A method of constructing a mouse tumor model using a single tumor cell

Abstract

The present invention relates to a method for constructing a mouse tumor model using a single tumor cell. In particular, the method according to the present invention comprises the step of transplanting a fibrin gel comprising a single tumor cell into a mouse; preferably, the method comprises the step of transplanting a fibrin gel comprising a single tumor cell into the subcutis of a mouse.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. Specifically, this invention relates to a method for establishing a mouse tumor model using a single tumor cell and the use of this model in studying the mechanisms of tumor occurrence and development and screening anti-tumor drugs. Background Technology

[0002] Malignant tumors, as a serious threat to human life and health, still lack effective treatments, primarily because the mechanisms of tumor development and progression remain unclear. The scientific community generally agrees that in the process of tumorigenesis, a single tumor-initiating cell, under suitable in vivo microenvironment, gradually divides and proliferates, eventually forming pathologically visible tumor tissue. Therefore, the occurrence, development, recurrence, and metastasis of tumors are closely related to individual tumor-generating cells within the tumor tissue that possess self-renewal and regenerative capabilities, also known as tumor stem cells. Furthermore, these tumor-generating cells often play a crucial role in the process of metastasis and recurrence. Therefore, establishing single-cell tumorigenesis models is an important and effective means of simulating and studying this process. However, current research methods still cannot establish biological tumor models from a single tumor cell. In basic tumor research, suitable animal models are paramount for studying the mechanisms of tumorigenesis, development, and treatment. Therefore, establishing a single-cell mouse subcutaneous ectopic tumor model is of great significance for studying the cellular characteristics and development process of a single tumor-initiating cell, as well as for understanding the occurrence and development of tumors.

[0003] In current experimental research, researchers establish stable animal tumor models through transplantation of multiple tumors (>10) or chemical induction. These two methods cannot effectively simulate the initial tumor development and progression of a single tumor-initiating cell, hindering researchers' studies on tumor development and progression. Establishing an animal tumor model initiating from a single tumor cell is an urgent need in contemporary tumor research. Summary of the Invention

[0004] In a first aspect, the present invention provides a method for constructing a mouse tumor model, the method comprising the step of transplanting fibrin glue containing individual tumor cells into a mouse; preferably, the method comprises the step of transplanting fibrin glue containing individual tumor cells subcutaneously into a mouse.

[0005] Specifically, the present invention provides a method for constructing a mouse tumor model, the method comprising the following steps:

[0006] 1) Mix the culture medium containing tumor cells with fibrinogen and spread it in a 96-well plate; preferably, the concentration of the fibrinogen is 0.1 to 10 μg / ml, more preferably 4-8 μg / ml; more preferably, the fibrinogen is human fibrinogen or salmon fibrinogen;

[0007] 2) Add thrombin to the 96-well plate from step 1) and incubate at 37°C for 60 minutes;

[0008] 3) Add culture medium to the wells and incubate at 37°C for 6 to 12 hours;

[0009] 4) Select fibrin glue containing individual tumor cells, remove the culture medium, and

[0010] 5) The fibrin glue containing single tumor cells obtained in step 5) is transplanted into mice, preferably into the subcutaneous tissue of mice.

[0011] In a preferred embodiment of the method according to the present invention, the single tumor cell is selected from melanoma, liver cancer, breast cancer, colorectal cancer, myeloma, small cell lung cancer, non-small cell lung cancer, head and neck cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, large B-cell lymphoma rich in T-cells / histocytes, multiple myeloma, myeloid leukemia, etc. Tumor cells associated with various cancers, including leukemia-1 protein, myelodysplastic syndrome, gastric cancer, ovarian cancer, lymphoblastic leukemia, lymphocytic leukemia, endometrial cancer, prostate cancer, thyroid cancer, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, bone cancer, Ewing's sarcoma, cervical cancer, brain cancer, bladder cancer, clear cell renal cell carcinoma, pharyngeal cancer, hepatobiliary cancer, central nervous system cancer, esophageal cancer, malignant pleural mesothelioma, systemic light chain amyloidosis, lymphoplasmacytic lymphoma, myelodysplastic syndrome, myeloproliferative tumors, neuroendocrine tumors, Merkel cell carcinoma, testicular cancer, and skin cancer.

[0012] In a second aspect, the present invention provides a method for preparing fibrin glue containing single tumor cells, the method comprising the following steps:

[0013] 1) Mix the culture medium containing tumor cells with fibrinogen and spread it in a 96-well plate; preferably, the concentration of the fibrinogen is 0.1 to 10 μg / ml, more preferably 4-8 μg / ml; more preferably, the fibrinogen is human fibrinogen or salmon fibrinogen;

[0014] 2) Add thrombin to the 96-well plate from step 1) and incubate at 37°C for 60 minutes;

[0015] 3) Add culture medium to the wells and incubate at 37°C for 6 to 12 hours; and

[0016] 4) Select the fibrin gel containing a single tumor cell, remove the culture medium, and obtain the fibrin gel containing a single tumor cell.

