CN113875690B - Lewis lung cancer mouse model based on tumor microenvironment and construction method thereof - Google Patents

Abstract

The invention discloses a Lewis lung cancer mouse model based on a tumor microenvironment and a construction method thereof, belonging to the technical field of tumor models. The Lewis lung cancer mouse model contains Lewis lung cancer cells, peripheral blood mononuclear cells and fibroblasts. The ratio of Lewis lung cancer cells to peripheral blood mononuclear cells is (1-15): 1. The Lewis lung cancer mouse model constructed by the present invention can construct a tumor immune microenvironment closer to a tumor patient in the mouse body, thereby providing a better immune system for tumors. Mechanistic studies of treatment provide more suitable and more accurate and effective models.

Description

A mouse model of Lewis lung cancer based on tumor microenvironment and its construction method

technical field

The invention relates to the technical field of tumor models, in particular to a Lewis lung cancer mouse model based on tumor microenvironment and a construction method thereof.

Background technique

At present, lung cancer ranks first in the morbidity and mortality of malignant tumors in China and the world, and it is the first type of malignant tumor that threatens human health. Current treatments for lung cancer include surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. In recent years, the study of tumor microenvironment mechanism (tumor immune microenvironment) has become a research hotspot.

Animal models have become important preclinical experimental models for studying tumor immune mechanisms and clinical therapeutic efficacy. Common animal models include: heterotopic transplantation animal models, orthotopic transplantation animal models, induced animal models, and transgenic animal models.

The current common animal models are shown in Table 1.

Table 1 Common tumor mouse models

As far as we know, the tumor microenvironment (TME) is composed of tumor cells, extracellular matrix (ECM), tumor-associated fibroblasts (CAFs), endothelial cells, immune cells and other cellular components, as well as blood vessels. It plays an important role in invasion, metastasis, angiogenesis, metabolism, immunosuppression and drug resistance.

Tumor-infiltrating lymphocytes (TILs) are a heterogeneous lymphocyte population within the TME, mainly including T cells, B cells, dendritic cells (DCs), natural killer cells (NK), and macrophages. Among them, there are three main subtypes of T lymphocytes: CD8+, CD4+, and regulatory T cells (Treg cells). The human immune system consists of dendritic cells (DC), natural killer cells (NK), CD8+ T cells, regulatory T cells (Treg), tumor-associated macrophages (TAM), and myeloid-derived suppressor cells (MDSCs). ) etc.

At present, the more commonly used animal model: the heterotopic transplantation animal model, its obvious shortcomings are: the infiltrated lymphocytes in the tumor tissue are few, and the blood vessels in the tumor tissue are not rich, as shown in Figure 1.

As can be seen from the comparison in Figure 1, the current mouse models of lung cancer cannot truly simulate the tumor microenvironment of lung cancer patients, and the research results of tumor immune mechanisms are also unconvincing.

Therefore, it is urgent to construct a mouse model that is closest to the tumor immune microenvironment of tumor patients.

SUMMARY OF THE INVENTION

One of the objectives of the present invention is to provide a Lewis lung cancer mouse model based on the tumor microenvironment to solve the above problems.

In order to achieve the above object, the technical solution adopted in the present invention is as follows: a mouse model of Lewis lung cancer based on tumor microenvironment, wherein the mouse model of Lewis lung cancer contains Lewis lung cancer cells, peripheral blood mononuclear cells and fibroblasts The ratio of Lewis lung cancer cells to peripheral blood mononuclear cells was (1-15):1.

As a preferred technical solution: the ratio of Lewis lung cancer cell number to peripheral blood mononuclear cell number is 5:1.

In this application, unless otherwise specified, the above ratio refers to the ratio of LLC to PBMC cells. Currently, only tumor cells are injected into the existing tumor xenograft model, but in order to better simulate the orthotopic tumor microenvironment and improve the tumor formation rate , this application builds the above model, and mixing the three for inoculation is the invention point of this application, and the specific ratio is only one of the verification results of this project. Among them, LLC, PBMC and L929 cells were mixed, and it was found that the ratio of LLC, PBMC and L929 was ⁵ :1, the best effect, and L929 was described in the following description.

The second purpose of the present invention is to provide a method for constructing the above-mentioned Lewis lung cancer mouse model based on the tumor microenvironment. and fibroblasts.

