Immune humanized liver cancer patient-derived xenotransplantation mouse model and construction method and application thereof

Abstract

The invention relates to an immune humanized liver cancer patient-sourced xenotransplantation mouse model and a construction method and application thereof, and belongs to the technical field of medical research and biologication.The immune humanized liver cancer PDX model is innovatively constructed by optimizing transplantation of tumor tissues and introducing and screening immune cells, and the model has the advantages of being high in specificity, high in sensitivity and high in reliability. The defects of a traditional PDX model in immune system and immunotherapy evaluation are overcome. The model has tumor characteristics of liver cancer patients and a functional human immune system, has reliable immune response and anti-cancer treatment evaluation capability, and provides scientific and reliable model support for research and screening of novel liver cancer immunotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of medical research and biotechnology, and relates to a method for constructing a liver cancer research model. The model aims to provide a scientific basis for immunotherapy, drug screening and anti-tumor efficacy evaluation by constructing an immune humanized liver cancer PDX mouse model. Background Art

[0002] Currently, patient-derived xenograft (PDX) models have been widely used in cancer research. The PDX model directly transplants tumor patient tissues into immunodeficient mice, retaining the genetic and phenotypic characteristics of the patient's tumor. It is an important tool for studying cancer biology, screening drugs, and evaluating the effects of chemotherapy. Especially in liver cancer research, the PDX model can effectively simulate the patient's tumor characteristics and help study the molecular mechanisms and drug sensitivity of the tumor. However, the traditional PDX model has a significant defect: because the transplanted tumor tissue grows in immunodeficient mice, the model lacks the human immune system and cannot evaluate the effect of immunotherapy.

[0003] Disadvantages and shortcomings of existing technology:

[0004] 1. Lack of immune system: The immunodeficient mice used in traditional PDX models (such as NOD / SCID mice or B-NDG mice) do not have a functional human immune system and lack mature T cells, B cells, and natural killer cells. Therefore, this model cannot simulate the complex interaction between the human immune system and tumors, limiting its application in immunotherapy evaluation.

[0005] Limitations of immunotherapy evaluation: With the rise of immunotherapy such as immune checkpoint inhibitors (such as PD-1 / PD-L1 antibodies) and tumor vaccines, cancer immunotherapy has gradually become an important means of tumor treatment. However, traditional PDX models cannot be used to evaluate the efficacy of these immunotherapies, which greatly limits their potential for application in new anti-tumor therapies.

[0006] 2. Risk of xenotransplant rejection: In order to introduce human immune cells to achieve "humanization", researchers have tried to transplant human peripheral blood mononuclear cells (PBMC) or CD34+ hematopoietic stem cells into PDX models to construct PDX models with a human immune system (i.e., immune humanized PDX models). However, existing humanized PDX models also have problems such as xenogeneic graft-versus-host disease (GvHD), especially when using PBMC transplantation, and the treatment effect can usually only be observed in the short term.

[0007] 3. Limited model stability and experimental window: The stability of existing humanized PDX models is not high, especially after the occurrence of graft-versus-host reaction, the experimental window is shortened, affecting the long-term use of the model and the stability of the evaluation of treatment effects. For the evaluation of treatment effects that require a long time, such as chronic immune activation or persistent tumor suppression, the experimental window of existing technologies is short, which limits the scope of application of the model.

[0008] In summary, existing technologies have made some progress in constructing PDX models, but they are obviously insufficient in immune system reconstruction and immunotherapy evaluation. Therefore, there is an urgent need for an improved immune humanized liver cancer PDX model that can reconstruct a functional human immune system while retaining the characteristics of liver cancer tumors, so as to evaluate the efficacy of immunotherapy and extend the experimental window, providing more scientific and reliable model support for immunotherapy research. Summary of the invention

[0009] In view of the above problems, the present invention aims to provide a method for constructing an immunohumanized hepatocellular carcinoma patient-derived xenograft (PDX) mouse model for immunotherapy evaluation. By introducing the human immune system into the traditional PDX model, the model not only retains the genetic and phenotypic characteristics of the patient's tumor, but also can reconstruct a functional human immune system, which can more realistically simulate the interaction between human immunity and tumors, and provide an innovative and reliable model for immunotherapy drug screening and efficacy evaluation.

[0010] The task of the present invention is:

[0011] 1. Constructing a model with both human liver cancer characteristics and a functional human immune system: The present invention constructs an immune-humanized liver cancer PDX model that retains the molecular characteristics of liver cancer patient tumors and includes the human immune system, so as to more accurately reflect the immune microenvironment of human liver cancer.

