CN109481666B

CN109481666B - Method for establishing PDX model of human blood tumor

Info

Publication number: CN109481666B
Application number: CN201811568072.0A
Authority: CN
Inventors: 朱学军; 田芳; 姜鹏君; 吴坚; 孔祥图; 于慧; 于菊华; 徐祖琼; 代兴斌; 陈晓丽; 庞洁; 李峻; 张文曦; 倪海雯; 陈健一; 陈玉根
Original assignee: Jiangsu Provincial Hospital of Chinese Medicine
Current assignee: Jiangsu Provincial Hospital of Chinese Medicine
Priority date: 2018-12-21
Filing date: 2018-12-21
Publication date: 2020-11-03
Anticipated expiration: 2038-12-21
Also published as: CN109481666A

Abstract

The invention discloses a method for establishing a human blood tumor PDX model, which comprises the steps of extracting blood tumor cells of a patient, adding rabbit anti-human thymocyte immunoglobulin ATG and patient autologous serum, mixing, incubating, after incubation is finished, resuspending the obtained cells, and inoculating the cells into a mouse body; the vaccinated mice were fed cyclosporin CsA for 5-10 days starting 2 days before the hematological tumor cells were vaccinated. The invention adopts a novel high-immunodeficiency NCG mouse independently developed in China to establish a human leukemia PDX model for the first time, eliminates and inhibits T cells from a donor source through 'long-term oral administration of a human thymocyte immunoglobulin pretreatment sample and cyclosporine', combines the toxic effect of ATG on lymphocytes and the functional blocking effect of CsA on T lymphocytes, can persistently inhibit the immune function of the T lymphocytes and obviously improve the success rate of PDX modeling of blood tumors.

Description

Method for establishing PDX model of human blood tumor

Technical Field

The invention relates to a construction method of a blood tumor experimental animal model, in particular to a construction method of a human blood tumor PDX model.

Background

According to the latest statistics, the morbidity of hematological tumors in China is about 3-4 people/10 ten thousand, and the 6 th (male) and 8 th (female) in the mortality rate of malignant tumors, and the first in children and adults under 35 years old. A xenograft (PDX) model of patient tumor tissue is a preclinical study and drug screen animal model created for malignancies. The model directly transplants fresh tumor tissues of a patient into an immunodeficient mouse body, establishes a tumor tissue cell bank capable of being stably passaged, and provides important support for tumor-related basis and clinical research. However, this model also has certain problems at present. In one aspect, PDX model construction requires the use of immunodeficient mice. Although the existing immunodeficient mouse variety can meet the requirements of establishing a solid tumor PDTX model of certain types, the establishment of a leukemia PDX model still has great difficulty and challenge, and stable passage is difficult to achieve. In fact, if the PDTX model cannot be successfully transferred to 3 rd generation and 4 th generation, it will not be widely used in related disease research and clinical drug development. Foreign reports apply a new generation of high immunodeficiency NOG or NSG mice to model multiple solid tumors PDX, the success rate is up to more than 70%, and the success rate of PDX modeling of blood system tumors is lower than 30%. On the other hand, the applicant and the foreign researchers observed that a small amount of patient-derived T lymphocytes contained in a leukemia sample are greatly amplified in a mouse body during the PDX modeling process, which is currently considered to be another main reason for the low efficiency of PDX modeling of a hematological tumor. Previous researchers have attempted to eliminate T lymphocytes from patients' peripheral blood or bone marrow-derived monocytes via a "CD 3 magnetic bead sorting" protocol. However, not all T lymphocytes express the extracellular antigen CD3, T cell precursors (including Pro-T, Pre-T) mainly express CD44, CD25 and c-kit, and CD4/CD8 double positive T cells only weakly express CD 3. Therefore, the "CD 3 magnetic bead sorting method" cannot remove T lymphocytes efficiently, is high in cost and has certain limitations, and needs further improvement.