As a preferred technical solution: the mice are C57BL/6 male mice, 6-8 weeks old, weighing 20-22 g.

Peripheral blood mononuclear cells (PBMC), Lewis lung cancer cells (LLC), fibroblasts (L929)

Compared with the prior art, the present invention has the advantages that: the Lewis lung cancer mouse model constructed by the present invention can construct an immune microenvironment closer to a tumor patient in the mouse body, thereby providing a more suitable and accurate mechanism for the study of tumor therapy. valid model.

Description of drawings

Fig. 1 is the comparison chart of tissue scanning of Lewis mouse model and lung cancer patient in the prior art;

Fig. 2 is the model construction technology roadmap of the present invention;

Figure 3 is the change curve of the tumor formation rate of each group of mice at different times;

Figure 4 is the change curve of Lewis lung cancer transplanted tumor volume;

Figure 5 is the volume of transplanted tumor in mice at the end of the experiment (mean ± SD);

Figure 6 is a histogram of changes in the size of Lewis lung cancer xenograft tumor volume (mean ± SD);

Figures 7-9 show the expression results of effector T cells in peripheral blood of tumor-bearing mice in different groups;

Figure 10-11 shows the expression results of effector Treg cells in peripheral blood of tumor-bearing mice in different groups

Figure 12 shows the results of HE staining of tumor-bearing mice in different groups;

Figure 13 is a scanning electron microscope image of immunohistochemical FAPα of different groups of tumor-bearing mice (×200);

Figure 14 is a comparison chart of the percentage of immunohistochemical FAPα in different groups of tumor-bearing mice;

Figures 15-17 are multiple immunofluorescence images of different groups of tumor-bearing mice;

Figure 18 shows the results of immunofluorescence CD8+ in different groups of tumor-bearing mice;

Figure 19 shows the results of immunofluorescence CD39+ in different groups of tumor-bearing mice;

Figure 20 shows the results of immunofluorescence PD-1 in different groups of tumor-bearing mice;

Figures 21-23 are graphs of tumor volume changes in different groups of tumor-bearing mice after radiotherapy;

Figure 24 shows the percentage of CD4 cells/CD3 cells in different groups of tumor-bearing mice;

Figure 25 shows the percentage of CD8 cells/CD3 cells in different groups of tumor-bearing mice;

Figure 26 shows the expressions of CD8, CD39 and PD1 in different tumor tissues.

Detailed ways

The present invention will be further described below with reference to the accompanying drawings.

Example 1

A method for constructing a Lewis lung cancer mouse model based on tumor microenvironment, see Figure 2, wherein the Lewis lung cancer mouse model contains Lewis lung cancer cells ("LLC" for short), peripheral blood mononuclear cells ("PBMC" for short) ") and fibroblasts (the fibroblasts in this example were selected from L929),

Cell lines: Lewis lung cancer cells (LLC), fibroblasts (L929)

Experimental animals: maternally homologous inbred C57BL/6 male mice, 6-8 weeks old, weighing 20-22 g.

According to whether they are homologous or not, there are 6 animals in each group, as follows,

Group A: LLC+PBMC (inactivated lymphocyte)

Group B: LLC+PBMC (inactivated lymphocyte)+L929

Group C: LLC+PBMC (LLC:PBMC=1:1)

Group D: LLC+PBMC+L929 (LLC:PBMC=1:1)

Group E: LLC+PBMC (LLC:PBMC=5:1)

Group F: LLC+PBMC+L929 (LLC:PBMC=5:1)

Group G: LLC+PBMC (LLC:PBMC=10:1)

Group H: LLC+PBMC+L929 (LLC:PBMC=10:1)

In the above groups, the number of LLC cells inoculated in each mouse is: 5 × 10 ⁵ , and the number of L929 cells is: 1 × 10 ⁵ , the ratios described are the ratio of the number of cells between LLC and PBMC, For example, "LLC:PBMC=1:1" in group C means that the number of LLC cells is 5×10 ⁵ and the number of PBMC is 5×10 ⁵ ;

The method of inactivating lymphocyte by PBMC is as follows: using mitomycin with a concentration of 25 μg/ml, inactivating PBMC extracted from peripheral blood at 37°C for 30 min;

On the 0th day, 0.1 ml of different tumor cell suspensions were subcutaneously inoculated into the right hindlimb of the mice, and the state, tumor formation and tumor volume of the mice were observed and recorded after tumor inoculation. Tumor volume V(mm3)=axb2/2. The tumor-bearing mice were treated 28 days after the tumor was implanted, and laboratory detection methods were carried out. Among them, flow cytometry: PBMCs were isolated from the peripheral blood of mouse eyeballs, and effector T cells (CD3+CD8+, CD3+CD4+ expression), Treg cells (CD4+ CD25+, CD4+FOXP3+ expression).

