Preparation method and application of lung cancer anti-PD-1 antibody drug-resistant cell strain

Abstract

The invention discloses a preparation method and application of a lung cancer anti-PD-1 antibody drug-resistant cell strain, and relates to the technical field of biological medicine, and the preparation method comprises the following operation steps: constructing a mouse subcutaneous tumor model by using a mouse-derived lung cancer cell line LLC; performing mPD-1 antibody treatment on the mouse subcutaneous tumor model, and detecting the tumor size to obtain a mouse which cannot be slowly released; extracting lung cancer cells, which are not slowly released, in tumor tissues of the mouse, and constructing a new subcutaneous tumor model of the mouse; then receiving mPD-1 antibody treatment; and repeating the steps until the tumor size of the mPD-1 antibody treatment group is compared with the tumor size of the control group and has no significant difference or even exceeds the tumor size of the control group, so that the lung cancer mPD-1 antibody drug-resistant mouse model is successfully constructed. The method comprises the following steps: mechanically separating tumor tissues on a lung cancer mPD-1 antibody drug-resistant mouse model, extracting primary cells of the drug-resistant mouse tumor tissues, and subculturing the primary cells to obtain the lung cancer anti-PD-1 antibody drug-resistant cell strain.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically to a method for preparing a lung cancer anti-PD-1 antibody resistant cell line and its application. Background Art

[0002] Lung cancer is one of the most common malignant tumors in the world, and its mortality rate ranks first among all tumors. According to the World Health Organization, the incidence and mortality of lung cancer have increased significantly in both men and women, especially in smokers. The main treatments for lung cancer include surgical resection, radiotherapy, chemotherapy, and targeted therapy. However, with the in-depth study of the biological characteristics of lung cancer, immunotherapy has gradually become an emerging and effective treatment method.

[0003] In recent years, immune checkpoint inhibitors have made significant progress in the treatment of lung cancer. Among them, programmed cell death-1 (PD-1) antibody, as an important immunotherapy drug, has been widely used in the clinical treatment of non-small cell lung cancer (NSCLC). PD-1 interacts with its ligand PD-1 and can inhibit the activity of T cells, thereby causing tumor cells to escape immune surveillance. By blocking the PD-1 / PD-1 signaling pathway, PD-1 antibodies such as Nivolumab and Pembrolizumab can effectively restore the function of T cells and enhance the body's immune response to tumor cells.

[0004] Although PD-1 antibodies have shown good therapeutic effects in many patients with advanced lung cancer, they also face the problem of drug resistance in clinical applications. This drug resistance phenomenon means that some patients still cannot obtain significant survival benefits even after receiving PD-1 antibody treatment, and the mechanism of drug resistance is not yet fully understood. Therefore, the establishment of PD-1 antibody-resistant mouse models and resistant cell lines will help to further explore the molecular mechanism of lung cancer immune escape and provide new ideas for solving the problem of drug resistance.

[0005] Currently, research on PD-1 antibody resistance is mainly focused on discovering resistance-related gene mutations, abnormal activation of signaling pathways, and changes in the tumor microenvironment. However, the lack of systematic animal models and cell lines limits our understanding of the resistance mechanism. Therefore, the development of PD-1 antibody-resistant mice and cell lines will not only help to elucidate the resistance mechanism in lung cancer, but also provide an experimental basis for the development of new treatment strategies. Through these models, we can evaluate the effectiveness of different combination treatment regimens and provide possible solutions to overcome PD-1 antibody resistance. Therefore, the preparation of PD-1 antibody-resistant mice and cell lines has important scientific significance and clinical value. This will provide a powerful tool for studying immunotherapy for lung cancer, thereby promoting the progress of personalized treatment and improving patients' survival rate and quality of life. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing a lung cancer anti-PD-1 antibody resistant cell line and its application, so as to solve the problems raised in the above background technology.

