A method for constructing a PDTX model of brain glioma and its application

Abstract

The present invention belongs to the technical field of tumor animal models, and specifically relates to a method for constructing a glioma PDTX model and its application. The present invention has formulated a tumor screening method suitable for constructing a glioma PDTX model, by screening tumor samples with high transcription levels of GFAP, SOX2, SLC1A3 and OLIG2 genes as ideal tumor sources through transcriptome sequencing of tumor tissues, and constructing a model through a new glioma PDTX model construction method provided by the present invention, which can effectively retain the original microenvironment characteristics of the tumor by transplanting the screened glioma tissues into immunodeficient mice, and significantly improve the success rate of glioma PDTX model construction.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor animal models, and in particular relates to a method for constructing a PDTX model of brain glioma and an application thereof. Background Art

[0002] In the prior art, the construction of brain glioma tumor models mainly adopts the following three methods: (1) preparing fresh tumor tissue from patients into single cell suspension, and injecting a certain amount of cell suspension into the mouse skull to form an orthotopic transplanted tumor; (2) preparing the patient-derived tumor into a single cell suspension or mincing it into tumor fragments, and transplanting an appropriate amount of cells or tumor fragments of appropriate size into the subcutaneous part of the mouse using a syringe or a cannula needle to form a xenograft tumor; (3) selecting a commercial tumor cell line for modeling, culturing the cells in vitro, and then injecting the cells into the animal body.

[0003] However, there are still many defects in the current conventional tumor model construction methods. For cell transplantation, it is difficult to form the same tumor microenvironment as before, and the microenvironment of brain tumors is often more complex. For the whole brain tumor xenotransplantation, it is difficult for the transplanted tumor to form a blood supply. The related tumor cells are more fragile than other tumors and require more complex nutrients, and have higher requirements for the transplantation environment, which leads to slow growth and low transplantation success rate. At the same time, simple tumor transplantation cannot replicate the state and microenvironment of the original tumor, including the original matrix cells (fibroblasts, lymphocytes, macrophages and endothelial cells), immune cells (T and B lymphocytes, etc.) and extracellular components (cytokines, growth factors, hormones, extracellular matrix ECM, etc.). There are also a large number of nerve cells in brain tumors, and their interactions with tumor cells and tumor microenvironment are also relatively complex. Ordinary tumor transplantation cannot completely ensure the good coexistence of the three. Summary of the invention

[0004] In view of the defects and shortcomings of the current existing technologies, the present invention provides a method for constructing a glioma PDTX model and its application, so as to better reflect the overall structure and microenvironment of a real glioma, and at the same time improve the success rate of constructing a glioma PDTX model.

[0005] To achieve the above objectives, the technical solutions adopted by the present invention include:

[0006] In a first aspect, the present invention provides a method for constructing a PDTX model of brain glioma, the method comprising the following steps:

[0007] S1. Screen tumor tissues with high transcription levels of brain tumor-related genes to obtain human tumor living tissues;

[0008] S2, washing the human tumor living tissue and cutting it into tissue blocks, and immersing the tissue blocks in a pretreatment solution for storage;

[0009] S3, inoculating the tissue blocks soaked in the pretreatment solution into mice for tumor transplantation;

[0010] S4. After inoculation, the mice were injected with human VEGFA and glutamine every week for culture, and then the constructed model was evaluated to obtain a successfully constructed brain glioma PDTX model;

[0011] The brain tumor-related genes include at least four of GFAP, P2RY12, SOX2, RBFOX3, NEFH, SLC17A7, NPY, SLC1A3, CSPG4 and OLIG2;

[0012] The high transcription level of the brain tumor-related gene means that the TPM value of the brain tumor-related gene is higher than the median TPM value of the corresponding brain tumor-related gene in the sample library.

[0013] Existing glioma tumor models are difficult to perfectly reflect the overall structure and complex microenvironment of real gliomas, especially they cannot fully reproduce the various cell types and interactions in the tumor microenvironment. In order to make up for this deficiency, the present invention provides a new method for constructing a glioma PDTX (patient-derived tumor xenograft) model, which can effectively retain the original microenvironment characteristics of the tumor, including key components such as glial cells, brain neurons, and neural stem cells, by transplanting patient-derived glioma tissue into immunodeficient mice. The PDTX model can not only simulate the growth dynamics of the tumor, but also more realistically reproduce the interaction between the tumor and the host tissue, providing a more physiologically relevant research platform, which is helpful for in-depth research on the biological characteristics of gliomas, and provides a more reliable experimental basis for new drug screening and efficacy evaluation.

