Lung cancer PDL1 immunotherapy drug-resistant cell strain LLC-R and application thereof

Abstract

The invention discloses a lung cancer PDL1 immunotherapy drug-resistant cell strain LLC-R and application thereof, and relates to the technical field of cell biology. The cell strain LLC-R is preserved in the China Center for Type Culture Collection on November 6, 2024, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: C2024374. The invention provides a reliable experimental model for researching the drug resistance mechanism of lung cancer on PDL1 immunotherapy and related signal channels, so that technicians in the field can more deeply understand the reaction of lung cancer cells on PDL1 immunotherapy, which is helpful for exploring new biomarkers and researching and developing new combined treatment strategies, and has a wide application prospect. The treatment effect is improved; the occurrence of drug resistance is reduced. The invention has important scientific and clinical significance for deeply understanding the lung cancer PDL1 immunotherapy drug resistance and improving the clinical treatment effect of patients.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and in particular to a cell line LLC-R resistant to immunotherapy of lung cancer PDL1 and an application thereof. Background Art

[0002] Primary lung cancer is the most common malignant tumor with the highest morbidity and mortality. Among them, non-small cell lung cancer accounts for about 85%. The overall 5-year survival rate of patients with advanced lung cancer is about 20%.

[0003] At present, the treatment methods of lung cancer mainly include surgical resection, radiotherapy, chemotherapy, targeted therapy and immunotherapy. Programmed cell death 1ligand 1 (PDL1) is a type I transmembrane protein that can inhibit the activation of T cells and maintain immune homeostasis by binding to programmed cell death receptor 1 (PD-1). PDL1 inhibitors, as representatives of immune checkpoint inhibitors (ICIs), have become one of the mainstream methods for treating patients with non-small cell lung cancer. Anti-PD-1 / PDL1 therapy restores the vitality of exhausted T cells by blocking the interaction between PD-1 and PDL1, thereby inhibiting tumor growth. However, clinical data show that the response rate of anti-PD1 / PDL1 treatment is limited, and many patients are prone to resistance to immunotherapy due to innate or acquired factors, suffering from primary resistance and acquired resistance. At present, these potential resistance mechanisms are still unknown, and new research methods and experimental techniques are urgently needed to further clarify the resistance mechanisms of immunotherapy.

[0004] There is no lung cancer cell line resistant to PDL1 immunotherapy in the existing technology, making it difficult to conduct experiments. Therefore, constructing a lung cancer cell line resistant to PDL1 immunotherapy is necessary to find the key molecules that cause resistance to immunotherapy, draw a more detailed map of the resistance mechanism, and further provide a theoretical basis for the treatment strategy after resistance, which is very necessary to guide the precise treatment of lung cancer in the future and expand the response rate. Summary of the invention

[0005] The purpose of the present invention is to provide a cell line LLC-R resistant to immunotherapy of PDL1 in lung cancer and its application to solve the problems existing in the above-mentioned prior art. The present invention provides a reliable experimental model for studying the resistance mechanism of lung cancer to PDL1 immunotherapy and related signaling pathways, so that those skilled in the art can have a deeper understanding of the response of lung cancer cells to PDL1 immunotherapy, which is helpful to explore new biomarkers, study and develop new combined treatment strategies, improve treatment effects and reduce the occurrence of drug resistance.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a cell line resistant to immunotherapy of PDL1 lung cancer, wherein the cell line is a mouse (Mus musculus) lung cancer cell LLC-R, which was deposited in the China Center for Type Culture Collection on November 6, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: C2024374.

[0008] The present invention also provides the use of the above cell line in studying the drug resistance mechanism of PDL1 immunotherapy.

[0009] The present invention also provides the use of the above cell line in preparing an animal model of lung cancer PDL1 immunotherapy resistance.

[0010] The present invention also provides a method for constructing an animal model of lung cancer PDL1 immunotherapy resistance, comprising the steps of inoculating the above-mentioned cell line subcutaneously into an experimental animal to construct the animal model of lung cancer PDL1 immunotherapy resistance.

[0011] Furthermore, the experimental animal is a mouse.