[0012] 2. Evaluate the effect of immunotherapy: This model can be used to evaluate the efficacy of different immunotherapy regimens (such as PD-1 / PD-L1 antibodies, tumor vaccines, etc.), provide an effective tool for preclinical research on immunotherapy, and help screen the optimal treatment plan for liver cancer.

[0013] 3. Extend the experimental window and optimize the model stability: By improving the humanization method, the present invention strives to reduce the incidence of graft-versus-host disease, thereby extending the experimental window, improving the stability of the model, and meeting the needs of long-term research.

[0014] Support the development and screening of anticancer drugs and combination therapies: The immunohumanized liver cancer PDX model of the present invention can be used to evaluate the effectiveness of new immune drugs and explore their combined effects with existing therapies, providing a scientific basis for the development of precision medicine and personalized treatment plans for liver cancer.

[0015] Through the construction of this model, the present invention aims to fill the gap in the existing technology in the research of liver cancer immunotherapy, improve the accuracy and applicability of the cancer model, and bring breakthrough progress to the treatment development of liver cancer patients.

[0016] The specific scheme adopted by the present invention is:

[0017] In the first aspect, the present invention claims a method for constructing an immune humanized xenograft mouse model derived from a liver cancer patient, comprising the following steps: Step 1: Obtaining and processing patient tumor tissue: The fresh liver cancer tissue samples are strictly screened and processed, and cut into small pieces after washing and removing necrotic tissue to ensure tumor activity and genomic integrity; Step 2: Construction of liver cancer PDX model: Transplant the liver cancer tissue treated in step 1 into severely immunodeficient mice, and establish a passage model to obtain a stable liver cancer PDX model; Step 3: Introduction of human immune system: After the PDX model has grown stably, humanization is achieved by transplanting human peripheral blood mononuclear cells (PBMCs). PBMC cell suspension is injected into the tail vein of mice to reconstruct human immune cells in the mice, thus obtaining a liver cancer PDX model with both human liver cancer characteristics and immune function. Step 4: Monitoring of immune function of humanized models: Flow cytometry was used to regularly detect immune cell markers including CD45+, CD3+, and CD8+ in peripheral blood and tumor tissues to monitor the reconstruction of human immune cells in mice. Specific markers were used to determine the success of the model construction and ensure that the model had reliable immune response function.

[0018] As a further optimization of the above technical solution, in the above step 3, the preparation method of the PBMC cell suspension is as follows: add 15 mL of cell separation solution to a centrifuge tube, then add 17 mL of a mixture of equal volumes of peripheral blood and PBS, keep the centrifuge tube vertical, put it in a centrifuge, and centrifuge it at 1200×g for 10 minutes at room temperature; take out the centrifuge tube, and quickly pour the supernatant into a new 50 mL centrifuge tube; use PBS containing 2% fetal bovine serum to wash the obtained supernatant twice, centrifuge it at 300×g for 8 minutes for the first time, discard the supernatant, add a new PBS containing 2% fetal bovine serum, wash it for the second time, centrifuge it at 120×g for 10 minutes to remove platelets, and discard the supernatant; resuspend the obtained cell pellet with serum-free PBS, and adjust the cell concentration to 5×10 8 / mL to obtain PBMC cell suspension.

[0019] As a further optimization of the above technical solution, in the above step three, CD34+ hematopoietic stem cells are used to replace PBMC for humanization.

[0020] As a further optimization of the above technical solution, in the above step 4, a specific proportion of CD45+ human immune cells is considered to be a sign of successful model construction; by detecting activation markers of CD8+ T cells including GrB and INF-γ, the T cell activity of the immune humanized PDX model is evaluated to ensure that the model has a reliable immune response function.

[0021] In a second aspect, the present invention seeks to protect an immune humanized xenograft mouse model derived from liver cancer patients constructed using the above-mentioned construction method.

[0022] In a third aspect, the present invention seeks to protect the use of the above-mentioned immunohumanized xenograft mouse model derived from liver cancer patients in the evaluation of liver cancer immunotherapy.

[0023] As a further optimization of the above application, immune checkpoint inhibitors and tumor vaccines were used alone or in combination to observe the effects of different treatment regimens on model tumor growth in a successfully constructed immunohumanized xenograft mouse model derived from liver cancer patients.

[0024] Furthermore, flow cytometry and histopathological analysis were used to evaluate the infiltration and functional changes of immune cells in each treatment group to verify the efficacy of immunotherapy.

[0025] Furthermore, the immune checkpoint inhibitor is a PD-1 / PD-L1 inhibitor, a CTLA-4 inhibitor or a LAG-3 inhibitor; the tumor vaccine is a CSF-Vac Gel Tumor vaccines. Instead of CSF-VacGel tumor vaccines, other immune activators (such as STING agonists, TLR agonists) can be selected for tumor immunity research to enhance the anti-tumor immune effect and verify the applicability of the model.