[0007] A method for preparing a lung cancer anti-PD-1 antibody resistant cell line, comprising the following steps:

[0008] S1.1: (1) Use the mouse lung cancer cell line LLC to construct a mouse subcutaneous tumor model; treat the mouse subcutaneous tumor model with mPD-1 antibody, detect the tumor size after mPD-1 antibody treatment, and obtain mice that failed to release; (2) Extract lung cancer cells from the tumor tissue of the mice that failed to release, and construct a new mouse subcutaneous tumor model; then treat the mouse subcutaneous tumor model with mPD-1 antibody and detect the tumor size;

[0009] S1.2: Repeat step S1.1 until the tumor size of the mPD-1 antibody-treated group is not significantly different from or even exceeds that of the control group, and the mPD-1 antibody-resistant mouse model of lung cancer is successfully established;

[0010] S2.1: Mechanically separate the tumor group in the lung cancer mPD-1 antibody-resistant mouse model, extract the primary cells of the resistant mouse tumor tissue, and subculture them to obtain the lung cancer anti-PD-1 antibody-resistant cell line.

[0011] More optimally, the anti-PD-1 antibody-resistant cell line for lung cancer has been deposited in the China Center for Type Culture Collection on December 17, 2024, with the deposit number being CCTCC NO: C2024423.

[0012] In the plan, the lung cancer anti-PD-1 antibody-resistant cell line was named Mouse Lung Cancer Immune-Resistant Primary Cell Res-LLCMus musculus; the depositor was the General Hospital of the Eastern Theater Command of the People's Liberation Army of China; the preservation unit was the China Center for Type Culture Collection; and the viability of the culture was tested by the China Center for Type Culture Collection on December 23, 2024, and the result was survival.

[0013] The more optimized specific extraction method of the lung cancer anti-PD-1 antibody resistant cell line is:

[0014] The tumor tissue in the lung cancer mPD-1 antibody-resistant mouse model was mechanically separated and washed with PBS to obtain the tumor tissue; the tumor tissue was cut into pieces, resuspended and centrifuged with sterile PBS, the precipitate was resuspended in pre-cooled digestion solution, shaken in a constant temperature shaker for 70 to 90 minutes, filtered and centrifuged, and added to DMEM culture medium for culture to obtain a lung cancer anti-PD-1 antibody-resistant cell line.

[0015] In the scheme, the resistant cell line was cultured in vitro and verified to be able to continue to grow rapidly; after treatment with mPD-1 antibodies, the subcutaneous tumor still showed a relatively fast growth rate, showing obvious drug resistance. Currently, the resistant cell line has been properly frozen.

[0016] More optimally, the PBS washing contains 10 wt % penicillin-streptomycin.

[0017] More optimally, the digestion solution contains 0.1% IV collagenase.

[0018] Preferably, the rotation speed of the constant temperature shaker is 220 rpm and the temperature of the constant temperature shaker is 37°C.

[0019] Preferably, the mice are 5-6 week old C57BL / 6 mice; the injection volume of the mouse lung cancer cell line LLC is 5×10 6 / 100 μL; the antibody used in the control group was IgG; the IgG and mPD-1 antibodies were administered at 200 μg / time every 3 days.

[0020] A method for preparing a lung cancer anti-PD-1 antibody resistant cell line is disclosed for preparing a lung cancer anti-PD-1 antibody resistant cell line.

[0021] Application of a lung cancer anti-PD-1 antibody resistant cell line in screening or preparing anti-tumor drugs

[0022] A lung cancer anti-PD-1 antibody resistant cell line is used in screening and evaluating or preparing a detection reagent for evaluating anti-tumor effects.

[0023] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0024] This technical solution provides an important experimental tool for in-depth understanding of the resistance mechanism of lung cancer to immune checkpoint inhibitors by constructing PD-1 antibody-resistant mice and cell lines; at the same time, these models help to screen and evaluate new combination treatment strategies, promote the progress of lung cancer immunotherapy, and improve patients' cure rate and quality of life. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Flow chart for constructing mouse subcutaneous tumor model;

[0026] Figure 2 This is a diagram showing the tumor response of mice subcutaneous tumors after drug administration;

[0027] Figure 3 This is the mean proliferation graph of tumor volume size for the isotype control IgG group, mPD-1 antibody treatment-resistant group, and parental mPD-1 antibody treatment-sensitive group;