[0012] Furthermore, the cell line is inoculated subcutaneously in the experimental animal in the form of a cell suspension.

[0013] The present invention also provides an application of an animal model constructed according to the above method in studying the drug resistance mechanism of PDL1 immunotherapy.

[0014] The present invention discloses the following technical effects:

[0015] The present invention constructs a cell line resistant to immunotherapy of lung cancer PDL1, namely, mouse lung cancer cell LLC-R, which provides a reliable experimental model for studying the mechanism of lung cancer resistance to immunotherapy of PDL1 and related signaling pathways, enabling those skilled in the art to have a deeper understanding of the response of lung cancer cells to immunotherapy of PDL1, and is helpful for exploring new biomarkers, researching and developing new combined treatment strategies, improving treatment effects and reducing the occurrence of drug resistance. The present invention has important scientific and clinical significance for in-depth understanding of immunotherapy resistance of lung cancer PDL1 and improving the clinical treatment effect of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 Flow chart of the experimental design for constructing a subcutaneous tumor model using LLC cells and C57BL / 6J mice and screening for PDL1-resistant cell lines for lung cancer;

[0018] Figure 2 The results of the experiments to verify the drug resistance of the cell line LLC-R resistant to lung cancer PDL1; A is the proliferation curve of the subcutaneous tumor of the tumor-bearing mice of the cell line LLC-R resistant to lung cancer PDL1; B is the volume of the subcutaneous tumor of the tumor-bearing mice of the cell line LLC-R resistant to lung cancer PDL1; C is the statistical graph of the subcutaneous tumor weight of the tumor-bearing mice of the cell line LLC-R resistant to lung cancer PDL1 in the C57BL / 6J model; D is the survival time analysis graph of the subcutaneous tumor-bearing mice of the cell line LLC-R resistant to lung cancer PDL1;

[0019] Figure 3 Figure 2 is a graph showing the changes in the proportion of immune cell subsets in the microenvironment of LLC-R tumor tissues resistant to anti-PDL1 antibodies and LLC tumor tissues in the control group; A is a graph showing the overall changes in the proportion of major myeloid and lymphoid immune cells in LLC-R and LLC tumor tissues; B is a graph showing the changes in the proportion of Granzyme B + and IFN-γ + Waiting in CD8 + Proportional changes in T cells; C is CD8 + / CD45 + Flow cytometry gate diagram; D is the quantification diagram of C; E is the IFN-γ + / CD8 + Flow gate diagram of ; F is the quantization diagram of E;

[0020] Figure 4 The results of detecting the in vitro proliferation and clone formation ability of the lung cancer PDL1-resistant cell line LLC-R and the control wild-type cell line LLC; A is the image under the microscope; B is the plate clone morphology; C is the statistical chart of the clone number results;

[0021] Figure 5 A schematic diagram of the commonly upregulated genes in two lung cancer PDL1-resistant cell lines;

[0022] Figure 6 This is the KEGG pathway enrichment analysis diagram of the commonly upregulated genes in two lung cancer PDL1-resistant cell lines;

[0023] Figure 7 This is the GO functional enrichment analysis diagram of the commonly upregulated genes in two lung cancer PDL1-resistant cell lines;

[0024] Figure 8 The diagram shows the changes in the JAK-STAT Signaling Pathway of two lung cancer PDL1-resistant cell lines;

[0025] Fig. 9 This is a comparison of the enrichment of immune-related signal pathways in bulk sequencing in the PDL1-resistant cell line LLC-R of lung cancer and the wild-type LLC tumor tissue of the control; A is a change diagram of immune response signals; B is a change diagram of the activation pathway of immune response; C is a change diagram of lymphocyte-mediated immune pathways; D is a change diagram of granzyme-mediated programmed cell death signal pathways; E is a change diagram of leukocyte-mediated immune signal pathways;

[0026] Fig.10 This is the GO functional enrichment analysis diagram of bulk sequencing in the lung cancer PDL1-resistant cell line LLC-R and the control wild-type LLC tumor tissue;

[0027] Fig.11 Volcano plot of significantly upregulated and downregulated differentially expressed genes in the PDL1-resistant lung cancer cell line LLC-R and the control wild-type LLC cell line. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] The culture medium formula used in the following examples is as follows:

[0034] Complete culture medium: 89% DMEM basal medium + 10% fetal bovine serum (FBS) + 1% double antibody (penicillin and streptomycin).