[0017] In a third aspect, the present invention provides fibrin glue containing single tumor cells obtained according to the method of the present invention.

[0018] In a fourth aspect, the present invention provides the use of fibrin glue containing single tumor cells obtained according to the method of the present invention in the construction of mouse tumor models.

[0019] In a fifth aspect, the present invention provides the use of mouse tumor models constructed according to the method of the present invention in screening antitumor drugs. Attached Figure Description

[0020] Figure 1 The image shows the tumor formation of single B16 tumor cells after transplantation into the subcutaneous tissue of wild-type C57 mice in 3D fibrin glue and collagen hydrogel. In the 3D fibrin glue group, 9 out of 10 mice developed tumors on day 30, while in the collagen hydrogel group, 0 out of 10 mice developed tumors on day 30.

[0021] Figure 2 The image shows the tumor formation of single MP-1 tumor cells transplanted into the subcutaneous tissue of NSG mice in 3D fibrin glue and collagen hydrogel. In the 3D fibrin glue group, 7 out of 10 mice developed tumors on day 30, while in the collagen hydrogel group, 0 out of 10 mice developed tumors on day 30.

[0022] Figure 3 The image shows the tumor formation of single H22 tumor cells transplanted into the subcutaneous tissue of BABL / c mice in 3D fibrin glue and collagen hydrogel. In the 3D fibrin glue group, 9 out of 16 mice developed tumors on day 30, while in the collagen hydrogel group, 0 out of 16 mice developed tumors on day 30. Detailed Implementation

[0023] The present invention is described below through specific embodiments. It should be understood that this is merely for the purpose of better understanding the invention and not for the purpose of limitation. Those skilled in the art will understand that any modifications, substitutions, or replacements to the steps of the present invention will fall within the scope of the present invention, as long as they can achieve the functions described in the present invention.

[0024] It is well known in the art that fibrin glue is a degradation product prepared by the action of thrombin on fibrinogen. In short, fibrinogen consists of three pairs of different polypeptide chains: α, β, and γ. Under the action of thrombin, fibrinogen forms fibrin monomers, which covalently bind to each other to form fibrin polymers. The α chains interlock and covalently crosslink to form a stable, transparent, amorphous, fibrous, elastic gel-like degradation product—that is, fibrin glue. As used herein, the term "fibrin glue" refers to fibrin glue formed by the breakdown of fibrinogen by thrombin, which also contains individual tumor cells; preferably, the tumor cells are primary cultured and / or secondary cultured tumor cells; preferably, the tumor cells are selected from melanoma, liver cancer, breast cancer, colorectal cancer, myeloma, small cell lung cancer, non-small cell lung cancer, head and neck cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma. Tumors, including large B-cell lymphomas rich in T-cells / histocytes, multiple myeloma, myeloid leukemia-1 protein, myelodysplastic syndrome, gastric cancer, ovarian cancer, lymphoblastic leukemia, lymphocytic leukemia, endometrial cancer, prostate cancer, thyroid cancer, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, bone cancer, Ewing's sarcoma, cervical cancer, brain cancer, bladder cancer, clear cell renal cell carcinoma, pharyngeal cancer, hepatobiliary cancer, central nervous system cancer, esophageal cancer, malignant pleural mesothelioma, systemic light chain amyloidosis, lymphoplasmacytic lymphoma, myelodysplastic syndrome, myeloproliferative tumors, neuroendocrine tumors, Merkel cell carcinoma, testicular cancer, and skin cancer.

[0025] As used herein, the term "collagen hydrogel" refers to a hydrogel containing collagen and individual tumor cells.

[0026] The present invention provides a method for constructing a mouse tumor model using fibrin glue containing individual tumor cells, the method comprising the step of transplanting fibrin glue containing individual tumor cells into mice; preferably, the method comprises the step of transplanting fibrin glue containing individual tumor cells subcutaneously into mice.

[0027] Specifically, the present invention provides a method for constructing a mouse tumor model using fibrin glue containing single tumor cells, the method comprising the following steps:

[0028] 1) Mix the culture medium containing tumor cells with fibrinogen and spread it in a 96-well plate;

[0029] 2) Add thrombin to the 96-well plate from step 1) and incubate at 37°C for 60 minutes;

[0030] 3) Add culture medium to the wells and incubate at 37°C for 6 to 12 hours;

[0031] 4) Select fibrin glue containing individual tumor cells, remove the culture medium, and

[0032] 5) The fibrin glue containing single tumor cells obtained in step 5) is transplanted into mice, preferably into the subcutaneous tissue of mice.

