. 3 The tissue blocks. S2 specifically includes: based on the number of tissue blocks formed from the tumor sample, preparing at least two immunodeficient mice for each patient to construct the PDX model; selecting inoculation sites from both sides of the lower limbs and / or both sides of the upper limbs of each mouse. By establishing tumor-bearing models through multiple sites, the limited number of tumor samples and experimental animals can be utilized to the maximum extent, rapidly expanding the number of PDX models and improving modeling efficiency and cost.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tumor animal models, in particular to a method for constructing an osteosarcoma PDX model and application thereof. BACKGROUND

[0002] Patient Derived Tumor (PDT) is derived from patients, and tumor tissues are diverse, heterogeneous and complete, which has important scientific research and clinical application value. With the development of immunodeficient mice, it is possible to inoculate patient-derived tumor tissues into immunodeficient mice, which provides a very good model for the development of tumor drugs, the development of immunotherapy and the testing of various targeted drugs. PDX model (Patient-Derived Xenograft Model, PDX) is a new generation of human tumor xenograft model established by inoculating surgically resected patient tumor tissues into immunodeficient mice. This model retains the histological and genetic characteristics of primary tumors and maintains the heterogeneity of patient tumors, and the pharmacodynamic results have a high correlation with clinical results.

[0003] The existing method for constructing an osteosarcoma PDX model is as follows: after a patient tumor specimen is washed with RPMI 1640 medium (Corning, New York, USA), it is cut into 3-5 mm 3 small pieces, then sucked into a 25g puncture cannula, the cannula is used for bluntly separating connective tissue, and then the tissue cannula is pushed to inoculate subcutaneously (s.c.) in the groin of the lower limbs of an immunodeficient mouse; the volume of the subcutaneous tumor is measured once a week, and the tumor is removed when it reaches 500 mm 3 for passage and cryopreservation. The passage method is consistent with the method for constructing an osteosarcoma PDX model, which includes tissue washing, cutting into 3-5 mm 3 small pieces, and inoculating subcutaneously in the groin using a puncture cannula. The cryopreservation method is as follows: first, the tumor sample is cut into 3 mm 3 small pieces, soaked in a suitable preservation solution, 60% of which is RPMI 1640 medium, 30% of which is fetal bovine serum (Hangzhou Skeying Biological Engineering Materials Co., Ltd., Hangzhou, China), and 10% of which is dimethyl sulfoxide (Ameresco, Framingham, USA); then the tumor is placed in a Nalgene programmed cooling box (New York, USA) at-80°C overnight, and stored in liquid nitrogen; using a constant temperature water bath, the water temperature is adjusted to 37°C, the frozen tube stored in liquid nitrogen is put into the water bath for thawing, and the frozen tumor is incubated at 37°C until it is completely melted; then, the subcutaneous model is re-established according to the foregoing method.

[0004] The existing osteosarcoma PDX model construction method has the following disadvantages: multiple people are required to cooperate when using a trocar, and the subcutaneous puncture technique needs to be mastered; the tumor sample volume size and inoculation quantity are strictly limited, because the tissue trocar diameter is limited, which makes it difficult to operate on large tumor tissues; the tumor is frozen using traditional programmed cryopreservation, which takes a long time. SUMMARY

[0005] To solve the above technical problems, the present application provides a method for constructing an osteosarcoma PDX model, comprising: S1, pretreatment of the tumor sample of an osteosarcoma patient, and S2, subcutaneous inoculation of mice. S1 specifically comprises: S11, first washing the tumor sample with normal saline, then storing and transporting the tumor sample in a low-temperature culture solution at 0-4℃, and then washing the tumor sample with PBS at room temperature, wherein the culture solution comprises more than 85% PBS solution and 10% fetal bovine serum; S12, cutting the tumor sample into 2-3mm 3 tissue blocks by mechanical method. S2 specifically comprises: S21, based on the number of tissue blocks formed by the tumor sample of the patient, preparing at least 2 immunodeficient mice for each patient to construct a PDX model; S22, selecting inoculation sites from both sides of the lower limbs and / or both sides of the upper limbs of each mouse.