[0026] Compared with the prior art, the present invention has significant advantages and beneficial effects in the following aspects:

[0027] 1. Reconstruct the human immune system and improve the accuracy of immunotherapy research

[0028] The present invention constructs a humanized liver cancer model with human immune response by introducing the human immune system into the liver cancer PDX model. Traditional PDX models lack an immune system and are difficult to use for immunotherapy evaluation. The immune humanized model of the present invention can effectively simulate the immune microenvironment of human liver cancer, making the evaluation of the effect of immunotherapy (such as PD-1 / PD-L1 inhibitors) more scientific and realistic, and providing a more clinically relevant experimental basis for preclinical research.

[0029] 2. High model stability and extended experimental window

[0030] The present invention effectively reduces the incidence of xenogeneic graft-versus-host disease (GvHD) and improves the survival and stability of humanized cells in mice by optimizing PBMC screening and transplantation methods. The extension of the experimental window is not only convenient for observing short-term immune responses, but also suitable for evaluating the effects of chronic immune activation and long-term anti-cancer treatment, overcoming the problem of limited model experimental time in the prior art.

[0031] 3. Support the evaluation of multiple immunotherapy combinations

[0032] The model provided by the present invention can be applied to evaluate the effects of single and combined application of various immunotherapy methods such as PD-1 / PD-L1 inhibitors and tumor vaccines (such as CSF-VacGel), making it a powerful tool for exploring combined immunotherapy strategies and helping to screen the best immunotherapy for liver cancer. The potential of this combined application will significantly enhance the applicability of the model in preclinical drug development.

[0033] 4. Comprehensive analysis of tumor and immune microenvironment

[0034] Through flow cytometry and histopathological analysis, the activation state and infiltration of key immune cells such as T cells and dendritic cells can be monitored in the model, including the expression of functional markers such as GrB and INF-γ of CD8+T cells. This comprehensive analytical method can accurately evaluate the impact of immunotherapy on the tumor microenvironment and further reveal the mechanism of action of the treatment.

[0035] 5. Promote the development of precision medicine and personalized treatment

[0036] Because the model retains the characteristics of the patient's liver cancer tissue and introduces a functional human immune system, it can more accurately predict an individual's response to immunotherapy, providing an effective tool for achieving precision treatment of liver cancer. This model will help researchers and drug developers better understand the differences in the efficacy of immunotherapy, thereby accelerating the development of personalized treatment plans.

[0037] Comprehensive Effect

[0038] On the basis of ensuring the stability of the model and the experimental window, the present invention reconstructs a liver cancer PDX model with a functional human immune system, which can be applied to the scientific evaluation of immunotherapy, filling the gap in the existing technology in the research of liver cancer immunotherapy. The wide applicability, reliability and innovation of the model significantly improve the accuracy and practicality of cutting-edge research on liver cancer immunotherapy, and is of great significance to promoting the clinical application of liver cancer immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The expression of human CD45 in mouse peripheral blood at different time points +Cell flow cytometry analysis.

[0040] Figure 2 The following are the results of the mouse treatment monitoring: (A) Point photos of mice in each group at different times. (B) Quantitative analysis of the changes in tumor volume in each group. (C) Changes in the weight of mice in each group. *: p<0.05, **: p<0.01. DETAILED DESCRIPTION

[0041] The present invention aims to solve the problems of immune system deficiency, immunotherapy evaluation limitations, xenograft rejection, insufficient model stability, etc. in the existing technology of xenograft (PDX) models derived from liver cancer patients. An improved construction method is proposed to provide an immune humanized liver cancer PDX mouse model with both human immune system and liver cancer tumor characteristics, so as to truly simulate human immune response, improve model stability and experimental window, and provide a new solution for liver cancer immunotherapy research.

[0042] The present invention achieves technical improvement through the following steps:

[0043] 1. Acquisition and processing of patient tumor tissue:

[0044] Fresh liver cancer tissue samples were obtained and strictly screened and processed. The samples were washed, necrotic tissue was removed, and cut into small pieces to ensure tumor activity and genomic integrity, so as to retain the molecular characteristics of the patient's tumor after transplantation into mice.

[0045] 2. Construction of liver cancer PDX model:

[0046] The treated liver cancer tissue is transplanted into severely immunodeficient mice (such as B-NDG mice) and a subculture model is established to obtain a stable liver cancer PDX model. Each generation of the model retains the heterogeneity and growth characteristics of the tumor, laying the foundation for subsequent immune humanization operations.