The field is competitive on intellectual property, so that the research strength of the disease animal model field in China is increased in many aspects, and the research pace is accelerated. In the present stage, the disease animal model modeling strategy mainly refers to a genetically modified animal model, and spontaneous disease animal models, environmental induction, physical factor induction and the like are involved, and in recent years, researchers continuously make efforts to achieve certain results. For example, a leukemia model or the like is established in zebrafish by using a gene editing technique. However, the variety of animal models of leukemia is limited compared to other solid tumors, and at present, sufficient quantities for preclinical studies have not been provided, which has prevented the prediction of clinical efficacy and the identification of heterogeneous responses in patients. Therefore, the establishment of an efficient and stable leukemia PDX model can generate great economic and social benefits.

A series of breakthrough progresses are made in the beginning of PDX model establishment in 2002, and an NOD/SCID-based NOG (NOD/Shi-SCID, IL-2R gamma null) mouse model is established by the cultivation of a Mamoruito research group of a Japanese laboratory animal institute, and a mouse strain with serious immunologic function insufficiency is successfully established by hybridizing NOD/SCID mice and gamma-chain IL-2 receptor knockout mice. In 2005, the american jackson laboratory related researchers have also issued NOG-based NSG improvement models. Compared with NOD/scid mice, NOG and NSG mice have no T and B lymphocyte immune leakage, have longer service life, have obviously improved human cell and tissue transplantation survival rate, and can be implanted into approximately 70-80% of normal or cancerated human cells and tissues. However, many experimental institutions including China do not have the proprietary intellectual property rights of the two types of mice, and therefore, the experimental institutions need to be imported at high price so as to carry out relevant experimental research, the experimental cost is high, the time and the number of the obtained mice cannot be guaranteed, and the establishment of large-scale disease models is not facilitated.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for establishing a human blood tumor PDX model with high success rate and stable passage aiming at the defects of the prior art.

Clinically, the use of inhibitors against T lymphocytes before or early after allograft transplantation can successfully reduce the incidence of acute rejection and the mortality of patients associated with transplantation. Rabbit anti-human thymocyte immunoglobulin (ATG) and cyclosporine (CsA) are often used clinically to treat graft-versus-host disease (GVHD), which provides a new idea for our research. The rabbit anti-human thymocyte immunoglobulin is an anti-lymphocyte immunosuppressant, and can inhibit the function of T (thymus-dependent) lymphocytes under the participation of complement (C1-C4). It can recognize active substances on the surface of thymus-derived T cells such as: CD2, CD3, CD4, CD8, CD11a, CD25, HLA-DR and class i HLA, T cells are complement dependent lysed and cleared from the circulation by Fc-dependent opsonic mechanisms formed by monocyte and phagocyte interactions. Therefore, ATG is an immunosuppressant which can comprehensively recognize antigens and can eliminate the most effective T lymphocytes in various developmental stages from thymus. Cyclosporine (CsA) is an immunosuppressant with high specificity and strong inhibitory effect, and can selectively act on the initial stage of T lymphocyte activation without affecting the normal hematopoietic function of bone marrow, and has little effect on B cells, granulocytes and macrophages. Has the characteristics of no toxicity to hematopoietic tissues, no increase of infection chances, convenient use and the like, and can greatly improve the survival rate of transplantation.

The specific scheme of the invention is as follows:

a method for establishing a PDX model of a human blood tumor comprises the following steps:

(1) extracting blood tumor cells of a patient;

(2) adding rabbit anti-human thymocyte immunoglobulin and patient autologous serum into the blood tumor cells obtained in the step (1), mixing and incubating;

(3) after incubation is finished, the obtained cells are re-suspended and inoculated in a mouse body;

(4) the vaccinated mice were fed cyclosporin for 5-10 days starting 2 days before the hematological tumor cells were vaccinated.

In the step (1), the extraction mode is to collect 5-50ml of blood or bone marrow of a patient and carry out gradient centrifugation by using Ficoll separating medium; the isolated mononuclear cells were washed with physiological saline and then resuspended in 0.5-2ml of physiological saline.