[0033] The method for constructing mouse tumor models using fibrin glue containing single tumor cells provided by this invention is applicable to a variety of tumors, including but not limited to melanoma, liver cancer, breast cancer, colorectal cancer, myeloma, small cell lung cancer, non-small cell lung cancer, head and neck cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, and large B-cell lymphoma rich in T-cells / histocytes. Lymphoma, multiple myeloma, myeloid leukemia-1 protein, myelodysplastic syndrome, gastric cancer, ovarian cancer, lymphoblastic leukemia, lymphocytic leukemia, endometrial cancer, prostate cancer, thyroid cancer, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, bone cancer, Ewing's sarcoma, cervical cancer, brain cancer, bladder cancer, clear cell renal cell carcinoma, pharyngeal cancer, hepatobiliary cancer, central nervous system cancer, esophageal cancer, malignant pleural mesothelioma, systemic light chain amyloidosis, lymphoplasmacytic lymphoma, myelodysplastic syndrome, myeloproliferative neoplasms, neuroendocrine tumors, Merkel cell carcinoma, testicular cancer, and skin cancer.

[0034] The mouse tumor model constructed according to the method of the present invention can stably form subcutaneous tumors within 15 to 60 days, and provides an important tool for exploring the mechanisms of tumor formation, development and progression, as well as for screening anti-tumor drugs.

[0035] In this invention, the inventors used collagen hydrogels commonly used in the art as a control to further demonstrate the significant effects of our fibrin gel system.

[0036] In a preferred embodiment of the method for constructing a mouse tumor model according to the present invention, fibrin glue containing single tumor cells is formed by mixing fibrinogen, single tumor cells, and thrombin. Preferably, the fibrinogen is animal-derived, and more preferably, fibrinogen derived from salmon or humans. In a preferred embodiment of the method for constructing a mouse tumor model according to the present invention, 50 μl of fibrin glue formed from a mixture containing fibrinogen, single tumor cells, and thrombin is transplanted subcutaneously into mice.

[0037] In a preferred embodiment of the method for constructing a mouse tumor model according to the present invention, the final concentration of fibrinogen in the mixture containing fibrinogen, single tumor cells and thrombin is 0.1-10 μg / ml, preferably 4-8 μg / ml.

[0038] The mouse tumor model constructed according to the method of the present invention can be used for the study of tumor development and progression mechanisms, the efficacy evaluation of antitumor drugs, the pharmacological safety evaluation of antitumor drugs, and clinical tumor assessment.

[0039] The following examples are used to explain the purpose of implementing the present invention and should not be construed as limiting the present invention.

[0040] Example

[0041] Example 1. Tumor cell culture

[0042] B16 cells (a melanoma cell line), MP-1 cells (a patient tissue-derived melanoma cell line), and H22 cells (a liver tumor cell line) were obtained from the Cell Resource Center of Peking Union Medical College and cultured at 37°C in suitable culture medium in 10cm cell culture dishes.

[0043] Cells were treated with trypsin for 2 minutes at room temperature, digested in culture medium, and then centrifuged at 600 rpm for 5 minutes. After centrifugation, the cells were resuspended in 1640 medium, and the cells were counted using a cell counter. The cell suspension was further diluted to a concentration of 5 cells / μl of culture medium based on the cell concentration.

[0044] Example 2. Preparation of fibrin glue containing a single tumor cell

[0045] The 3D fibrin glue reaction system (taking a suspension volume of 250 μl as an example) contains:

[0046] Fibrinogen 50μl

[0047] 200 μl of culture medium containing tumor cells (containing 5 cells)

[0048] Thrombin (catalyst) 1 μl (spotted in a 96-well plate).

[0049] Specifically, 50 μl of human fibrinogen (Reagent Proteins, 40 μg / ml) or salmon fibrinogen (Reagent Proteins, 20 μg / ml) and 200 μl of culture medium containing tumor cells were thoroughly mixed and set aside. 1 μl of thrombin (Reagent Proteins, SEA-135, 100 U / ml) was added to the center of a sterile 96-well plate. Then, 50 μl of the aforementioned fibrinogen and tumor cell-containing culture medium was added to the sterile 96-well plate and thoroughly mixed. The 96-well plate was incubated at 37°C for 60 minutes. After that, 200 μl of 1640 medium was added to each well, and the plate was incubated for another 6 hours. Wells containing a single tumor cell were then observed under a microscope and labeled for later use.