[0006] Preferably, the low-temperature culture solution at 0-4℃ contains penicillin 100-500 U / ml and streptomycin 100-500 ug / ml.

[0007] Preferably, the total storage and transportation time of the tumor sample in the low-temperature culture solution at 0-4℃ is not more than 30 minutes.

[0008] Preferably, the shape of the tissue block is square flake.

[0009] Preferably, the immunodeficient mouse is a 6-8 week old T, B, and NK cell triple extremely severe immunodeficient mouse.

[0010] Preferably, at most 4 inoculation sites are selected for each mouse, and the 4 inoculation sites are located on both sides of the lower limbs and both sides of the upper limbs of the mouse.

[0011] Preferably, the inoculation sites are preferentially selected from both sides of the lower limbs of the mouse.

[0012] Specifically, when the inoculation site is on the upper limb side, an opening of 0.5 cm is made above the xiphoid process in the upper abdominal position using ophthalmic scissors, and the tumor tissue is delivered to the corresponding inoculation site along the subcutaneous separation channel using forceps. The forceps are left in the site for about 10 seconds, and after the tumor tissue no longer slides with the forceps, the incision is sutured. When the inoculation site is on the lower limb side, an opening of 0.5 cm is made about 1 cm below the xiphoid process on the abdominal midline, the subcutaneous tissue is bluntly separated using forceps, and the sample tissue block is delivered to the mouse lower limb groin position using a blunt tip. After staying for about 5 seconds, the forceps are removed, and the incision is sutured using simple interrupted suture.

[0013] Further, it further comprises: S3, model passage. Specifically, it comprises: S31, when the tumor volume in the mouse to be inoculated reaches about 1000 mm 3 After that, the tumor tissue of the mouse is removed to obtain P0 primary; S32, the P0 primary is grouped, and one group is used for passage to expand the scale of the model.

[0014] The application also provides a use of the osteosarcoma PDX model obtained by the foregoing method in screening drugs for preventing or treating osteosarcoma.

[0015] The features and advantages of the application include:

[0016] (1) The upper abdominal and / or lower abdominal incision is adopted, which is particularly suitable for multiple sample and multiple site inoculation. Compared with the existing tissue sleeve needle, the pretreatment of the tumor of the patient and the mode of animal inoculation are simpler, and single-person operation can be realized.

[0017] (2) The ex vivo treatment time of the tumor sample is strictly limited within 30 minutes, which can greatly improve the success rate of tumor tissue inoculation.

[0018] (3) The tumor-bearing model established by multiple sites can maximize the use of limited tumor samples and limited experimental animals, rapidly expand a large number of PDX models, and improve the modeling efficiency and cost. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present disclosure, and other drawings can be obtained by those skilled in the art without creative labor.

[0020] Figure 1 is a schematic diagram of the selection of the tumor transplantation site when the mouse PDX modeling according to the application;

[0021] Figure 2 Figure 1 is a pathological tissue section diagram of each generation of tumor tissue or PDX model according to the present application;

[0022] Figure 3 Figure 2 is a schematic diagram of body weight change after drug sensitivity experiment on the PDX model constructed by the method of the present application;

[0023] Figure 4 Figure 3 is a schematic diagram of tumor size change after drug sensitivity experiment on the PDX model constructed by the method of the present application. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present disclosure will be described clearly and completely below in combination with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are only part of the embodiments of the present disclosure, rather than all the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present disclosure.

