Multiple myeloma model with extramedullary lesion and construction method and application thereof

Abstract

The invention relates to a multiple myeloma model with extramedullary lesion as well as a construction method and application thereof. The multiple myeloma model is obtained by injecting bone marrow mononuclear cells (BMMC) of a multiple myeloma patient into an hIL-6NSG mouse. The invention provides a new model of multiple myeloma with extramedullary lesion, which not only is beneficial to research on gene pathway and mechanism of the multiple myeloma, but also lays a foundation for research on tumor recurrence, preclinical drug screening, effect evaluation and the like, and has great significance in promoting disease healing.

Description

Technical Field

[0001] The present invention belongs to the field of animal models, and particularly relates to a multiple myeloma model with extramedullary lesions, a construction method thereof, and an application thereof. Background Art

[0002] Multiple myeloma (MM) is a malignant disease characterized by the clonal proliferation of abnormal plasma cells in the bone marrow. The incidence rate accounts for 10% of hematological tumors, and it usually causes clinical symptoms such as anemia, bone pain, renal insufficiency, hypercalcemia, etc. For most MM patients, plasma cell proliferation is limited to the bone marrow. However, due to the presence of extramedullary clonal plasma cells, a small number of MM patients can develop MM with extramedullary lesions (EMD), that is, plasmacytoma involves the bone marrow and organs outside the bone marrow, such as skin, liver, spleen, brain, and lymph nodes, etc. Compared with MM, the prognosis of EMD patients is worse. Although the combined application of drugs such as proteasome inhibitors (PI), immunomodulatory drugs (IMiD), and monoclonal antibodies has improved the survival and prognosis of patients, MM still cannot be completely cured, and the prognosis of patients with multi-drug refractory recurrence is very poor. The chimeric antigen receptor T (CAR-T) cell product cilta-cel targeting B cell maturation antigen (BCMA) has an effective rate of 89.6% in Chinese patients with relapsed / refractory multiple myeloma (RRMM), but nearly half of the patients still face recurrence after 2 years. Therefore, there is an urgent need to seek new therapeutic targets and drugs. The research and development of new drugs and the evaluation of various treatment methods are inseparable from animal models close to clinical practice. However, there is still a lack of an effective and widely available multiple myeloma mouse model.

[0003] Currently, the reported multiple myeloma models mainly include SCID-hu / SCID-synth-hu (implanted fetal human bone or synthetic bone model), C57Bl / KaLwRij, MIS(KI)TRG6, and NSG mouse models, etc. However, the above models all have their own limitations: it is difficult to obtain embryonic bones in the implanted fetal human bone model, C57Bl / KaLwRij only allows the survival of MM cell lines, the purchase method of MIS(KI)TRG6 mice has not been made public, the femoral injection method applied in the NSG mouse model destroys the bone marrow microenvironment, and tumor cells can only grow in the femur, while patient tumor cells mainly survive in the vertebrae. The above factors all limit their wide application. Therefore, there is a need for a simple, efficient, repeatable, and visually monitored model to overcome the defects of previous models, serve the research of MM gene pathways and mechanisms, drug research and development and screening, preclinical treatment, and other aspects of research while retaining the growth and metastasis characteristics of primary tumors. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a multiple myeloma model with extramedullary lesions, its construction method and application. This model not only helps to carry out research on its gene pathways and mechanisms, but also lays a foundation for studying tumor recurrence, preclinical drug screening and efficacy evaluation, etc., and has great significance in promoting the cure of diseases.

[0005] The present invention provides a multiple myeloma model with extramedullary lesions, which is obtained by injecting bone marrow mononuclear cells (BMMC) of multiple myeloma patients into hIL-6 NSG mice.

[0006] Preferably, the hIL-6 NSG mouse is a NOD.CB17-Prkdc scid II2rg tm1 Il6 tm1(IL6) / Bcgen mouse. The hIL-6 secreted by the NOD.CB17-Prkdc scid II2rg tm1 Il6 tm1(IL6) / Bcgen mouse plays a crucial role in the proliferation and survival of myeloma cells.

[0007] The present invention also provides a construction method of a multiple myeloma model with extramedullary lesions, including the following steps:

[0008] Using NOD.CB17-Prkdc scid II2rg tm1 Il6 tm1(IL6) / Bcgen mouse as the model mouse, injecting busulfan into the mouse by intraperitoneal injection; 24 hours after injection, injecting BMMC of multiple myeloma patients into the mouse by intracanthal venous plexus injection, and the multiple myeloma model with extramedullary lesions is obtained.

[0009] Preferably, the NOD.CB17-Prkdc scid II2rg tm1 Il6 tm1(IL6) / Bcgen mouse is an 8-10 week-old mouse.