In the step (2), blood tumor cells are taken and stored in physiological saline, and then 50-400 mu g of rabbit anti-human thymocyte immunoglobulin and 1ml of patient autologous serum are added according to the inoculation amount of each mouse.

The complement in the serum of part of leukemia patients is low and needs to be supplemented with rabbit complement, 1ml of autologous serum is adopted, and the rabbit complement to be supplemented accounts for 20-50%.

Preferably, the incubation conditions are: incubating at 37 deg.C for 0.5-1.5 h.

In the step (3), the inoculation amount is a mixed system obtained after 0.1-0.25ml of heavy suspension is inoculated to each mouse, and the inoculation amount of the blood tumor cells in the mixed system is 2 multiplied by 10⁵-1×10⁶The inoculation mode is intravenous injection through rat tail.

The mice are female NCG mice, 6-8 weeks old, and purchased from Nanjing university model animal institute.

In the step (4), the injection/feeding dosage of the cyclosporine is 15-35 mg.kg^-1·d^-1And (6) metering.

Has the advantages that:

1. the invention adopts a novel high-immune-deficiency NCG mouse independently developed in China to establish a human leukemia PDX model for the first time. Compared with the existing and widely used NOG mice (from the Japanese institute of animals) and NSG mice (from the Jackson laboratory in America), the mouse has the advantages of purer background, more serious immune system defect, longer service life, suitability for long-term experimental observation and medication, high survival rate of xenograft, highest tumor formation rate of the existing immune defect mice, and particular suitability for establishment of non-solid tumors, namely blood system tumors.

2. For mononuclear cells from peripheral blood or bone marrow of a patient, a human thymocyte immunoglobulin pretreatment sample is combined with cyclosporine for long-term oral administration to clear and inhibit T cells from a donor, and the toxic effect of ATG on lymphocytes is combined with the functional blocking effect of CsA on T lymphocytes, so that the immune function of the T lymphocytes can be persistently inhibited, and the success rate of PDX modeling of blood tumors is remarkably improved.

Drawings

The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.

FIG. 1 is a B-ALL/NCG mouse model of the present invention.

FIG. 2 is a peripheral blood and bone marrow smear from P1 generation B-ALL/NCG mice.

FIG. 3 shows the HE staining and immunohistochemistry indicating infiltration of human CD45 leukemia cells into organs such as spleen, liver and kidney.

FIG. 4 shows the proliferation of 3 mouse peripheral blood CD45/CD19 human leukemia cells in model process by flow test of P2 generation.

FIG. 5 is an experimental observation of drug susceptibility of B-ALL NCG mice of P3 generation: tumor cell growth rate in the saline control group, fludarabine combination and bortezomib + methotrexate administration group.

Detailed Description

The invention will be better understood from the following examples.

Firstly, leukemia sample pretreatment of a patient:

1. collecting 5-50ml blood or 5-50ml bone marrow of a patient, performing gradient centrifugation by using a Ficoll separating medium, separating single nuclear cells of the patient, washing the single nuclear cells twice by using normal saline, then re-suspending the single nuclear cells by using the normal saline with the volume of 0.5-2ml, and counting the cells.

2. ATG + complement incubation group: a part of the patient sample cells is added with ATG (200 mug/cell) and 1ml of patient autologous serum (the serum contains complement, if the patient's autologous complement is insufficient, a certain amount of rabbit complement can be supplemented), mixed evenly and incubated at 37 ℃ for 1 hour. One part of the product is reserved for flow detection to identify the content of T lymphocytes, and the T lymphocytes are resuspended and counted every 1 × 10⁶Individual cells/0.2 ml mixture/mouse, mice were inoculated by tail vein injection.

Secondly, NCG mice are raised and inoculated:

1) mouse feeding conditions: female NCG mice, 6-8 weeks old. The breeding is carried out in cages under the condition of meeting the SPF grade. Keeping the room temperature at 18-25 deg.C and relative humidity at 40-60%, and sterilizing special mouse cage, padding, feed and drinking water at 121 deg.C for 30 min. Dunnage is changed at least 1 time per week.