[0025] Example 1 Construction of osteosarcoma PDX model

[0026] 1.1 Preprocessing of patient samples

[0027] Osteosarcoma tissues were obtained by surgical operation. The osteosarcoma tissues were selected from ten osteosarcoma patients who underwent surgical treatment in Tianjin Cancer Hospital from January 2023 to October 2023. The osteosarcoma PDX model construction method provided by the present application will send the tumor tissue samples obtained after the patient undergoes surgical resection of the tumor to the experimental animal center for PDX model inoculation as soon as possible. The pathological tissue sections of the patient tumor tissues obtained by the present application are shown in Figure 1 F0. Figure 2

[0028] ​The osteosarcoma tissue is collected in a culture solution for inoculation. The culture solution comprises a suitable amount of phosphate buffered saline solution (PBS solution) and fetal bovine serum FBS (Gibco, Thermo Fisher Scientific Ltd), wherein the PBS solution accounts for at least 85% and the fetal bovine serum accounts for 10%. In some embodiments, the PBS solution accounts for 90%. Specifically, after the postoperative osteosarcoma tissue sample is washed with normal saline, it is transferred to a destination, such as a laboratory, in about 20 ml of the culture solution stored at 0-4°C in a 50 ml centrifuge tube. In particular, the time for transporting the tissue sample is strictly limited to 30 minutes, which helps to ensure the activity of the tissue sample. According to the volume of the tissue block and the storage time of the tissue block in the culture solution, a corresponding volume of the culture solution is selected for storage. Preferably, the culture solution for transportation and storage contains 1%-5% of antibiotics, for example, contains penicillin 100-500 U / ml and streptomycin 100-500 ug / ml. The tumor sample is first pre-washed in PBS at room temperature, and then cut into tissue blocks with a size of 2-3 mm 3 by a mechanical method, in the shape of a square flake, because such size and shape of the tissue block facilitates the penetration of the cryoprotective solution, thereby more fully protecting the tumor tissue block from damage and maintaining the integrity of the tissue structure.

[0029] In some embodiments, the osteosarcoma tissue sample washed with normal saline can be divided into two groups, the first group is used for PDX modeling, and the processing and preservation method is as described above. The other group is used for comparison with the modeling results, and can also be used for frozen storage for subsequent inoculation. The obtained second group of fresh samples are quickly frozen in liquid nitrogen and stored in a liquid nitrogen tank or placed in a 10% neutral formalin solution for storage.

[0030] 1.2 Mouse inoculation

[0031] After obtaining the fresh specimen, prepare the mice for transplantation, and prepare 2-4 mice of varying quantities for each patient according to the size of the sample. Use 6-8 week old T, B, and NK cell triple severe immunodeficient mice (NOG), and each patient's tumor sample will construct a corresponding PDX model. The mice used to construct the model will be carefully cared for. The mice are raised in a specific pathogen-free (SPF) environment with high-efficiency filtration, constant temperature and humidity under positive pressure. All mice are inoculated after being anesthetized by isoflurane inhalation.

[0032] The PDX model construction method provided by the application adopts a multi-point inoculation method to maximize the preservation of precious patient-derived tumor tissues. According to the tumor sample amount, the number of points for inoculating each mouse is determined. Generally, each mouse can be inoculated with a maximum of 4 tissue blocks, one tissue block for each point, and each mouse can be inoculated with 4-6 2-3mm 3 samples. The 4 inoculation sites are located on both sides of the upper limbs and both sides of the lower limbs, as shown in Figure 1 . The number of inoculation sites is selected according to the tumor sample amount of a single patient. Generally, 8-16 tissue blocks for inoculation are obtained from a single patient, so 2-4 mice are needed for inoculation for a single patient, and the lower limbs of the mice are preferentially selected as the inoculation sites.

[0033] When the lower limbs (3, 4 sites) are selected as the inoculation sites, a 0.5cm opening is cut about 1cm below the xiphoid process on the midline of the abdomen, the subcutaneous tissue is bluntly separated with forceps (blunt ophthalmic forceps), and the sample tissue block is delivered to the mouse groin (3) position with the blunt end, and after staying for 5 seconds, the forceps are removed, and the same operation is performed on the other side (4 sites). After inoculation, the incision is sutured by simple interrupted suture.