[0010] Preferably, the dosage of busulfan is 30 mg / kg. Injecting busulfan into the mouse to thoroughly clear the bone marrow of the mouse makes enough space for the transplanted bone marrow cells to clone, proliferate and transform, which is a necessary condition for successfully inducing a multiple myeloma model in the mouse.

[0011] More preferably, 11.7 g / l neomycin sulfate water is given immediately after busulfan injection, and the water is changed once a week for 21 consecutive days. Applying antibiotic water can prevent the infection caused by injection in this immunodeficient mouse.

[0012] Preferably, the method for preparing mononuclear cells of the multiple myeloma patient is as follows: collect the bone marrow fluid of the multiple myeloma patient, count after Ficoll density gradient centrifugation, and resuspend with PBS + 10% FBS to 5×10 6 / 100 μl. Injecting the bone marrow mononuclear cells of the multiple myeloma patient instead of the sorted multiple myeloma cells into the mouse is beneficial to retaining the unique bone marrow microenvironment of the patient and promoting the growth and development of tumor cells.

[0013] Specifically, the method of injecting the medial canthal venous plexus is as follows: fix the mouse, gently force the eyeball to protrude from the orbit, insert the needle at 2 mm above the medial canthus and 1 mm inside the skin margin, and slowly insert the injection needle tip (with the sharp opening facing inward to protect the eyeball from being scratched) at a 45° angle to the direction of the mouse's nose tip into the medial canthus. A distinct sense of falling through will be felt with a slight force, and the insertion depth is 3 - 5 mm. Injecting the medial canthal venous plexus is easy to operate, direct, fast, efficient, does not require anesthesia of the mouse, and has a high success rate.

[0014] Furthermore, after injecting BMMC into NOD.CB17 - Prkdc scid II2rg tm1 Il6 tm1(IL6) / Bcgen mice, monitor the body weight and vitality of the mice weekly, take the mouse serum for ELISA experiments monthly to detect the expression level of human monoclonal immunoglobulin G (IgG). After the mice develop the disease, take the femurs, tibias and fibulas, spines and spleens of the mice for flow cytometry detection. In the model constructed by the present invention, ELISA analysis shows that the mouse serum contains multiple myeloma - specific human monoclonal immunoglobulin G. Through flow cytometry detection, it is found that in the femurs, tibias and fibulas, spines and spleens of the mice, myeloma cells with specific markers CD38 and CD138 expression and immune cells such as CD4+ T cells, CD8+ T cells, B cells and NK cells are significantly observed.

[0015] The present invention also provides an application of a multiple myeloma model with extramedullary lesions, including one or more of the following applications:

[0016] (1) For the study of the pathogenesis and recurrence mechanism and gene pathway of multiple myeloma;

[0017] (2) For the study of pre - clinical drug screening and efficacy evaluation;

[0018] (3) For the efficacy evaluation of the combination of chemotherapy, immunotherapy and CAR - T and other treatments.

[0019] Beneficial effects

[0020] (1) The modeling mice of the present invention are easy to obtain, have a high modeling efficiency and good repeatability;

[0021] (2) The method of injecting the internal canthal vein plexus in the present invention is simple and easy to perform, without the need for anesthesia. Compared with injecting into the tibia and fibula, it not only does not damage the original bone marrow cavity of the mouse but also has a higher success rate.

[0022] (3) Multiple myeloma cells grow and develop in the femur, tibia and fibula, spine and spleen of the mice in the present invention, solving the problem that multiple myeloma cells in the model of femoral injection are difficult to spread to organs other than the femur.

[0023] (4) The present invention injects bone marrow BMMCs of multiple myeloma patients into mice, while retaining tumor heterogeneity, and also provides a unique bone marrow microenvironment for the growth and development of multiple myeloma cells.

[0024] (5) The present invention provides a new model of multiple myeloma with extramedullary lesions, which not only helps to carry out research on its gene pathways and mechanisms, but also lays a foundation for research on tumor recurrence, preclinical drug screening and efficacy evaluation, etc., and has great significance in promoting the cure of the disease. Brief Description of the Drawings

[0025] Figure 1 It is the ELISA detection of monoclonal gamma globulin G secreted by the model of the present invention (n = 12).

[0026] Figure 2 is the flow cytometry detection of tumor cells in the model of the present invention; among them, Figure A is the flow cytometry schematic diagram of the mouse spleen; Figure B is the flow cytometry schematic diagram of the mouse spine; Figure C is the flow cytometry schematic diagram of the mouse femur and tibia.