2) NCG mice were grouped: before the experiment, 20 mice were weighed, recorded, randomly divided into 2 groups of 10 mice each, and inoculated according to the cell treatment method described above.

3) And (3) feeding and treating cyclosporine: the control group was matched with normal drinking water without treatment, and the ATG + complement incubation group was dosed with cyclosporine (CsA).

Thirdly, successfully evaluating indexes of the system building:

1) general observation indexes are as follows: after the xenotransplantation, the mice of each group are observed whether the mice have the expression of mental withered, anorexia, emaciation, arch back, diarrhea, depilation, skin ulcer, hemiplegia, blindness and the like and whether the mice have the growth of lumps, the weight of the mice of each group is regularly given, data is recorded, and the mice are observed until 60 days after the transplantation.

2) Tail vein blood sampling: the morphology of peripheral cells of the mice and the leukemia immunophenotype were monitored weekly to determine whether the transplantation was successful and whether the T lymphocytes were greatly expanded in the mice.

3) Mice with successful transplantation inoculation were selected for sacrifice: collecting peripheral blood, bone marrow, spleen and liver of a mouse, making into a smear or a print, observing cell morphology by Swiss staining, and calculating the percentage of tumor cells; spleen cell suspensions were analyzed by flow cytometry for immunophenotyping.

4) Pathological examination of each tissue and organ: the PDX mice are dissected before or after dying, the rest mice are uniformly sacrificed 60 days after the xenograft is observed in a laminar flow frame, and tumor tissues of the mice, livers, spleens, lung tissues, kidneys, brain tissues, bone marrow, skins and the like of the mice are taken. Conventional fixation, paraffin embedding and slicing are adopted. The tissue corresponding to normal mouse NCG was used for histopathological control. And (3) staining the taken tissue by adopting a hematoxylin-eosin staining method, and observing the morphology of the tumor-bearing tissue. Different tissues of PDX mice were examined for human CD45 antigen expression using immunohistochemical staining.

5) Continuously inoculating and passaging leukemia cells: blood was drawn from primary NCG mice, or spleen suspensions of primary mice were thawed quickly using frozen stock, washed with normal saline, at 1 × 10⁶The cells were added with 0.2ml of physiological saltResuspend in water and inoculate the next generation of NCG mice by intravenous injection at the tail.

Fourthly, experimental results:

1. treatment of NCG mice with the "ATG + CsA" protocol for side effects

In order to preliminarily verify the scientificity of the theoretical basis of 'ATG + CsA' and evaluate the toxic and side effects of the combined immunization of the two medicines, firstly, ATG + complement (200 mu g/mouse, tail vein injection) and CsA (35mg/kg, drinking water feeding) are adopted for NCG mouse experiments (total 6 mice), and the experiment lasts for 2 months, so that the mice have no influence on diet and drinking water, normal hair color and activity, no adverse reactions such as hematopoietic dysfunction, infection and the like.

2. The B-ALL/NCG mouse model constructed by the scheme of 'ATG + CsA' is successful

FIG. 1 is a B-ALL/NCG mouse model: the mice have the phenomena of dull fur color, listlessness, reduced activity and the like 8 to 9 weeks after the injection of the blood tumor cells. The mice were dissected to show severe splenomegaly (black arrows in figure 1).

The inventors further isolated a case of leukemia cells derived from peripheral blood of a B-ALL patient suffering from relapsing refractory B-cell acute lymphoblastic leukemia, and established P1 generation B-ALL/NCG mice, which had a large amount of tumor cell infiltration in peripheral blood and spleen cells. FIG. 2 is a peripheral blood and bone marrow smear observation of P1 generation B-ALL/NCG mice: a large number of leukemia cells are visible.