[0034] When the upper limbs are selected as the inoculation sites, an opening is made with ophthalmic scissors 0.5cm above the xiphoid process on the upper abdomen, Figure 1 , and the 1, 2 sites are used as the reference, the subcutaneous tissue of one side of the upper limb is bluntly separated with forceps, the separated tumor tissue is clamped with forceps, and then sent to the corresponding inoculation site (1, 2) along the subcutaneous separation channel. After the target site is sent, the forceps are left in the site for about 10 seconds, and after the tumor tissue no longer slides with the forceps, the incision is sutured, and the tumor tissue inoculation is completed. When the number of inoculation sites is ≤2, the 3, 4 sites are preferentially selected because they have a rich blood vessel network and subcutaneous fat, which can improve the success rate of PDX model inoculation.

[0035] After xenotransplantation, the growth of the mice is observed, and the observation is continued for at least 60 days after transplantation. Because the tumor growth rate of each mouse is not the same, and the tumor in the mouse body grows too large, so according to experience, when the tumor volume of the inoculated mouse reaches 1000 mm 3 , the mouse is handled in batches, euthanized, and the mouse tumor tissue (P0 primary) is removed. The removed tumor tissue needs to be divided into three groups, the first group is quick-frozen for subsequent genome sequencing. The second group is fixed with formalin for pathological sectioning and immunohistochemical preparation. The third group is passaged for tumor tissue inoculation in mice to expand the model scale.

[0036] 1.3 Modeling monitoring

[0037] The rapid and stable growth of PDX tumors can be monitored in several ways.

[0038] A. Tumor growth monitoring: By regularly measuring the size of the tumor, calculating the growth rate and doubling time of the tumor, and evaluating the growth of the model. Regularly measure the weight of the mouse, observe the health status of the mouse, and evaluate the progress of the model. Non-invasive monitoring techniques such as CT can be used.

[0039] B. Histopathological examination: Through histopathological examination, observe the tissue structure and cell morphology of the tumor, and evaluate the similarity and difference of the model.

[0040] C. Drug response evaluation: Evaluate new drugs or treatment regimens, observe the efficacy and adverse reactions of drugs on the model, and predict their efficacy and safety in humans.

[0041] Through the above monitoring methods, the growth, biological characteristics, and drug response status of the PDX model can be comprehensively evaluated, providing strong support for new drug research and development, tumor diagnosis and treatment.

[0042] Example 2: Subculture of osteosarcoma PDX model

[0043] S1. Prepare tools and materials: sterile surgical instruments, gloves, masks, sterile operating table, physiological saline, sterile gauze, sterile PBS culture solution, culture dishes, centrifuge tubes, etc.

[0044] S2. Take material: Take tumor tissue from the already tumor-bearing PDX model, rinse with physiological saline, and remove necrotic tissue.

[0045] S3. Cut the tumor tissue: Cut the tumor tissue into small pieces, about 2mm in size 3 .

[0046] S4. PBS cleaning: Put the cut tumor tissue into a centrifuge tube, add appropriate amount of PBS, and clean for 5 minutes to remove blood and other impurities from the tumor tissue.

[0047] S5. Implantation: Implant the cleaned tumor tissue into the subcutaneous tissue of immunodeficient mice, and inoculate tumor pieces of a certain size in each part.

[0048] S6. Observation: Regularly observe the tumor formation of immunodeficient mice and record the growth of the tumor.

[0049] S7. Data analysis: According to the tumor growth curve, latent period, tumor formation rate and other data, evaluate the stability and consistency of the model.

[0050] S8. Passage: When the tumor grows to a certain size, the tumor tissue can be taken out for re-passaging to establish the next generation of PDX model.

[0051] Example 3 Verification of osteosarcoma PDX model

[0052] 3.1 Success rate of modeling

[0053] Referring to Table 1, F1 is the osteosarcoma tissue obtained after surgery, P0 is the primary model obtained after inoculation based on the tumor tissue of the patient after surgery, and P1 is the tumor model obtained after inoculation and passage based on the tumor model of P0. According to the modeling method provided by the present application, the tumor survival rate is about 80%.