[0035] Example 1

[0036] 1. Construction of PDL1-resistant lung cancer cell lines

[0037] The present invention uses a mouse lung cancer cell line LLC and C57BL / 6J mice to construct a subcutaneous tumor model, separates primary tumor cells from anti-PDL1 antibody-resistant mouse tumors, cultures and passages them, and obtains an anti-PDL1 antibody-resistant lung cancer cell line LLC-R (see Figure 1 ), the construction method includes the following steps:

[0038] (1) Prepare DMEM complete medium, take LLC cells out of liquid nitrogen, revive them in a 37°C water bath as soon as possible, resuspend them, add LLC cells to 5 mL complete medium, centrifuge at 1000 rpm at room temperature for 5 min, remove the supernatant, add 1 mL DMEM complete medium, gently pipette evenly, transfer to a 10 cm culture dish loaded with 9 mL DMEM complete medium containing 10% fetal bovine serum, and place in a 37°C, 5% CO2 incubator for sterile culture.

[0039] (2) Collect LLC tumor cells in the logarithmic growth phase according to the cell passaging method, resuspend the cells in sterile 1× PBS and count them, and adjust the cell concentration to 1×10 7The LLC cell suspension was placed on ice for later use.

[0040] (3) Use a 1 mL syringe to inoculate 100 μL of LLC cell suspension into the subcutaneous tissue of the back of C57BL / 6J mice to construct a subcutaneous tumor model.

[0041] (4) After 7 days of tumor growth, the long diameter a and short diameter b of the tumor were measured using a vernier caliper. The volume was calculated according to the formula V = 0.5236ab 2 Tumor volume was calculated.

[0042] (5) Select tumors with a volume of 60 to 70 mm 3 Mice within the range were given anti-PDL1 monoclonal antibody by intraperitoneal injection. The anti-PDL1 monoclonal antibody was prepared in advance with 0.9% saline at a concentration of 1 mg / mL and stored on ice for later use. Each mouse was given 200 μL / time, and the drug was administered once on the 1st, 4th, 7th, 10th, and 14th days, for a total of 5 times.

[0043] (6) The tumor volume of mice was measured and calculated every 2 days. After 20 days of tumor bearing, the mouse tumor with the largest volume in the anti-PDL1 monoclonal antibody treatment group was considered to have anti-PDL1 resistance potential. The mouse tumor tissue was isolated on a sterile operating table and transferred to a 10 cm cell culture dish, washed three times with sterile 1× PBS, and the waste liquid was discarded.

[0044] (7) Add 1 mL of DMEM complete medium containing 10% fetal bovine serum to the culture dish and cut the tumor tissue into pieces of approximately 1 mm in size using sterile scissors. 3 The small pieces were supplemented with DMEM complete medium to 10 mL, placed in a 37°C, 5% CO2 incubator, cultured aseptically for 48 hours, and then the adherent cells were passaged and frozen.

[0045] (8) The primary cells obtained above were collected according to the cell passage method in the logarithmic growth phase of the tumor cells, resuspended in sterile 1× PBS and counted, and the cell concentration was adjusted to 1×10 7 / mL, and place on ice for later use. Use a 1mL syringe to inoculate 100μL of cell suspension into the subcutaneous tissue of the back of C57BL / 6J mice to re-establish the C57BL / 6J mouse subcutaneous tumor model. Repeat the above screening process at least 3 times until the tumor of LLC-R tumor-bearing mice with resistant cell line can still grow rapidly after treatment with anti-PDL1 antibody, and there is no significant difference with the control treatment group.