[0047] 3. Introduction of human immune system (immune humanization operation):

[0048] After the PDX model has grown stably, humanization is achieved by transplanting human peripheral blood mononuclear cells (PBMC). PBMC cell suspension is injected into the tail vein of mice to reconstruct human immune cells in the mice, thereby obtaining a liver cancer PDX model with both human liver cancer characteristics and immune function.

[0049] In order to reduce the risk of xenogeneic graft-versus-host disease (GvHD), the present invention optimizes the purification and screening steps of PBMC to ensure that the human immune cells after cell injection can survive stably for a long time in the mouse body.

[0050] 4. Monitoring of immune function in humanized models:

[0051] Flow cytometry is used to regularly detect human CD45+, CD3+, CD8+ and other immune cell markers in peripheral blood and tumor tissues to monitor the reconstruction of human immune cells in mice. A specific proportion (such as more than 25%) of CD45+ human-derived immune cells is considered a sign of successful model construction.

[0052] By detecting activation markers such as GrB and INF-γ of CD8+ T cells, the T cell activity of the immune humanized PDX model was evaluated to ensure that the model had reliable immune response function.

[0053] 5. Evaluation of immunotherapy effect:

[0054] In the successfully constructed immune humanized liver cancer PDX model, immune checkpoint inhibitors (such as PD-1 / PD-L1 antibodies) and tumor vaccines were used alone or in combination to observe the effects of different treatment regimens on model tumor growth.

[0055] The present invention utilizes flow cytometry and histopathological analysis to evaluate the infiltration and functional changes of immune cells (such as dendritic cells and CD8+T cells) in each treatment group, specifically including the expression levels of effector molecules such as GrB and INF-γ, to verify the effect of immunotherapy.

[0056] 6. Model stability and extended experimental window:

[0057] The present invention effectively reduces the occurrence of GvHD and prolongs the experimental window by optimizing the humanization operation and human immune cell transplantation method. Based on the stable humanized PDX model, longer immune efficacy observation can be achieved, thereby improving the applicability of the model in liver cancer immunotherapy research.

[0058] This invention innovatively constructs an immune humanized liver cancer PDX model by optimizing the transplantation of tumor tissues, the introduction and screening of immune cells, and overcomes the deficiencies of traditional PDX models in the immune system and immunotherapy evaluation. The model combines the tumor characteristics of liver cancer patients with a functional human immune system, and has reliable immune response and anti-cancer treatment evaluation capabilities, providing scientific and reliable model support for the research and screening of new liver cancer immunotherapies.

[0059] The technical solution of the present invention can effectively solve the key problems of the prior art in the research of liver cancer immunotherapy and has significant application value in preclinical research.

[0060] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0061] In the following examples, unless otherwise specified, the materials or reagents used are commercially available, and the operations used are conventional technical means.

[0062] 1 Construction of liver cancer PDX mouse model

[0063] 1.1 Experimental animals

[0064] Four-week-old male B-NDG mice were housed in an SPF environment.

[0065] 1.2 Tumor model construction

[0066] Fresh liver cancer tissue samples were obtained from patients undergoing liver cancer resection surgery. The tissue blocks were placed in 5 ml EP tubes, added with serum-free DMEM culture medium containing 2% penicillin-streptomycin, stored at 4°C, and quickly transported to the animal center. The modeling time was controlled to be completed within 2 hours. First, the tumor block was washed with new culture medium (containing 2% penicillin-streptomycin), and the necrotic tissue was removed with ophthalmic scissors, and then divided into two parts. The first part of the tissue was fixed with 4% paraformaldehyde tissue fixative for 24 hours, then dehydrated and paraffin-embedded, and the tissue source was registered.

[0067] The second part of tissue was placed in a culture dish, and the tumor tissue was trimmed into tissue blocks of about 3 mm in size using ophthalmic scissors. The blocks were washed twice and immersed in new serum-free DMEM culture medium (containing 2% penicillin-streptomycin) at 4°C for later use.

[0068] According to the above method, after induction and internal anesthesia of B-NDG mice by gas anesthesia machine, they were fixed on the operating table in supine position, and the hair around the right thigh was shaved with a shaver, and the residual hair was thoroughly removed with a depilatory cream. The skin was disinfected twice with iodine tincture, and an opening of about 5 mm in length was cut in the abdominal skin with ophthalmic scissors. Only the skin was cut, and the peritoneum and muscle layer were not cut. Then, the liver cancer tissue was implanted subcutaneously in the upper left, lower left, upper right and lower right of the abdomen with tissue forceps, the skin opening was sutured, iodine tincture was disinfected, and the modeling date, tumor sample number and mouse number were marked. After inoculation, the mice were intraperitoneally injected with antibiotics for 3 days.