[0014] In addition, the present invention has developed a tumor screening method suitable for constructing a PDTX model of brain glioma, by performing transcriptome sequencing (RNA-seq) on tumor tissue to detect the transcription of brain tumor-related genes, including GFAP, P2RY12, SOX2, RBFOX3, NEFH, SLC17A7, NPY, SLC1A3, CSPG4, OLIG2, which play an important role in the occurrence, development and microenvironment of brain glioma. The present invention screens out tumor samples (greater than 3) with high transcription levels of multiple genes by analyzing the transcription of the above genes in the samples, so as to serve as an ideal tumor source for the construction of PDTX model.

[0015] Preferably, the brain tumor-related genes are GFAP, SOX2, SLC1A3 and OLIG2.

[0016] Experimental research found that GFAP, SOX2, SLC1A3 and OLIG2 are the best gene combination for screening brain glioma tissue samples. The high transcription levels of the four genes can better reflect the cell type and tumor microenvironment of brain glioma, so that the transplanted tumor tissue can retain the original microenvironment characteristics of the tumor tissue, and have characteristic brain glioma characteristics such as glial cells, brain neurons, and neural stem cells.

[0017] Preferably, the screening conditions for high transcription levels of GFAP, SOX2, SLC1A3 and OLIG2 are: TPM value of GFAP>100, TPM value of SOX2>100, TPM value of SLC1A3>100, TPM value of OLIG>25.

[0018] Preferably, the pretreatment solution comprises Hibernate A culture medium and Matrigel, and the mass ratio of the Hibernate A culture medium to the Matrigel is (1-2): (1-2).

[0019] Preferably, the pretreatment solution further comprises 1×B27, 1.5-2.5 mM glutamine, 45-55 U / mL Pen-Strep, 0.5-1.5 U / mL heparin, 15-25 ng / mL bFGF and 15-25 ng / mL EGF.

[0020] In the construction of the PDTX model of brain glioma, the pretreatment solution provides a strong guarantee for the survival, proliferation and subsequent tumor establishment of tumor tissue by providing necessary nutrients, supporting factors and growth environment, ensuring that the construction of the PDTX model can be successful and efficient.

[0021] Preferably, in step S4, the injection concentration of the human VEGFA is 40-60 μg / mouse, the injection concentration of the glutamine is 90-110 μg / mouse, and the human VEGFA is injected via the tail vein.

[0022] The present invention effectively promotes the formation of tumor microvessels by injecting a specific concentration of vascular endothelial growth factor A (VEGFA) into the tail vein of mice inoculated with tumor tissue every week. This strategy aims to simulate the blood supply dynamics of tumors and enhance the growth and metastasis potential of tumors. In addition, in order to support the growth and maintenance of nerve cells in tumor tissues, the present invention also injects a specific concentration of glutamine into the tumor inoculation site every week. Glutamine, as an important neurotrophic factor, can promote the survival and proliferation of nerve cells, thereby better simulating the nerve cell components in brain gliomas. The combination of the above strategies helps to accurately reproduce the growth characteristics and microenvironment of brain gliomas, and promote and improve the success rate of PDTX models.

[0023] Preferably, in step S3, the vaccination is carried out at a subcutaneous site.

[0024] Preferably, the specific method for subcutaneous site vaccination is: remove the hair on the right side of the mouse, clamp the tissue block and place it in the vaccination needle sleeve, cut a 1.5-2.5 mm opening on the upper right side of the exposed skin of the mouse, insert the vaccination needle into the opening and advance it 2.5-3.5 cm subcutaneously, and push the sleeve to complete the subcutaneous site vaccination.

[0025] Preferably, the mouse is an immunodeficient mouse, which lacks mature T, B and NK immune cells, has reduced complement activity, and has weakened phagocytosis of human cells by macrophages.

[0026] Preferably, the evaluation criteria for the successfully constructed glioma PDTX model include the three items shown in (1)-(3):

[0027] (1) Volume evaluation: The tumor was successfully explanted when a bulge was observed at the inoculation site;

[0028] (2) Pathological examination: The constructed PDTX tumor tissue was subjected to histomorphological identification and confirmed as brain glioma;

[0029] (3) Immunohistochemistry: Immunohistochemistry was performed on PDTX tumor tissues, and the protein expression of brain tumor-related genes was at a high level, confirming the presence of a brain glioma microenvironment;

[0030] If all the evaluation criteria (1)-(3) are met, the construction of the PDTX model of brain glioma is determined to be successful.