[0054] Table 1

[0055] Number of patients Number of F1 tissue pieces (pre-P0 inoculation) Number of P0 surviving tissue pieces Number of P1 pre-inoculation tissue pieces Number of P1 surviving tissue pieces 10 people 160 129 774 620

[0056] 3.2 HE staining analysis

[0057] The second group of preserved osteosarcoma tissues (human tumor), P0 generation tumor tissues and P1 generation tumor tissues were fixed with 10% formalin for 24 h and embedded with paraffin. The aforementioned three kinds of tumor tissues were cut into 4 μm slices and subjected to hematoxylin-eosin staining (HE). Hematoxylin is alkaline, which makes the chromatin in the nucleus and the nucleic acid in the cytoplasm purple blue; eosin is an acidic dye, which makes the components in the cytoplasm and extracellular matrix red. HE staining method is the most basic and widely used technique in histology, embryology and pathology teaching and scientific research. By observing the pathological sections and comparing the tumor structures, the tumor structure of the osteosarcoma model can be compared with that of the clinical patient.

[0058] After rapid observation under a microscope, it can be judged whether the staining is sufficient or excessive. If the staining is insufficient, it needs to be re-dyed in hematoxylin. If the staining concentration is too strong, the slice shows background staining, and an acidic solution can be used to lighten the staining to achieve differential staining of the slice. After this step, blue dye needs to be used again and washed under tap water.

[0059] The next step is eosin staining. Eosin is a counterstain for hematoxylin, which can differentially stain red blood cells, collagen, and smooth muscle through pink color. Differentiation also requires washing excess eosin with water according to the condition of the tissue. Then the stained slide is immersed in gradient ethanol and then in xylene for dehydration of the slide. After staining and dehydration, a mounting medium is used to encapsulate the slice.

[0060] Bright-field microscopy allows for the observation of hematoxylin and eosin (HE) stained slides. Hematoxylin stains cell nuclei blue-purple, while eosin stains cytoplasm and connective tissue light pink. Staining allows pathologists to easily distinguish different tissue types, such as muscle or connective tissue, and to detect abnormalities or irregularities in the sections. This helps identify changes reflecting specific pathologies, such as infection, chronic inflammation, or malignancy. The visible information allows for the observation of structural changes within the tissue, thereby determining the nature and progression of the disease.

[0061] See also Table 2 and Figure 2 F0 is a pathological section of osteosarcoma tissue obtained from the patient after surgery, F1 is a section of osteosarcoma tissue after the first generation (P0) inoculated into mice, and F2 is a section of osteosarcoma tissue after the second generation (P1) inoculated into mice.

[0062] Table 2

[0063] Tumor tissue generation number F0 F1 (P0) F2 (P1) Proportion of interstitial infiltration between mice (%) 0 <5% <20%

[0064] 3.3 Drug sensitivity test

[0065] Five successfully established P0 generation osteosarcoma PDX models were selected and subjected to drug sensitivity tests. The treatment regimens for osteosarcoma included: methotrexate, doxorubicin and cisplatin group; doxorubicin and cisplatin group; and drug solvent (vehicle) control group.

[0066] See Figure 3 and Figure 4 In the three groups, the drug solvent served as the control group (Vehicle), showing no significant effect on mouse body weight, although weight gradually increased over time, without any tumor inhibition. The other two groups served as clinical chemotherapy regimens for osteosarcoma, verifying the concordance between the osteosarcoma PDX model and clinical patient treatment regimens. The methotrexate, doxorubicin, and cisplatin group showed results consistent with clinical treatment, exhibiting less impact on mouse body weight compared to the doxorubicin and cisplatin group, indicating less influence on mouse condition, while also showing the most significant tumor inhibition. The other experimental group, the doxorubicin and cisplatin group, had a greater impact on body weight; although possessing tumor inhibition ability, its inhibition efficiency was lower than that of the methotrexate group. Using the above drug regimens, the methotrexate, doxorubicin, and cisplatin group can be selected as the optimal clinical treatment regimen to benefit patients.