[0069] The growth of tumor tissue was observed twice a week. After the transplanted tissue mass grew, the mouse was marked as the P0 generation PDX model. When the P0 generation PDX tumor grew to 1000mm 3 When the tumor grows up, the tumor tissue is removed and the above modeling process is repeated to obtain P1 and P2 generation PDX models. After each generation of tumor grows up, a piece of tissue is cut out, fixed and dehydrated, and embedded in paraffin for preservation.

[0070] This experiment used the P3 generation. The model construction method was basically the same as before, but the tumor tissue was implanted subcutaneously above the right thigh. The condition of the mice was observed on the second day after the model was constructed, and immune humanization operations were performed.

[0071] 1.3 Immune humanization of PDX models

[0072] On the second day after mouse tumor implantation, immunohumanization was performed. PBS (containing 2% fetal bovine serum) and Return to room temperature. Draw human peripheral blood, anticoagulate with sodium heparin, and transfer to a biosafety cabinet. Mix human peripheral blood and room temperature PBS (containing 2% fetal bovine serum) in equal volumes for later use. Add 15 mL of Add another 17 mL of a mixture of peripheral blood and PBS, and keep the centrifuge tube vertical. Subsequently, place the centrifuge tube in a centrifuge and centrifuge at 1200 × g for 10 minutes at room temperature. Take out all centrifuge tubes and quickly pour the supernatant into a new 50 mL centrifuge tube. Wash the supernatant twice with PBS (containing 2% fetal bovine serum). Centrifuge at 300 × g for 8 minutes for the first time and discard the supernatant. Add new PBS (containing 2% fetal bovine serum) for the second wash, centrifuge at 120 × g for 10 minutes to remove platelets, and discard the supernatant. Resuspend the resulting cell pellet with serum-free PBS, count using a cell counter and adjust the cell concentration to 5 × 10 8 / mL. The cells were placed in an ice box and quickly transferred to the animal center. The mice implanted with tumor tissue the day before were taken out and fixed on the tail vein injection instrument. The PBMC cell suspension was injected through the tail vein, 100μL per mouse. After the injection, the mice were returned to the cage and the status of the mice was monitored twice a week.

[0073] After cell injection, peripheral blood human CD45 in mice was monitored weekly. + Cell ratio, human CD45 + The humanized model was successfully constructed when the cells were greater than 25%. About 0.5 cm of the length was subtracted from the distal end of the mouse tail, and blood was collected in a sodium heparin anticoagulation tube. The obtained blood was diluted with 1 mL of PBS, centrifuged at 800 × g for 5 minutes at 4°C, and the supernatant was discarded. Then 1 mL of red blood cell lysis buffer was added to each tube of sample, and the sample was resuspended evenly and incubated at room temperature for 10 minutes, centrifuged at 800 × g for 5 minutes at 4°C, and the supernatant was discarded. 95 μL of flow staining buffer was added to each tube of sample, and 1 μL of mouse CD16 / 32 antibody and human CD16 antibody were added respectively. After incubation at room temperature for 10 minutes, 5 μL of APC human CD45 antibody was added to each tube of sample, and incubated at 4°C in the dark for 30 minutes. Take out the sample tube, add 200 μL of flow staining buffer, centrifuge at 800 × g for 5 minutes at 4°C, and detect human CD45 under flow cytometer. + The proportion of cells.

[0074] 2. Evaluation of treatment response

[0075] 2.2.1 Analysis of T cells and dendritic cells in tumor tissue

[0076] (1) Drug treatment of mice

[0077] Observe the mice in which the immune humanized PDX model was successfully constructed, and select mice in good condition with no scab and necrosis in tumor tissue. The mice were randomly divided into 4 groups, 3 in each group. The mice were treated with corresponding drugs according to the grouping: ① Control group (Control): no drug treatment. ② aPD-L1 group: intraperitoneal injection of human aPD-L1 solution (1 mg / mL) into the mouse body (10 mg / kg). ③ Tumor vaccine group: the vaccine solution (concentration of 1 mg / mL) was injected subcutaneously into the dorsal side of the right hind leg of the mouse, 100 μL / mouse. ④ Combined treatment group: intraperitoneal injection of aPD-L1 solution (1 mg / mL) into the mouse body (10 mg / kg), and at the same time, 100 μL of tumor vaccine solution was subcutaneously injected. 3 in each group. The mice were returned to the cage and raised in an SPF environment for 3 days, and the status of the mice was observed. After 3 days, after blood was collected from the orbital vein, the mice were anesthetized and cervical dislocation was dislocated, and the tumor tissue and spleen were removed for subsequent flow cytometry.