However, when an expanded number of P2 generation mice were inoculated with spleen suspension of P1 generation mice, only a part of the P2 generation mice (3 out of 6) had leukemia cells expanded, and further 3 mice exhibited a large amount of human T cells in peripheral blood and spleen without leukemia cells, and could not be passaged stably. After the spleen suspension of the P1 generation mouse is incubated with ATG and complement at 37 ℃ for 1h, 6 mice of the P2 generation are inoculated again, and the CsA is fed, the flow detection result shows that a large amount of leukemia cells appear in the peripheral blood of all the mice in one month, and the expansion phenomenon of T lymphocytes is not found. HE staining and immunohistochemistry are adopted to show that human CD45 leukemia cells infiltrate organs such as spleen, liver and kidney, and the like, as shown in figure 3; the spleen, the liver venous vascular area and the kidney glomerular area are infiltrated by a large amount of tumor cells, and hematoxylin and eosin staining and the human antibody CD45 show dark positive expression.

The flow detection shows that the proliferation condition of human B-ALL leukemia cells expressing CD45/CD19 in the peripheral blood of 3 mice of P2 generation in the modeling process. Tail vein blood sampling and flow detection of human-derived CD45/CD19 positive cells were performed on 10, 20, and 30 days, respectively, of modeling, see fig. 4. The result shows that the tumor cells can be stably passaged and greatly amplified in the B-ALL mouse model, and the proportion of the tumor cells in the peripheral blood is suitable for screening later-stage medicines.

This protocol was equally effective for 18P 3 generation mice, and P3 generation mice could be successfully used in subsequent drug susceptibility experiments. FIG. 5 is a general drug susceptibility experimental observation of P3 generation B-ALL/NCG mice: the method comprises the steps of carrying out a drug sensitivity experiment by utilizing a P3 generation B-ALL mouse which is stably established and expanded to a certain amount, dividing the mouse into a normal saline control group and a bortezomib and methotrexate combined administration group, and respectively detecting the growth rate of in vivo tumor cells. The above results demonstrate that the ATG plus complement combined with cyclosporine protocol is effective for modeling this example of B-ALL mice. In conclusion, by using the scheme, various blood system PDX mouse models such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia CML, myeloma MM and the like are successfully constructed, stable passage can be realized, and the method can be successfully applied to a later-stage drug susceptibility test.

The present invention provides a method and a method for establishing a PDX model of a human hematological tumor, and a plurality of methods and ways for implementing the method and the method are provided, and the above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, a plurality of improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.

Claims

1. A method for establishing a leukemia xenograft model is characterized by comprising the following steps:

(1) extracting leukemia tumor cells of a patient;

(2) adding rabbit anti-human thymocyte immunoglobulin and patient autologous serum into the leukemia tumor cells in the step (1), mixing and incubating;

(3) after incubation is finished, the obtained cells are re-suspended and inoculated in a mouse body through rat tail intravenous injection;

(4) the inoculated mice are fed with cyclosporine for 5-10 days starting 2 days before the leukemia tumor cells are inoculated;

in the step (2), storing the leukemia tumor cells in physiological saline, and then adding 50-400 mu g of rabbit anti-human thymocyte immunoglobulin and 1ml of patient autologous serum according to the inoculation amount of each mouse; (ii) the patient's autologous serum needs to be supplemented with 20-50 vt% rabbit complement;

in the step (3), the inoculation amount is a mixed system obtained after 0.1-0.25ml of heavy suspension is inoculated to each mouse, wherein the inoculation amount of leukemia tumor cells contained in the mixed system is 2 multiplied by 10⁵-1×10⁶A plurality of;

in the step (3), the mice are female NCG mice, 6-8 weeks old and purchased from Nanjing university model animal institute;

in the step (4), the dosage of the cyclosporine is 15-35 mg.kg^-1·d^-1And (6) metering.

2. The method for establishing a leukemia xenograft model according to claim 1, wherein in the step (1), the extraction method comprises collecting 5-50ml of patient blood or bone marrow, and performing gradient centrifugation by using Ficoll separating medium; the isolated mononuclear cells were washed with physiological saline and then resuspended in 0.5-2ml of physiological saline.

3. The method for establishing a leukemia xenograft model according to claim 1, wherein in the step (2), the incubation conditions are: incubating at 37 deg.C for 0.5-1.5 h.