[0028] Figure 4 This is a comparison chart of tumor weights of the isotype control IgG group, the mPD-1 antibody sensitive group, and the insensitive group; the horizontal axis is time (unit: day), and the vertical axis is tumor weight (unit: gram);

[0029] Figure 5 A schematic diagram for evaluating the drug resistance effect of mPD-1 antibody-resistant lung cancer cell model Res-LLC subcutaneous tumor;

[0030] Figure 6 This is a picture of the subcutaneous tumor of mPD-1 antibody-resistant lung cancer cell Res-LLC. The experiment was divided into three groups. The isotype control was the parent LLC cells treated with mPD-1 antibody, the sensitive group Sen-mPD-1, the resistant control group Res-IgG, and the resistant mPD-1 antibody-treated group Res-mPD-1;

[0031] Figure 7 This is the mean proliferation trend diagram of the subcutaneous tumor volume of mPD-1 antibody-resistant tumor cells Res-LLC;

[0032] Figure 8 Figure 2 shows the tumor weights in different groups. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0034] In the following specific embodiments, parts are by mass. In this embodiment, it should be noted that there is no special restriction on the purchase manufacturers of all raw materials involved in the present invention, and exemplarily include: the drug-resistant cell line described in the present invention is named LLC mouse lung cancer cell Res-LLC (PD-1 antibody resistant LLC, Res-LLC); the mouse lung cancer cell line LLC cells used in the embodiment were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, and the C57BL / 6 mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. The mice were 5 to 6 weeks old and weighed between 18 and 20 g; the mPD-1 antibody used in the embodiment has a product number of BE0146, and the control IgG antibody has a product number of BE0260, both of which are commercially available products of BioXcell; the collagenase type IV has a product number of BS165, which was purchased from Biosharp Biotechnology Co., Ltd.

[0035] Example 1: LLC cell C57BL / 6 mouse subcutaneous tumor implantation model and drug resistance detection;

[0036] S1.1: (1) Take out the LLC cell cryopreservation tube frozen in liquid nitrogen, quickly place it in a 37°C constant temperature water bath and shake until it is completely thawed, and transfer it to an ultra-clean workbench for operation; use a 1mL pipette to transfer it to a 15mL centrifuge tube, add 7mL of complete culture medium, centrifuge at 1000rpm for 3 minutes, aspirate the supernatant, add 1mL of complete culture medium to resuspend, transfer it to a 10cm culture dish, add 7mL of complete culture medium, shake it using the "cross method" to evenly distribute the cells, and place it at 37°C and 5% CO 2 Culture in a cell culture incubator;

[0037] (2) After digestion and centrifugation at least twice according to the cell passaging method, LLC cells in the logarithmic growth phase were digested and centrifuged, resuspended in sterile PBS and counted; the cell concentration was adjusted to 5×10 6 / 100 μL to obtain a cell suspension; inject 100 μL of the cell suspension into the subcutaneous part of the mouse abdomen through a 1 mL sterile syringe to construct a mouse subcutaneous tumor model;

[0038] S1.2: Starting from the 7th day, 20 C57BL / 6 mice were randomly divided into two groups, 10 mice in each group. The control group was given the immune checkpoint mPD-1 antibody isotype control IgG group, and the experimental group was given the mPD-1 antibody, injected once every 3 days, 200 μg each time; the tumor size was detected every 3 days, and the long diameter a and short diameter b of the tumor were recorded using a vernier caliper. According to the formula V = 1 / 2ab 2 Calculate the tumor volume; monitor the drug resistance of mPD-1 antibody by observing the tumor growth of mice in different groups, and obtain mice that fail to release the drug; Figure 1 shown.