[0078] (2) T cell flow cytometry

[0079] ① Preparation of single cell suspension

[0080] Prepare a single cell suspension of tumor tissue as described above, add an appropriate amount of PBS (containing 2% FBS) and gently pipette to mix, place on ice, and prepare for counting.

[0081] ②Cell staining

[0082] a. Adjust the cell concentration in each tube to about 2×10 7 / mL, and the dispersion medium is flow staining buffer.

[0083] b. Single staining tube: Take 5 tubes of 95μL cell suspension, add 1 antibody to each tube, and add 5μL of each antibody: PerCPanti-human CD45, APC anti-human CD3, FITC anti-human CD8a, PE / Cyanine7 anti-human IFN-γ and PE Anti-human GrB antibodies. PE / Cyanine7 anti-human IFN-γ and PEAnti-human GrB antibodies need to be permeabilized and fixed before staining.

[0084] c. Cell staining: According to the grouping, add 85μL of cell suspension to each sample tube, 3 tubes per group, a total of 12 tubes. Add 1μL of mouse CD16 / 32 monoclonal antibody and human CD16 antibody, and incubate at room temperature for 10 minutes. Subsequently, add 5μL of each antibody to each tube, a total of 3 antibodies: PerCP anti-human CD45, APC anti-human CD3 and FITC anti-human CD8a. After mixing, place at 4℃, incubate in the dark for 30 minutes, and shake every 10 minutes. After 30 minutes, add 200μL of flow staining buffer, centrifuge the cell suspension at 800×g for 5 minutes, and discard the supernatant. Treat the sample with an intracellular factor fixation permeabilizer and fix it at room temperature for 1 hour. Centrifuge the cell suspension at 800×g for 5 minutes and discard the supernatant. Add 90 μL flow staining buffer, resuspend evenly, add 5 μL PE / Cyanine7 anti-human IFN-γ and PE Anti-human GrB antibodies respectively, mix well and incubate at 4°C in the dark for 30 minutes, add 200 μL flow staining buffer, centrifuge the cell suspension at 800×g for 5 minutes, and discard the supernatant. Finally, add 200 μL flow staining buffer for flow cytometry detection.

[0085] (3) Dendritic cell flow cytometry

[0086] ① Use the tumor tissue single cell suspension prepared above to detect dendritic cells. Adjust the cell concentration in each tube to about 2×10 7 / mL, and the dispersion medium is flow staining buffer.

[0087] ② Single staining tube: Take 4 tubes of 95 μL cell suspension, add one antibody to each tube, and add 5 μL of each antibody: PerCP anti-human CD45, FITC Lin-1 antibody (CD3 / 14 / 16 / 19 / 20 / 56), APC anti-human HLA-DR and PE anti-human CD11c antibody.

[0088] c. Cell staining: According to the grouping, add 80μL of cell suspension to each sample tube, 3 tubes per group, a total of 12 tubes. Add 1μL of mouse CD16 / 32 monoclonal antibody and incubate at room temperature for 10 minutes. Subsequently, add 5μL of each antibody to each tube, a total of 4 antibodies: PerCP anti-human CD45, FITC Lin-1 mixed antibody (CD3 / 14 / 16 / 19 / 20 / 56), APC anti-humanHLA-DR and PE Anti-human CD11c antibody. After mixing, place at 4℃, incubate for 30 minutes in the dark, and shake every 10 minutes. After 30 minutes, add 200μL flow staining buffer, centrifuge the cell suspension at 800×g for 5 minutes, and discard the supernatant. Add 200μL flow staining buffer for flow detection.

[0089] 2.2.2 Blood CD4 + and CD8a + T cell quantitative analysis

[0090] (1) Preparation of blood samples

[0091] Prepare mouse blood samples as described above. After lysing red blood cells, add 95 μL of flow cytometry buffer to the single staining tube and 85 μL of flow cytometry buffer to the experimental tube.

[0092] (2) Cell staining

[0093] ① Single staining tube: add one antibody to each tube, 5μL of each antibody: PerCP anti-human CD45, APCanti-human CD3 and FITC anti-human CD8a antibodies. After permeabilization and fixation of the other two tubes, add 5μL of PE / Cyanine7 anti-human IFN-γ and PE Anti-human GrB antibodies respectively.