[0027] Figure 3 is the flow cytometry detection of immune cells in the model of the present invention; among them, Figure A is the flow cytometry schematic diagram of the mouse spleen; Figure B is the flow cytometry schematic diagram of the mouse spine; Figure C is the flow cytometry schematic diagram of the mouse femur and tibia. Detailed Embodiments

[0028] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

[0029] The NOD.CB17-Prkdc scid II2rg tm1 Il6 tm1(IL6) / Bcgen mice used in this experiment were all female, 8-10 weeks old, purchased from Beijing Biocytogen Co., Ltd., and were raised in the Experimental Animal Center of Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine.

[0030] Example 1

[0031] This example is an example of establishing a multiple myeloma model with extramedullary lesions, and the operation is carried out according to the following steps:

[0032] (1) Mouse preparation

[0033] Specific pathogen-free (SPF)-level NOD.CB17-Prkdc scid II2rg tm1 Il6 tm1(IL6) / Bcgen female mice aged 8 - 10 weeks were used as standby model mice, and the mice were divided into two groups:

[0034] Group A (normal control group) and Group B (multiple myeloma experimental group).

[0035] (2) Modeling preparation

[0036] Busulfan was injected into NOD.CB17-Prkdc scid II2rg tm1 Il6 tm1(IL6) / Bcgen mice by intraperitoneal injection, and the dosage was 30 mg / kg.

[0037] (3) Tumor seeding

[0038] After NOD.CB17-Prkdc scid II2rg tm1 Il6 tm1(IL6) / Bcgen mice received intraperitoneal injection of busulfan for 24 h, bone marrow mononuclear cells (BMMC) from multiple myeloma patients were injected into Group B mice by the medial canthus venous plexus injection method.

[0039] Among them, the specific preparation method of BMMC from multiple myeloma patients:

[0040] 1) Take fresh anticoagulated bone marrow fluid, add 1×PBS washing solution according to a ratio of 1:1, dilute the bone marrow fluid (to reduce the viscosity of the bone marrow fluid), gently mix well, and set aside.

[0041] 2) Add an appropriate amount of mononuclear cell separation solution to a 15 ml sterile centrifuge tube, spread the diluted bone marrow fluid on top of the separation solution surface (separation solution: diluted whole bone marrow fluid = 1:2), and keep the interface of the two liquid surfaces clear.

[0042] 3) Centrifuge at 800 g for 20 min at room temperature (note: set a slower acceleration and deceleration, the acceleration is set to the third gear, and the deceleration is set to the zero gear).

[0043] 4) After centrifugation, aspirate and discard the plasma layer, carefully aspirate the BMMC layer (i.e., the buffy coat) and transfer it to a 15 mL centrifuge tube. Add 10 mL of 1×PBS washing solution to the centrifuge tube to resuspend the cells, centrifuge at 1800 rpm for 5 min at room temperature, discard the supernatant, and resuspend it in PBS + 10% FBS for later use.

[0044] Among them, the method of injecting the internal canthus venous plexus is as follows: Fix the mouse, gently force the eyeball to protrude from the orbit, insert the needle 2 mm above the internal canthus and 1 mm inside the skin margin, and the injection needle tip (with the sharp opening facing inward to protect the eyeball from being scratched) is slowly inserted into the internal canthus at an angle of 45° to the direction of the mouse's nose tip. There will be an obvious feeling of falling through with a slight force, and the insertion depth is 3 - 5 mm.

[0045] Example 2

[0046] This example is an ELISA detection example of human blood monoclonal gamma globulin G (hIgG) secreted by a multiple myeloma model with extramedullary lesions.

[0047] (1) Dilute the standard product in the ELISA kit and add the sample, and make a standard curve according to the concentration of the standard product.

[0048] (2) Take an appropriate amount of mouse serum required by the kit, add the sample and incubate according to the requirements of the kit.

[0049] (3) Measure the OD value with an enzyme - labeled instrument at the wavelength required by the kit.

[0050] (4) Determine the expression of hIgG in mice in this model according to the standard curve.

[0051] As Figure 1 shown, the mice in group B that had been injected with patient BMMC secreted hIgG, while the control mice in group A did not secrete hIgG. It is proved that this model has the typical clinical characteristics of multiple myeloma that secretes hIgG.

[0052] Example 3

[0053] This example is a detection example of the tumor burden of a multiple myeloma model with extramedullary lesions by flow cytometry.

[0054] (1) Sacrifice the normal mice in group A and the diseased mice in group B by cervical dislocation.

[0055] (2) Dissect the tissues such as femur, tibia - fibula, spine and spleen of the dead mice.

[0056] (3) Remove the excess muscle around the bones and gently grind them into single - cell suspension.

[0057] (4) After taking pictures of the mouse spleen, quickly grind the spleen into single - cell suspension with the frosted part of the end of the glass slide.

[0058] (5) Filter the ground cells through a 40-μm filter membrane into a 15-ml centrifuge tube, centrifuge at 300 g for 5 min at room temperature, and pour off the supernatant.