[0067] Example 4: Application of the osteosarcoma PDX model

[0068] The speed of PDX modeling indicates the prognosis of patients, and PDX models with high tumorigenicity often show high malignancy or invasiveness. Patients who can establish PDX models, the faster the PDX establishment speed, the higher the malignancy. Therefore, optimizing the timeliness of establishing osteosarcoma PDX models for evaluating the efficacy of osteosarcoma treatment drugs has very practical significance for clinical drug guidance, especially targeted drugs.

[0069] By implanting and establishing PDX models, humanized mice can be obtained. Humanized PDX models can pair human tumors with human immune systems, more accurately reflecting human immunity and pathology. Researchers can use these models to evaluate the efficacy of immunotherapy (or combined immunotherapy regimens).

[0070] The PDX model of the present application can be widely used in new drug development, especially in the clinical trial patient screening and predictive biomarker research of target drugs. The PDX model can completely reproduce the characteristics of tumor pathology, tumor genetics, etc. In addition, the PDX model can also be used to accurately screen targeted and chemotherapy drugs to develop the best drug regimen. Real-time monitoring of drug treatment effect, and research on tumor drug resistance mechanism, etc.

[0071] The above only describes several embodiments of the present disclosure, and those skilled in the art can make various modifications or changes to the embodiments of the present disclosure according to the content disclosed in the application file without departing from the spirit and scope of the present disclosure.

Claims

1. A method of constructing an osteosarcoma PDX model, characterized by, Comprise: S1, tumor sample pretreatment of osteosarcoma patients, comprising: S11, first clean the tumor sample with normal saline, then store and transport the tumor sample in a low-temperature culture solution at 0-4 DEG C, and then clean the tumor sample with PBS solution at room temperature, wherein the culture solution comprises more than 85% PBS solution and 10% fetal bovine serum; S12, tumor sample is cut into 2~3mm tissue blocks by mechanical method; 3 S12, tumor sample is cut into 2~3mm tissue blocks by mechanical method; S2, subcutaneous inoculation of mice, comprising: S21, based on the number of tissue blocks formed by the tumor sample of the patient, prepare at least 2 immunodeficient mice for each patient for PDX model construction; S22, select inoculation sites from both sides of the lower limbs and / or both sides of the upper limbs of each mouse.

2. The method of constructing an osteosarcoma PDX model according to claim 1, wherein, The low-temperature culture solution at 0-4 DEG C contains penicillin 100-500 U / ml and streptomycin 100-500 ug / ml.

3. The method of constructing an osteosarcoma PDX model according to claim 2, wherein, The total duration of storing and transporting the tumor sample in the low-temperature culture solution at 0-4 DEG C is not more than 30 minutes.

4. The method of constructing an osteosarcoma PDX model according to claim 1, wherein, The shape of the tissue block is a square sheet.

5. The method for constructing an osteosarcoma PDX model according to claim 4, wherein the immunodeficient mice are 6-8 week old T, B and NK triple extremely severe immunodeficient mice.

6. The method of constructing an osteosarcoma PDX model according to claim 5, wherein, Up to 4 inoculation sites are selected for each mouse, and the 4 inoculation sites are located on both sides of the lower limbs and both sides of the upper limbs of the mouse.

7. The method of constructing an osteosarcoma PDX model according to claim 1, wherein, Further comprising: S3, model passage, comprising: S31, the tumor volume in the mouse to be inoculated reaches about 1000 mm 3 After that, the tumor tissue of the mouse is taken out to obtain P0 primary; S32, group the P0 primary, wherein one group is used for passage to expand the scale of the model.

8. The use of an osteosarcoma PDX model obtained by the method of any one of claims 1-7 in screening drugs for preventing or treating osteosarcoma.

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