[0094] ② Experimental tube: Add 1 μL of mouse CD16 / 32 monoclonal antibody and human CD16 antibody, incubate at room temperature for 10 minutes, then add 5 μL of each antibody to each tube, a total of 4 antibodies: PerCP anti-human CD45, APC anti-human CD3 and FITCanti-human CD8a antibody. Mix well and incubate at 4°C in the dark for 30 minutes, shaking every 10 minutes. After 30 minutes, add flow staining buffer, centrifuge the cell suspension at 800×g for 5 minutes, and discard the supernatant. Treat the sample with an intracellular factor fixation permeabilizer and fix at room temperature for 1 hour. Centrifuge the cell suspension at 800×g for 5 minutes and discard the supernatant. Add 90 μL flow staining buffer, resuspend evenly, then add 5 μL PE / Cyanine7 anti-human IFN-γ and PE Anti-human GrB antibodies respectively, mix well and incubate at 4°C in the dark for 30 minutes, add 200 μL flow staining buffer to resuspend the cells, and detect and analyze using a flow cytometer.

[0095] 2.9 Mouse Treatment Monitoring

[0096] Four weeks after model construction, tumors with a volume of approximately 100 mm 3 , weight and good condition of 16 mice were randomly divided into 4 groups: ① Control group (Control): no drug treatment. ② aPD-L1 group: intraperitoneal injection of aPD-L1 solution (1 mg / mL) was intraperitoneally injected into the mouse (10 mg / kg), once every 3 days, for a total of 3 times (1st, 3rd and 5th day). ③ Tumor vaccine group: The tumor vaccine solution (concentration of 1 mg / mL) was injected subcutaneously into the dorsal side of the right hind leg of the mouse, 60 μL / mouse, once every 3 days, for a total of 2 times (1st and 5th day). ④ Tumor vaccine group: intraperitoneal injection of aPD-L1 solution (1 mg / mL) was intraperitoneally injected into the mouse (10 mg / kg), once every 3 days, for a total of 3 times, and at the same time, 100 μL of tumor vaccine solution was subcutaneously injected, once every 3 days, for a total of 2 times. The long and short diameters of the tumor, as well as the weight of the mouse were measured every 2 days, and the tumor volume was calculated. And take pictures and record. After the last monitoring, all mice were anesthetized and killed by cervical dislocation. The tumor tissues were dissected and then fixed with 4% paraformaldehyde and sent for pathological examination. Figure 2 shown.

[0097] The present invention has the following technical points:

[0098] 1. Construction method of immune humanized liver cancer PDX model:

[0099] The specific steps of model construction include the whole process from obtaining liver cancer tissue samples from patients to processing, transplantation and passaging, ensuring that the transplanted model retains the characteristics of liver cancer patient tumors.

[0100] The method of choice for using severely immunodeficient mice (such as B-NDG mice) as model carriers, as well as specific steps for multiple generations to ensure model stability.

[0101] 2. Human immune system introduction technology:

[0102] Protocol for humanizing PDX models using human peripheral blood mononuclear cells (PBMCs), including technical details for purification, selection, and tail vein injection of PBMCs to reconstitute a functional human immune system in mice.

[0103] The success of immune humanization is monitored by specific markers (such as the proportion of CD45+ cells) to ensure effective reconstruction of the model's immune function.

[0104] 3. Technical improvements in model stability and experimental window extension:

[0105] Operational details for optimizing PBMC transplantation protocols to reduce the occurrence of graft-versus-host disease (GvHD), improve the stability of human immune cells, and extend the experimental window.

[0106] Specific immune monitoring technologies, such as flow cytometry to detect GrB and INF-γ expression levels of CD8+ T cells, ensure the immune response capacity of the model for long-term immunotherapy evaluation.

[0107] 4. Immunotherapy evaluation methods:

[0108] By applying immune checkpoint inhibitors (such as PD-1 / PD-L1 antibodies) and tumor vaccines alone and in combination on immune-humanized PDX models, their effects on tumor growth and immune cell function in the model are systematically evaluated.

[0109] Technical points for analyzing the activation and infiltration of dendritic cells and T cells (especially CD8+ T cells) in different treatment groups by flow cytometry and histopathology.

[0110] Alternative solutions:

[0111] 1. Different humanization methods:

[0112] CD34+ hematopoietic stem cells can be used to replace PBMCs for humanization to obtain a longer time window and more comprehensive human immune system reconstruction. CD34+ hematopoietic stem cells can avoid GvHD reactions and can differentiate into a variety of immune cells, but their collection and experimental procedures are complicated.

[0113] 2. Application of other immune checkpoint inhibitors or immunomodulators:

[0114] In addition to PD-1 / PD-L1 inhibitors, CTLA-4 inhibitors, LAG-3 inhibitors or other novel immune checkpoint molecules can also be used to evaluate the efficacy of immunotherapy. According to the mechanism of action of different drugs, the efficacy and immune response of the model under different immune regulation pathways can be evaluated.