[0059] (6) Add 500 μl of lysis buffer to each tube to lyse red blood cells (lyse spleen cells for 40 s and bone marrow cells for 20 s). Subsequently, wash the cells with 10 ml of PBS + 10% FBS, centrifuge at 300 g for 5 min at room temperature, pour off the supernatant, resuspend the cells in PBS + 10% FBS, count the cells, and divide the cells into two equal parts.

[0060] (7) Add anti-human CD138, CD38, CD45 antibodies and anti-mouse CD45 antibody to an appropriate amount of PBS + 10% FBS, mix well, add the cells to this PBS + 10% FBS, and incubate on ice for 30 min; meanwhile, add anti-human CD3, CD8, CD56, CD138, CD38, CD45, CD19, CD27 and other immune-related antibodies to an appropriate amount of PBS + 10% FBS, mix well, add the cells to this PBS + 10% FBS, and incubate on ice for 30 min.

[0061] (8) After the antibody incubation is completed, add an appropriate amount of PBS + 10% FBS to wash once, centrifuge at 300 g for 5 min, and discard the supernatant.

[0062] (9) Resuspend each tube of cells in an appropriate amount of PBS + 10% FBS.

[0063] (10) Detect using a BD LSRFortessa X20 flow cytometer.

[0064] As Figure 2A shown in -C, multiple myeloma cells with an immunophenotype of CD38+CD138+ exist in the femur, tibia-fibula, spine, and spleen, simulating the pathological characteristics of multiple myeloma with extramedullary lesions, indicating that the application of the present invention can successfully induce a multiple myeloma model with extramedullary lesions.

[0065] As Figure 3A shown in -C, the B group of mice has tumor cells of patients and immune cells such as CD4+ T cells, CD8+ T cells, B cells, and NK cells in the bone and spleen and the bone marrow microenvironment, retaining the tumor heterogeneity and bone marrow microenvironment, and can better reflect the interaction between multiple myeloma cells and the bone marrow microenvironment.

[0066] The above are only embodiments of the present invention. Specific structures and common knowledge such as characteristics that are well-known in the art are not described in detail herein. Those of ordinary skill in the art know all the common general technical knowledge in the technical field to which the invention pertains before the filing date or the priority date, can know all the prior art in this field, and have the ability to apply the conventional experimental means before this date. Those of ordinary skill in the art can, under the inspiration given in this application, combine their own abilities to complete and implement this solution. Some typical well-known structures or well-known methods should not become obstacles for those of ordinary skill in the art to implement this application. It should be noted that for those skilled in the art, without departing from the structure of the present invention, several deformations and improvements can still be made, and these should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent. The protection scope claimed in this application should be based on the content of its claims, and the specific implementation manners and the like described in the specification can be used to interpret the content of the claims.

Claims

1. A multiple myeloma model with extramedullary lesions, characterized in that: It was obtained by injecting bone marrow mononuclear cells (BMMC) of multiple myeloma patients into hIL-6 NSG mice.

2. The multiple myeloma model with extramedullary lesions according to claim 1, characterized in that: The hIL-6NSG mouse is a NOD.CB17-Prkdc scid II2rg tm1 Il6 tm1(IL6) / Bcgen mouse.

3. A method for constructing a multiple myeloma model with extramedullary lesions, comprising the following steps: Using NOD.CB17-Prkdc scid II2rg tm1 Il6 tm1(IL6) / Bcgen mice as model mice, injecting busulfan into the mice by intraperitoneal injection; 24 hours after injection, injecting mononuclear cells of multiple myeloma patients into the mice by injection into the medial canthal venous plexus, thus obtaining a multiple myeloma model with extramedullary lesions.

4. The construction method according to claim 3, wherein: The NOD.CB17-Prkdc scid II2rg tm1 Il6 tm1 (IL6) / Bcgen mice are 8 - 10 weeks old mice.

5. The construction method according to claim 3, wherein: The administration dose of busulfan is 30 mg / kg.

6. The construction method according to claim 3, characterized in that: The method for preparing mononuclear cells of the multiple myeloma patient is as follows: collect the bone marrow fluid of the multiple myeloma patient, count after Ficoll density gradient centrifugation, and resuspend with PBS + 10% FBS to 5×10 6 / 100 μl.

7. Use of a multiple myeloma model with extramedullary lesions as described in claim 1, characterized in that: It includes one or more of the following applications: (1) For the study of the pathogenesis and recurrence mechanism and gene pathways of multiple myeloma; (2) For the study of preclinical drug screening and efficacy evaluation; (3) For the efficacy evaluation of the combination of chemotherapy, immunotherapy and CAR-T therapy.

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