[0031] The present invention has formulated a set of characterization criteria for evaluating the success of the construction of the PDTX model of brain glioma. First, a preliminary assessment is made by the change in tumor volume to observe whether the tumor grows normally. Secondly, a case test is performed to confirm whether the characteristics of the tumor tissue are consistent with the primary tumor. Finally, the expression of brain tumor-related gene markers in the tumor is analyzed by immunohistochemistry to further verify the accuracy and biological relevance of the model. The three standards are combined to ensure that the construction of the PDTX model achieves the expected biological characteristics.

[0032] In a second aspect, the present invention provides the use of the glioma PDTX model prepared by the construction method in screening drugs for preventing or treating glioma.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention provides a new method for constructing a PDTX model of glioma, which can effectively retain the original microenvironment characteristics of the tumor by transplanting glioma tissue from patients into immunodeficient mice, and has characteristic glioma characteristics such as glial cells, brain neuron cells, and neural stem cells. The PDTX model can not only simulate the growth dynamics of the tumor, but also more realistically reproduce the interaction between the tumor and the host tissue, providing a more physiologically relevant research platform, which is helpful for in-depth research on the biological characteristics of glioma, and provides a more reliable experimental basis for new drug screening and efficacy evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a specific flow chart for constructing the PDTX model of glioma described in Example 1;

[0036] Figure 2 This is a HE staining result of the glioma PDTX model constructed from the tumor tissue of sample number P0105034 in Example 1;

[0037] Figure 3 This is a HE staining result of the glioma PDTX model constructed from the tumor tissue of sample number P0105114 in Example 1;

[0038] Figure 4 This is a GFAP staining result diagram of the glioma PDTX model constructed from the tumor tissue of sample number P0105034 in Example 1;

[0039] Figure 5 This is a GFAP staining result diagram of the glioma PDTX model constructed from the tumor tissue of sample number P0105114 in Example 1;

[0040] Figure 6 This is a diagram showing the changes in tumor volume when the PDTX model was successfully established using the tumor tissue of sample number P0105034 in both Example 1 and Comparative Example 1;

[0041] Figure 7 This is a diagram showing the changes in tumor volume when the PDTX model was successfully established using the tumor tissue with sample number P0105114 in both Example 1 and Comparative Example 1. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0043] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0044] The mice described in the examples are all T, B, and NK cell triple immunodeficient mice, which are from Saiye Biotechnology Co., Ltd., and the strain name is: NOD.Cg-Prkdc scid Iq em1cya / Cya, abbreviated as C-NKG.

[0045] Example 1

[0046] This embodiment provides a method for constructing a glioma PDTX model, comprising the following steps:

[0047] 1. Screening of brain glioma tumor tissue

[0048] The present invention detects the transcription of brain tumor-related genes by performing gene sequencing (RNA-seq) on tumor tissue. The present invention designs a high-level transcription gene combination, wherein the brain tumor-related genes include GFAP, P2RY12, SOX2, RBFOX3, NEFH, SLC17A7, NPY, SLC1A3, CSPG4, and OLIG2. According to the specific tumor type, samples with high transcription levels of the above multiple genes (more than 3) are screened as samples required for PDTX model construction. After determining the high-level transcription, human tumor living tissue is obtained.

[0049] According to the above method, the present invention screens tumor tissues with high transcription levels of specific genes (GFAP, SOX2, SLC1A3, OLIG2) by RNA-seq, and the screening conditions are TPM values: GFAP>100, SOX2>100, SLC1A3>100, OLIG>25 (this value is the median of the transcription level of glioma genes in the sample library rounded up). Two human tumor living tissues were screened out, and the tumor numbers were: P0105034 and P0105114, respectively. Both tumors were clinically identified as brain gliomas, and two tumor tissues were used to construct brain glioma PDTX models. Among them, the TPM values ​​of gene transcription of P0105034 and P0105114 are shown in Table 1.

[0050] Table 1

[0051]

[0052] 2. Construction of PDTX model of brain glioma

[0053] (1) Preparation of tissue pretreatment solution:

[0054] The main components of the tissue pretreatment solution are Hibernate A culture medium and matrix gel mixed in a 1:1 mass ratio, to which appropriate special cell growth factors and related components are added, including 1×B27, 2mM glutamine, 50U / mLPen-Strep, 1U / mL heparin, 20ng / mL bFGF and 20ng / mL EGF. All the above solutions are prepared in a biological safety cabinet and stored at 4°C.

[0055] (2) Tumor tissue processing:

[0056] The cryopreserved tubes containing the two tumor tissues obtained by screening were taken out of the liquid nitrogen environment, and the liquid nitrogen-frozen tumor tissues were placed in a 37°C water bath for rapid thawing and recovery. The cryopreserved tubes were transferred to a biosafety cabinet, and the tissues were taken out and washed in a pre-cooled PBS solution. Under a stereomicroscope, the tumor tissues were trimmed and cut into 2*2*2mm 3 Tissue blocks of different sizes are placed in the above-mentioned pretreatment solution and set aside for later use.