Paraffin block sections of pathological tissues of 20 lung cancer patients and tumor-bearing mice in each group:

HE staining: microscopic observation of pathological tissue structure (tumor cells, lymphocytes, blood vessel distribution)

Immunohistochemistry (SP method): expression of fibroblast activation protein (FAP) in tumor tissue

Multiple immunofluorescence: The expression of CD8, CD39, and PD-1 in tumor tissue was observed under a confocal microscope and images were collected, and the results were analyzed by imageJ software;

All experimental data were plotted and analyzed using Graph Pad Prism 8.0 software. Data are presented as mean ± SD. Statistical differences between the two groups were determined by Student's t test, and P<0.05 was considered statistically significant.

Experimental results:

(1) The change curve of tumor formation rate of mice in each group at different time is shown in Figure 3, and the change curve of Lewis lung cancer transplanted tumor volume (mean ± SD) is shown in Figure 4. It can be seen from the figure that LLC+PBMC (inactivated lymphocyte )+L929 group had the fastest tumor and the largest tumor volume; 1:1 LLC+PBMC group had the slowest tumor and the smallest tumor volume.

(2) At the end of the experiment, the volume of the mouse transplanted tumor (mean ± SD) is shown in Figure 5, and the histogram of the change in the volume of the Lewis lung cancer transplanted tumor (mean ± SD) is shown in Figure 6. Compared with inactivated lymphocyte), the tumor volume in LLC+PBMC (inactivated lymphocyte)+L929 group was larger, with statistical significance (P<0.01); the tumor volume in 1:1 LLC+PBMC group was smaller, with statistical significance (P<0.01). 0.001), in tumor-bearing mice, the mixed cell suspension containing L929 can promote tumor growth after inoculation, and the cell suspension containing lymphocyte can inhibit tumor growth.

Example 2

Radiotherapy Verification:

The method is as follows: 16 male C57BL/6 mice aged 6-8 weeks, 20-22g, are divided into 4 groups according to whether they are homologous or not. The specific groups are as follows.

Group A: LLC+PBMC (inactivated lymphocyte)

Group B: LLC+PBMC (inactivated lymphocyte)+8Gyx3f RT

Group C: LLC+PBMC+L929 (the optimum ratio of LLC and PBMC)

Group D: LLC+PBMC+L929 (the optimum ratio of LLC and PBMC)+8Gyx3f RT

After 2 weeks of tumor seeding, 8Gyx3f radiotherapy was performed every other day, and the volume changes of each group were observed.

On the second day after radiotherapy, flow cytometry was used to analyze the changes in the number of CD3+CD4+T, CD3+CD8+T lymphocytes and CD4+CD25+, CD4+FOXP3+Treg cells in the peripheral blood of tumor-bearing mice in each group.

Experimental results:

(1) The expression results of effector T cells in the peripheral blood of tumor-bearing mice in different groups are shown in Figure 7-9. It can be seen from the figure: CD3+CD4+T, CD3 CD3+CD4+T, CD3 The number of +CD8+T cells was higher than that of LLC+PBMC (inactivated lymphocyte) group, which was statistically significant.

(2) The expression results of effector Treg cells in peripheral blood of tumor-bearing mice in different groups are shown in Figure 10-11. It can be seen from the figure that CD4+CD25+, CD4+Fxop3 in peripheral blood of tumor-bearing mice in 5:1 LLC+PBMC+L929 group The number of +Treg cells was higher than that of LLC+PBMC (inactivated lymphocyte) group, which was statistically significant.