[0039] Example 2: Verification of drug resistance effect in lung cancer mPD-1 antibody resistant mouse model;

[0040] S1.1: (1) The mice that failed to release the tumor were anesthetized and the abdomen was depilated with a depilatory cream. The tumor tissues of the mice that failed to release the tumor were then mechanically separated and cut into 2 mm 3 (2) Use sterile sharp scissors to cut a 3 mm long shallow incision in the right thigh of the mouse to be infected with the tumor, and use straight toothless forceps to insert a 1 cm long subcutaneous tunnel, and insert a 2 mm 3 The tumor tissue was pushed to the root of the tunnel, and the mice were then observed until they recovered; (3) subcutaneous tumor-bearing mice whose parents were sensitive to mPD-1 were treated with mPD-1 as the control group;

[0041] S1.2: From day 7 onwards, mPD-1 antibody and IgG (control IgG group) were injected intraperitoneally with a sterile syringe, 200 μg each time, once every 3 days; the long diameter a and short diameter b of the tumor were recorded with a vernier caliper every 3 days, and the diameter was calculated according to the formula V = 1 / 2ab 2 The tumor volume was calculated to evaluate the drug resistance effect of the antibody. When the tumor size was the same as that of the control IgG group or even exceeded that of the control IgG group, the mPD-1 antibody-resistant mouse model of lung cancer was successfully established. Figures 2 to 4 As shown;

[0042] Compared with the isotype control IgG group (Res-IgG), the tumor volume of the resistant group (Res-mPD-1) was not significantly different, but it was statistically significant (p < 0.05) compared with the tumor volume of the parental PD-1 antibody-treated sensitive group (Sen-mPD-1). Figures 2-3 ); the tumor weight in the sensitive group was significantly lower than that in the control group, and significantly higher than that in the sensitive group (e.g. Figure 4 ); Statistical significance: ns means no statistical significance, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0043] Example 3: Construction and identification of lung cancer PD-1 antibody resistant mouse cell line;

[0044] S1.1: (1) Prepare 0.1% type IV collagenase tissue digestion solution. In a clean bench, add 50 mL of serum-free DMEM medium and 50 mg of type IV collagenase to a 50 mL centrifuge tube. Mechanically separate the tumor tissue from the lung cancer mPD-1 antibody-resistant mouse model. Place the tumor tissue in a 50 mL centrifuge tube containing pre-cooled PBS, wash it three times with 10× double-antibody (penicillin-streptomycin) PBS, and cut it into pieces of 2 to 4 mm. 3size, transferred to a 1.5 mL sterile EP tube, cut the tumor tissue into a paste, then resuspended and centrifuged with sterile PBS, resuspended the pellet in pre-cooled digestion solution containing 0.1 IV collagenase, and shaken in a constant temperature shaker at 220 rpm for 70 minutes;

[0045] (2) Centrifuge at 400G for 5 minutes, discard the supernatant, add 5mL DMEM medium to resuspend, filter with a 70μm filter to remove undigested tissue, randomly centrifuge at 400G for 5 minutes and discard the supernatant; add 2mL of red blood cell lysis buffer to lyse for 2 minutes, add 4mL DMEM medium to terminate lysis, centrifuge at 400G for 5 minutes to remove the supernatant, add DMEM medium to resuspend, filter with a 30μm filter to obtain a lung cancer anti-PD-1 antibody resistant cell line, and place at 37°C and 5% CO 2 Culture in a cell culture incubator;

[0046] S1.2: (1) Digest and centrifuge the anti-PD-1 antibody-resistant lung cancer cell line that has reached the logarithmic growth phase, resuspend it in sterile PBS, and count the cells and adjust the cell concentration to 5×10 6 / 100μL, use a 1mL sterile syringe to inject 100μL into the subcutaneous part of the mouse's rib cage; at the same time, subcutaneous tumor-bearing mice in the parental mPD-1 sensitive group were set up to receive mPD-1 treatment as a control; (2) Starting from the 7th day of tumor-bearing mice, use a sterile syringe to intraperitoneally inject mPD-1 antibody and control IgG group, 200μg each time, once every 3 days; record the long diameter a and short diameter b of the tumor every 3 days, according to the formula V = 1 / 2ab 2 Calculate the tumor volume and observe the difference between the PD-1 antibody resistance group and the control group; when the tumor volume of the mPD-1 antibody resistance group and the control IgG group is the same or even exceeds that of the control group, it indicates that the lung cancer anti-PD-1 antibody resistant cell line is resistant to the mPD-1 antibody;