[0057] (3) PDTX tumor inoculation:

[0058] The present invention adopts the method of subcutaneous site inoculation to carry out tumor transplantation to obtain tumor-bearing immunodeficient mice, and the specific method is as follows.

[0059] Subcutaneous inoculation: soak the pre-treated solution in a 2*2*2mm 3 The tumor tissue was inoculated into the subcutaneous area of ​​the middle right side of the mouse body. The specific operation is as follows: first, the surgical instruments are cleaned and disinfected for use, the hair on the right side of the mouse is shaved, a small piece of tumor tissue is carefully grasped with tweezers and placed in the inoculation needle sleeve, and a 2mm opening is cut in the upper right side of the exposed skin of the mouse with scissors, the inoculation needle is inserted into the opening and pushed subcutaneously for about 3cm, and the sleeve is pushed to complete the inoculation.

[0060] (4) Animal observation after vaccination:

[0061] After inoculation, the body weight and tumor volume growth of mice were observed weekly, and human VEGFA (50 μg / mouse) was injected into the tail vein of the inoculated mice weekly to promote microangiogenesis. Glutamine (100 μg / mouse) was injected into the tumor inoculation site weekly to promote nerve cell growth, and the mice were continuously cultured.

[0062] (5) Success criteria for PDTX tumor modeling of glioma:

[0063] The mouse transplanted tumors were evaluated, including tumor volume change curve (tumor growth curve), pathological examination (HE staining) and immunohistochemistry (specific highly expressed proteins), as follows:

[0064] a. Volume evaluation: The tumor is successfully implanted if a bulge is observed at the inoculation site (subcutaneous site);

[0065] b. Pathological examination: The constructed PDTX tumor tissue was identified by histomorphology and confirmed as brain glioma;

[0066] c. Immunohistochemistry: Immunohistochemistry is performed on PDTX tumor tissues to detect the expression of the above four proteins GFAP, SOX2, SLC1A3, and OLIG2. If the expression levels are significantly higher than those of normal tissues, it is considered "positive", confirming that the tissue has a brain glioma microenvironment;

[0067] In summary, if all three conditions are met, the PDTX model is considered to be successfully constructed. Figure 1 A specific flow chart for constructing the glioma PDTX model.

[0068] Comparative Example 1

[0069] This comparative example provides a method for constructing a glioma PDTX model. Two tumor tissues with tumor numbers P0105034 and P0105114 screened in Example 1 are used to construct a glioma PDTX model. The construction method is different from that in Example 1 only in that after the mice are inoculated, human VEGFA and glutamine are not injected into the mice, and the rest of the construction methods are the same as in Example 1.

[0070] Comparative Example 2

[0071] This comparative example provides a method for constructing a glioma PDTX model. According to the method described in Example 1, RNA-Seq was used to screen glioma samples with low transcription levels of GFAP, SOX2, SLC1A3 and OLIG2 as controls, numbered: P0105016, P0105033, wherein the TPM values ​​of P0105016 are: GFAP: 0.3, SOX2: 1.2, SLC1A3: 37.1, OLIG2: 0.1, and the TPM values ​​of P0105033 are: GFAP: 0.1, SOX2: 0.1, SLC1A3: 0.2, OLIG2: 0. The two screened glioma samples were used to construct a glioma PDTX model according to the method described in Example 1, wherein 5 transplants of glutamine + human VEGFA added / not added groups were designed after inoculation of mice for exploration, and the rest of the construction methods were the same as in Example 1.

[0072] Effect example

[0073] 1. Comparison of modeling success rate:

[0074] In both Example 1 and Comparative Example 1, two tumor tissues with tumor numbers P0105034 and P0105114 were selected, and in Comparative Example 2, two tumor tissues with tumor numbers P0105016 and P0105033 were selected to construct PDTX models of glioma, and finally the average number of successful tumor-out days and the number of tumor-out cases were statistically compared, and the specific results are shown in Table 2. As can be seen from the results in Table 2, in Example 1, a tumor screening method suitable for constructing a PDTX model of glioma was formulated, that is, tumor tissues with high transcription levels of specific genes (GFAP, SOX2, SLC1A3, OLIG2) were screened, and the two human tumor living tissues (P0105034 and P0105114) finally obtained can effectively ensure the successful tumor-out of glioma, and during the construction process, by injecting glutamine and human VEGFA into mice, it can help to accurately reproduce the growth characteristics and microenvironment of glioma, thereby improving the success rate of PDTX model and significantly shortening the successful modeling time. Meanwhile, tumor tissues (P0105016 and P0105033) with low transcription levels of specific genes (GFAP, SOX2, SLC1A3, OLIG2) did not develop tumors, indicating the importance of higher transcription levels of specific genes in improving the success rate of modeling.