[0046] (9) The largest tumor tissue in the anti-PDL1 antibody treatment group was separated and washed twice with sterile 1× PBS. The waste liquid was discarded, and 1 mL of DMEM complete medium was added and the tumor tissue was cut into pieces with sterile scissors with a volume of about 1 mm. 3The small pieces were supplemented with DMEM complete medium to 10 mL, and penicillin / streptomycin was added. The cells were cultured and passaged in a 37°C, 5% CO2 incubator to obtain a stable lung cancer PDL1-resistant cell line, which was named mouse lung cancer cell LLC-R.

[0047] 2. Cell preservation

[0048] Mouse lung cancer cells LLC-R were deposited in the China Center for Type Culture Collection on November 6, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: C2024374.

[0049] Example 2

[0050] 1. Verify the drug resistance effect of the lung cancer PDL1-resistant cell line LLC-R

[0051] (1) C57BL / 6J mouse subcutaneous tumor models were constructed using the lung cancer cell line LLC and the obtained lung cancer PDL1-resistant cell line LLC-R, respectively, and the construction method was as described in Example 1; anti-PDL1 antibody treatment was performed and the tumor volume change was detected to verify the drug resistance effect of the lung cancer PDL1-resistant cell line LLC-R.

[0052] (2) The experiment was divided into four groups: drug-resistant cell group LLC-R, drug-resistant cell treatment group LLC-R-PDL1, control cell group LLC, and control cell treatment group LLC-PDL1, with 5 mice in each group. The specific treatment method and tumor volume detection method were the same as in Example 1. Starting from the 7th day, the long diameter a and short diameter b of the tumor of each group of mice were measured every 3 days using a vernier caliper. According to the formula V = 0.5236ab 2 Calculate the tumor volume and draw the mouse tumor proliferation curve, with the horizontal axis representing time (unit: day) and the vertical axis representing tumor volume (unit: mm 3 ). The results are shown in Figure 2 Middle A.

[0053] (3) After 20 days of tumor bearing, the subcutaneous tumor tissues of each group of mice were isolated, the volumes were measured, and photographs were taken. The results are shown in Figure 2 Middle B.

[0054] (4) Weigh and record the weight of subcutaneous tumors in tumor-bearing mice to make relevant statistics. The horizontal axis represents the experimental group and the vertical axis represents the tumor weight (unit: grams). The results are shown in Figure 2 Middle C.

[0055] (5) In addition, the survival time of subcutaneous tumor-bearing mice in each group was recorded and statistically analyzed. The horizontal axis is time (unit: day) and the vertical axis is the remaining survival percentage. The results are shown in Figure 2 Middle D.

[0056] according to Figure 2 The results showed that the tumors of LLC-R tumor-bearing mice treated with anti-PDL1 antibodies could still grow rapidly, with no significant difference from the control group, indicating that the LLC-R tumor-bearing mice had anti-PDL1 antibody resistance (such as Figure 2 AC, P>0.05), and the survival time of LLC-R tumor-bearing mice treated with anti-PDL1 antibody was not significantly different from that of the control group (e.g. Figure 2 (D, P>0.05).

[0057] 2. Anti-PDL1 immunotherapy induces a suppressive tumor immune microenvironment after resistance

[0058] Flow cytometric staining of mouse tumor tissue includes the following steps:

[0059] (1) Add approximately 2 mL of 1640 buffer to each well of a six-well plate and place the isolated tumor tissue into each well.

[0060] (2) After the tumor is separated, aspirate the culture medium, leaving 0.1 mL, and mince the tumor tissue.

[0061] (3) After cutting, add 2 mL of digestion enzyme to each well (Note: 100× collagenase and 100× DNase mixture diluted 100 times with 5% FBSDMEM medium), preheat at 37°C, and place in a cell culture incubator for digestion for 30 min. Shake and mix every ten minutes, and add 2 mL of 1640 buffer to each well to terminate digestion.

[0062] 5% FBSDMEM culture medium: 95% DMEM basal medium + 5% fetal bovine serum (FBS).

[0063] (4) After digestion, take out the cells and place them on ice. Use a pipette to filter the liquid with the fragments through a 70 μm cellstrainer. Grind the tissue fragments on the filter with the back of a 1 mL syringe and rinse with 2 mL 1640 buffer to obtain a cell suspension.