[0050] 3D fibrin glue containing three types of cells—B16 cells, MP-1 cells, and H22 cells—was prepared according to this method.

[0051] Example 3. Preparation of collagen hydrogel containing a single tumor cell

[0052] The 3D collagen hydrogel reaction system (taking a suspension volume of 1000 μl as an example) includes:

[0053]

[0054] Specifically, phosphate buffer, collagen, and sodium hydroxide were added sequentially to the culture medium containing tumor cells. After thorough mixing, 50 μl of the mixture was evenly spread into each well of a 96-well plate. The 96-well plate was incubated at 37°C for 60 minutes. Then, 200 μl of 1640 medium was added to each well, and the plate was incubated for another 6 hours. Wells containing single tumor cells were then observed under a microscope and labeled for later use.

[0055] A 3D collagen hydrogel containing three cell types—B16 cells, MP-1 cells, and H22 cells—was prepared according to this method.

[0056] Example 4. Subcutaneous transplantation of tumor cells into mice

[0057] 1) Disinfection of surgical instruments

[0058] Surgical instruments required for high-temperature and high-pressure sterilization surgery include hemostats, needle holders, scissors, forceps, and gauze.

[0059] 2) Anesthesia

[0060] Wild-type C57 mice, BABL / c mice, and NSG mice were anesthetized with sodium pentobarbital at a concentration of 1%, administered intraperitoneally at a dose of 125 μL per mouse. After the mice were anesthetized, their fur was cleaned subcutaneously to prevent infection of the surgical wound.

[0061] 3) Skin preparation

[0062] Carefully make a 1 cm incision on the mouse's skin using hemostatic forceps and scissors, taking care to maintain aseptic technique and monitor the mouse's condition during the procedure.

[0063] 4) Processing 3D adhesive

[0064] First, the culture medium from the 3D gel in the 96-well plate was aspirated, and the wells were washed with physiological saline to remove any remaining culture medium and impurities. Then, the 3D fibrin gel and collagen hydrogel containing single B16 tumor cells, single MP-1 tumor cells, and single H22 tumor cells from the 96 wells were carefully transplanted into the skin incisions of wild-type C57 mice, NSG mice, and BABL / c mice, ensuring complete implantation. The mice were then quickly sutured using absorbable sterile sutures and a No. 5 needle. After suturing, the mice were returned to clean, sterile cages.

[0065] Observe the growth of tumor cells under the skin of mice in the 3D fibrin glue group and collagen hydrogel group (see) Figures 1 to 3 ).

[0066] The above results fully demonstrate that the method for constructing a mouse subcutaneous ectopic tumor model using a single tumor cell provided by this invention is applicable to a variety of tumor cells and can stably form subcutaneous tumors. It provides an important tool for scientific research on the formation, occurrence, and development mechanisms of tumors and has significant application prospects.

Claims

1. A method for constructing a mouse tumor model, the method comprising the step of transplanting fibrin glue containing individual tumor cells subcutaneously into a mouse, wherein the fibrin glue containing individual tumor cells is obtained through the following steps: 1) Mix the culture medium containing tumor cells with fibrinogen and spread it in a 96-well plate; wherein the fibrinogen is human fibrinogen or salmon fibrinogen; and the concentration of the fibrinogen is 4 to 8 μg / ml; 2) Add thrombin to the 96-well plate from step 1) and incubate at 37°C for 60 minutes; 3) Add culture medium to the 96-well plate and incubate at 37°C for 6 to 12 hours; and 4) Select the fibrin gel containing a single tumor cell and remove the culture medium.

2. The method for constructing a mouse tumor model according to claim 1, wherein the single tumor cell is selected from melanoma, liver cancer, breast cancer, colorectal cancer, myeloma, small cell lung cancer, non-small cell lung cancer, head and neck cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, large B-cell lymphoma rich in T-cells / histocytes, and multiple myeloma. Tumors, myeloid leukemia-1 protein, myelodysplastic syndrome, gastric cancer, ovarian cancer, lymphoblastic leukemia, lymphocytic leukemia, endometrial cancer, prostate cancer, thyroid cancer, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, bone cancer, Ewing's sarcoma, cervical cancer, brain cancer, bladder cancer, clear cell renal cell carcinoma, pharyngeal cancer, hepatobiliary cancer, central nervous system cancer, esophageal cancer, malignant pleural mesothelioma, systemic light chain amyloidosis, lymphoplasmacytic lymphoma, myelodysplastic syndrome, myeloproliferative tumors, neuroendocrine tumors, Merkel cell carcinoma, testicular cancer, and skin cancer.

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