Example 3

HE staining test:

The HE staining results of tumor-bearing mice in different groups are shown in Figure 12. It can be seen from the figure that PBMC in the mixed suspension of the tumor can increase the number of infiltrating lymphocytes in the tumor tissue of the tumor-bearing mice; while fibroblasts in the mixed suspension of the tumor L929 can promote angiogenesis in tumor tissue of tumor-bearing mice; 5:1LLC+PBMC+L929 experimental group has a tumor tissue structure closer to that of lung cancer patients under microscope, showing abundant tumor-infiltrating lymphoid cells more similar to those of lung cancer patients cells and abundant blood vessels.

Example 4

Immunohistochemical FAP test:

Fibroblast activation protein (FAP) is a specific marker of tumor-associated fibroblasts (CAFs). High expression of FAP in tumor tissue can promote immune escape by Treg cells and tumor-associated macrophages (TAM). It is also involved in tumor angiogenesis and plays an important role in tumor development, invasion and metastasis.

The test results are shown in Figure 13 and Figure 14. It can be seen from the figures that the expression of FAP in the tumor tissue of the tumor-bearing mice containing fibroblast L929 in the mixed suspension of the inoculated tumor is higher. The expression of FAP was increased in the groups containing L929 in the liquid; in Fig. 13, regardless of the ratio of LLC to LY (lymphocytes in monocytes), the number of inoculated L929 was ¹ x 105, although The expression is slightly different, but it is significantly different from the normal model, and it is closer to the tumor patient.

Example 5

Multiplex immunofluorescence CD8, CD39, PD-1 assay

The test results are shown in Figure 15, Figure 16, Figure 17, Figure 18, Figure 19 and Figure 20,

It can be seen from the figure that the expressions of CD8, CD39 and PD-1 in the tumor tissues of the experimental group and lung cancer patients of 5:1 LLC+lymphocytes+L929) were statistically significant compared with those of the control group LLC+PBMC (inactivated lymphocyte). It was further seen that the expressions of CD8, CD39, and PD-1 in 5:1 LLC+ lymphocytes+L929) were similar to those of lung cancer patients, so they could better simulate the tumor immune microenvironment of lung cancer patients.

Example 6

Radiotherapy verification test: 8Gy x 3f every other day,

(1) Changes in tumor volume

The results are shown in Figures 21-23. It can be seen from the figure that for 5:1 LLC+PBMC+L929, the tumor volume in the radiotherapy group grew slower than that in the non-radiotherapy group, with statistical significance; while LLC+PBMC (inactivated lymphocyte) , there was no significant difference in tumor volume between the two groups.

It can be seen that the tumor suppressor effect of radiotherapy on 5:1 LLC+PBMC+L929 is significantly better than LLC+PBMC (inactivated lymphocyte).

(2) The percentage of CD4 cells/CD3 cells in different groups of tumor-bearing mice and the percentage of CD8 cells/CD3 cells in different groups of tumor-bearing mice

The results are shown in Figure 24 and Figure 25. It can be seen from the figures that the number of CD3+CD4+ T cells in peripheral blood lymphocytes of the tumor-bearing mice in the LLC+PBMC+L929 (5:1) group decreased after radiotherapy (P<0.001). , the number of CD3+CD8+ T cells increased (P<0.001); the number of CD3+CD4+ T cells in LLC+PBMC (inactivated lymphocyte) group was significantly decreased (P<0.001), and there was no significant difference in the number of CD3+CD8+ T cells .

CD8, CD39, and PD-1 are important indicators of immune activation or immune suppression. As can be seen from Figure 26, the novel model, namely the "LLC+PBMC+L929 (5:1) group" of the present application, CD8, CD39 , PD-1 expression is closer to lung cancer patients.

The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

Claims (3)

1. A method for constructing a Lewis lung cancer mouse model based on a tumor microenvironment is characterized by comprising the following steps: the Lewis lung cancer mouse model contains Lewis lung cancer cells, peripheral blood mononuclear cells and fibroblasts, wherein the ratio of the number of the Lewis lung cancer cells to the number of the peripheral blood mononuclear cells is (1-15) to 1, and the construction method comprises the following steps: inoculating the Lewis lung cancer cells, the peripheral blood mononuclear cells and the fibroblasts into the mouse according to the proportion to obtain the mouse.

2. The method of claim 1, wherein: the ratio of the number of Lewis lung cancer cells to the number of peripheral blood mononuclear cells is 5: 1.

3. The method of claim 1, wherein: the mouse is a C57BL/6 male mouse, is 6-8 weeks old and has the weight of 20-22 g.

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