[0047] from Figures 6-7 It can be seen that the tumor volume of the drug-resistant group (Res-LLC-mPD-1) was not significantly different from that of the control IgG group (Res-LLC-IgG), but was statistically significant compared with the tumor volume of the parental PD-1 sensitive group (Sen-LLC-mPD-1) (p<0.05); Figure 8 It can be seen that there is no significant difference in tumor volume between the resistant group (Res-LLC-mPD-1) and the control IgG group (Res-LLC-IgG), but there is a statistically significant difference in tumor volume between the resistant group (Res-LLC-mPD-1) and the sensitive group (Sen-LLC-mPD-1) (p<0.05).

[0048] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. A method for preparing a lung cancer anti-PD-1 antibody resistant cell line, characterized in that: The steps include: S1.1: (1) Use the mouse lung cancer cell line LLC to construct a mouse subcutaneous tumor model; treat the mouse subcutaneous tumor model with mPD-1 antibody, detect the tumor size after mPD-1 antibody treatment, and obtain mice that failed to release; (2) Extract lung cancer cells from the tumor tissue of the mice that failed to release, and construct a new mouse subcutaneous tumor model; then treat the mouse subcutaneous tumor model with mPD-1 antibody and detect the tumor size; S1.2: Repeat step S1.1 until the tumor size of the mPD-1 antibody-treated group is not significantly different from or even exceeds that of the control group, and the mPD-1 antibody-resistant mouse model of lung cancer is successfully established; S2.1: Mechanically separate the tumor tissue from the lung cancer mPD-1 antibody-resistant mouse model, extract the primary cells from the resistant mouse tumor tissue, and subculture them to obtain the lung cancer anti-PD-1 antibody-resistant cell line.

2. The method for preparing a lung cancer anti-PD-1 antibody resistant cell line according to claim 1, characterized in that: The lung cancer anti-PD-1 antibody-resistant cell line was deposited in the China Center for Type Culture Collection on December 17, 2024, with the deposit number CCTCC NO: C2024423.

3. The method for preparing a lung cancer anti-PD-1 antibody resistant cell line according to claim 1, characterized in that: The specific extraction method of the lung cancer anti-PD-1 antibody resistant cell line is: The tumor tissue in the lung cancer mPD-1 antibody-resistant mouse model was mechanically separated and washed with PBS to obtain the tumor tissue; the tumor tissue was cut into pieces, resuspended and centrifuged with sterile PBS, the precipitate was resuspended in pre-cooled digestion solution, shaken in a constant temperature shaker for 70 to 90 minutes, filtered and centrifuged, and added to DMEM culture medium for culture to obtain a lung cancer anti-PD-1 antibody-resistant cell line.

4. The method for preparing a lung cancer anti-PD-1 antibody resistant cell line according to claim 3, characterized in that: In the PBS washing, the PBS contains 10 wt % of penicillin and streptomycin.

5. The method for preparing a lung cancer anti-PD-1 antibody resistant cell line according to claim 3, characterized in that: The digestion solution contains 0.1% IV collagenase.

6. The method for preparing a lung cancer anti-PD-1 antibody resistant cell line according to claim 3, characterized in that: The rotation speed of the constant temperature shaker is 220 rpm, and the temperature of the constant temperature shaker is 37°C.

7. The method for preparing a lung cancer anti-PD-1 antibody resistant cell line according to claim 1, characterized in that: The mice are 5-6 week old C57BL / 6 mice; the injection volume of the mouse lung cancer cell line LLC is 5×10 6 / 100 μL; the antibody used in the control group was IgG; the IgG and mPD-1 antibodies were administered at 200 μg / time every 3 days.

8. A lung cancer anti-PD-1 antibody resistant cell line prepared according to the method for preparing a lung cancer anti-PD-1 antibody resistant cell line according to any one of claims 1 to 7.

9. Use of the anti-PD-1 antibody-resistant cell line for lung cancer according to claim 8 in screening or preparing anti-tumor drugs.

10. Use of the anti-PD-1 antibody resistant cell line for lung cancer according to claim 8 in screening and evaluating or preparing a detection reagent for evaluating anti-tumor effects.

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