[0075] Figure 2-3 They are the results of HE staining of the PDTX model of glioma constructed with tumor numbers P0105034 and P0105114 in Example 1, marked as nerve cells, Figure 2-3 It can be seen that there is a neural microenvironment in the transplanted tumor obtained by the construction method described in Example 1 of the present invention; Figure 4-5 Figure 1 shows the GFAP staining in the PDTX models of glioma constructed by P0105034 and P0105114. Figure 4-5 It can be seen that GFAP is highly expressed in the transplanted tumor obtained by the construction method described in Example 1; the above results all indicate that the glioma PDTX model obtained in Example 1 is successfully constructed, and the new glioma PDTX model construction method provided by the present invention can effectively ensure the success rate of glioma PDTX model construction, and can effectively retain the original microenvironment characteristics of the tumor, and significantly shorten the time required for modeling.

[0076] Table 2

[0077]

[0078] 2. Tumor volume growth curve:

[0079] The changes in tumor volume of the successfully transplanted tumors in mice in Example 1 and Comparative Example 1 were recorded. The results are as follows: Figure 6-7As shown. The construction method described in Example 1 can effectively promote the growth of the target tumor by injecting glutamine and human VEGFA. Compared with the method described in Comparative Example 1 without injecting glutamine and human VEGFA, the tumor volume of the glioma PDTX model obtained in Example 1 is significantly increased under the same culture time.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for constructing a PDTX model of brain glioma, characterized in that: The construction method comprises the following steps: S1. Screen tumor tissues with high transcription levels of brain tumor-related genes to obtain human tumor living tissues; S2, washing the human tumor living tissue and cutting it into tissue blocks, and soaking the tissue blocks in a pretreatment solution; S3, inoculating the tissue blocks soaked in the pretreatment solution into mice for tumor transplantation; S4. After inoculation, the mice were injected with human VEGFA and glutamine every week for culture, and then the constructed model was evaluated to obtain a successfully constructed brain glioma PDTX model; The brain tumor-related genes are GFAP, SOX2, SLC1A3 and OLIG2; The high transcription level of the brain tumor-related gene means that the TPM value of the brain tumor-related gene is higher than the median TPM value of the corresponding brain tumor-related gene in the sample library; The mice are immunodeficient mice; The evaluation criteria for the successfully constructed glioma PDTX model include the three items shown in 1-3:

1. Volume evaluation: When a tumor protrudes from the inoculation site, it is considered that the tumor has been successfully explanted; 2. Pathological examination: The constructed PDTX tumor tissue was identified by histomorphology and confirmed as brain glioma; 3. Immunohistochemistry: Immunohistochemistry was performed on PDTX tumor tissues, and the protein expression of brain tumor-related genes was at a high level, confirming the presence of a brain glioma microenvironment; If the evaluation criteria 1-3 are all met, it is determined that the glioma PDTX model is successfully constructed.

2. The construction method according to claim 1, characterized in that: The pretreatment solution includes Hibernate A culture medium and matrigel, and the mass ratio of the Hibernate A culture medium to the matrigel is (1-2): (1-2).

3. The construction method according to claim 2, characterized in that: The pretreatment solution also includes 1×B27, 1.5-2.5 mM glutamine, 45-55 U / mL Pen-Strep, 0.5-1.5 U / mL heparin, 15-25 ng / mL bFGF and 15-25 ng / mL EGF.

4. The construction method according to claim 1, characterized in that: In the step S4, the injection concentration of the human VEGFA is 40-60 μg / mouse, the injection concentration of the glutamine is 90-110 μg / mouse, and the human VEGFA is injected through the tail vein.

5. The construction method according to claim 1, characterized in that: In step S3, the vaccination method adopts subcutaneous site vaccination, and the specific method of subcutaneous site vaccination is: remove the hair on the right side of the mouse, clamp the tissue block and place it in the vaccination needle sleeve, cut a 1.5-2.5mm opening on the upper right side of the exposed skin of the mouse, insert the vaccination needle into the opening and advance it 2.5-3.5cm subcutaneously, and push the sleeve to complete the subcutaneous site vaccination.

6. Use of the glioma PDTX model prepared by the construction method according to any one of claims 1 to 5 in screening drugs for preventing or treating glioma.

Images