[0115] 3. Choice of other tumor vaccines or immune activators:

[0116] Instead of CSF-VacGel tumor vaccine, other immune activators (such as STING agonists, TLR agonists) can be selected for tumor immunity research to enhance the anti-tumor immune effect and verify the applicability of the model.

[0117] In summary, the core protection technology of the present invention is to construct and optimize a liver cancer PDX model with a human immune system and apply it to the evaluation of immunotherapy. Although there are other alternatives, the technical path provided by the present invention has significant advantages in simplifying the experimental process, improving the stability of the model and extending the experimental window.

[0118] It should be noted that the above-described embodiments should be understood as illustrative rather than limiting the scope of protection of the present invention, and the scope of protection of the present invention shall be subject to the claims. For those skilled in the art, some non-essential improvements and adjustments made to the present invention still fall within the scope of protection of the present invention without departing from the essence and scope of the present invention.

Claims

1. A method for constructing an immune humanized xenograft mouse model derived from a liver cancer patient, characterized in that: The following steps are involved: Step 1: Obtaining and processing patient tumor tissue: The fresh liver cancer tissue samples are strictly screened and processed, and cut into small pieces after washing and removing necrotic tissue to ensure tumor activity and genomic integrity; Step 2: Construction of liver cancer PDX model: Transplant the liver cancer tissue treated in step 1 into severely immunodeficient mice, and establish a passage model to obtain a stable liver cancer PDX model; Step 3: Introduction of human immune system: After the PDX model has grown stably, humanization is achieved by transplanting human peripheral blood mononuclear cells (PBMCs). PBMC cell suspension is injected into the tail vein of mice to reconstruct human immune cells in the mice, thus obtaining a liver cancer PDX model with both human liver cancer characteristics and immune function. Step 4. Monitoring of immune function of humanized immune models: Use flow cytometry to regularly detect immune cell markers including CD45+, CD3+, and CD8+ in peripheral blood and tumor tissues to monitor the reconstruction of human immune cells in mice. Use specific markers to determine the success of model construction and ensure that the model has reliable immune response function.

2. The construction method according to claim 1, characterized in that: In step 3, the preparation method of the PBMC cell suspension is as follows: add 15 mL of cell separation solution to a centrifuge tube, then add 17 mL of a mixture of equal volumes of peripheral blood and PBS, keep the centrifuge tube vertical, put it in a centrifuge, and centrifuge it at 1200×g for 10 minutes at room temperature; take out the centrifuge tube, and quickly pour the supernatant into a new 50 mL centrifuge tube; use PBS containing 2% fetal bovine serum to wash the obtained supernatant twice, centrifuge it at 300×g for 8 minutes for the first time, discard the supernatant, add new PBS containing 2% fetal bovine serum, wash it for the second time, centrifuge it at 120×g for 10 minutes to remove platelets, and discard the supernatant; resuspend the obtained cell pellet with serum-free PBS, and adjust the cell concentration to 5×10 8 / mL to obtain PBMC cell suspension.

3. The construction method according to claim 1, characterized in that: In step three, CD34+ hematopoietic stem cells were used instead of PBMCs for humanization.

4. The construction method according to claim 1, characterized in that: In step 4, a specific proportion of CD45+ humanized immune cells is considered to be a sign of successful model construction; by detecting activation markers of CD8+T cells including GrB and INF-γ, the T cell activity of the immune humanized PDX model is evaluated to ensure that the model has reliable immune response function.

5. An immune humanized xenograft mouse model derived from liver cancer patients constructed by the construction method described in any one of claims 1 to 4.

6. Use of the immunohumanized xenograft mouse model derived from liver cancer patients according to claim 5 in the evaluation of liver cancer immunotherapy.

7. The use according to claim 6, characterized in that: The application refers to the use of immune checkpoint inhibitors and tumor vaccines alone or in combination for treatment on a successfully constructed immune humanized xenograft mouse model derived from liver cancer patients, to observe the effects of different treatment regimens on model tumor growth.

8. The use according to claim 7, characterized in that: Flow cytometry and histopathological analysis were used to evaluate the infiltration and functional changes of immune cells in each treatment group to verify the effect of immunotherapy.

9. The use according to claim 7, characterized in that: The immune checkpoint inhibitor is a PD-1 / PD-L1 inhibitor, a CTLA-4 inhibitor or a LAG-3 inhibitor; the tumor vaccine is a CSF-Vac Gel Tumor vaccines.

10. The use according to claim 7, characterized in that: An immune activator is used to replace the CSF-VacGel tumor vaccine, and the immune activator is a STING agonist or a TLR agonist.

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