[0064] (5) Transfer to a 15 mL centrifuge tube and centrifuge at 2000 rpm at 4°C for 5 min. Discard the waste liquid.

[0065] (6) The cells were resuspended in 1 mL of 2% PBS buffer (the specific amount of buffer added was adjusted according to the number of cells) and transferred to an EP tube. Centrifuged at 4000 rpm for 4 min and then the waste liquid was discarded. Note the serial number and keep a blank control group.

[0066] (7) Cell surface antibody staining:

[0067] The cells were incubated with Fcr antibody (cell suspension volume 2% PBS: Fcr antibody = 100:3) at 4°C for 15 min. The total liquid was prepared in advance and protected from light.

[0068] a. Add 1 mL of 2% PBS buffer, resuspend and centrifuge, and discard the waste liquid.

[0069] b. Add 0.5 μL of marker antibody mixture to every 100 μL of cell sample. Incubate at 4°C for 30 min. Prepare the total liquid in advance and keep it away from light.

[0070] c. Add 200 μL of 2% PBS buffer to each EP tube, mix well, and add 200 μL of 4% paraformaldehyde fixative for overnight storage.

[0071] d. Filter the cell suspension and detect using BD LSRFortessa instrument.

[0072] (8) Intracellular staining

[0073] a. Centrifuge the single cell suspension obtained in step (6) at 1500 rpm for 5 min, resuspend in 125 μL 2% 1640 culture medium, and incubate at 37°C, 5% CO2 for 30 min.

[0074] b. Add cell stimulation medium to a final concentration of 2 μL Cell Stimulation Cocktail (plus protein transport inhibitors) / 1 mL 1640 medium. Incubate at 37°C, 5% CO2 for 4 hours. Mix 3 times during the incubation.

[0075] c. Wash twice with 2% PBS buffer, centrifuge at 1500 rpm, 4°C for 5 min, and discard the supernatant. Block the FcR receptors of immune cells with anti-FcR and incubate at 4°C for 15 min.

[0076] d. Wash the cells once with 2% PBS buffer, add 100 μL of cell surface mixed antibodies to each sample, incubate at 4°C in the dark for 30 min, and mix once during the incubation process.

[0077] e. Wash twice with 300 μL 2% PBS buffer, centrifuge at 4°C, 1500 rpm for 5 min, discard the supernatant, and incubate with 200 μL fixation / permeabilization buffer (A:B=1:3) at 4°C for 1.5 hours.

[0078] f. Wash once with 300 μL 1× permeabilization buffer, centrifuge at 2000 rpm for 5 min at 4°C, and discard the supernatant.

[0079] g. Prepare a mixture of IFN-γ, TNF-α, and Granzyme B intracellular antibody using 1× permeabilization buffer, and stain each sample with 60-100 μL. Incubate at 4°C in the dark for 1 hour.

[0080] h. Wash twice with 300 μL of 1× Permeabilization Buffer, centrifuge at 4°C, 2000 rpm for 5 min, discard the supernatant, and finally resuspend with 300-500 μL of 1× Permeabilization Buffer.

[0081] i. Filter and analyze the samples using BD LSRFortessa.

[0082] In this part of the study, using an animal model of resistance to anti-PDL1 immunotherapy, the present invention compared the differences in the immune microenvironment in non-small cell lung cancer LLC tumor tissue and anti-PDL1 immunotherapy-resistant non-small cell lung cancer tumor tissue. The present invention found that the tumor microenvironment of mice resistant to anti-PDL1 immunotherapy was mainly a suppressive tumor immune microenvironment. The present invention detected the changes in the proportion of immune cell subpopulations in tumor tissues by flow cytometry and found that compared with the control group LLC, the changes in the immune cells in the resistant group LLC-R were mainly manifested in a decrease in the proportion of M1 macrophages, an increase in the proportion of M2 macrophages, an increase in the proportion of PMN-MDSCs cells, a decrease in the proportion of DC cell subpopulations, and a decrease in the proportion of CD4 + T cells, CD8 + The proportion of T cells decreased ( Figure 3 A), and Granzyme B + IFN-γ + and TIM3 + PD1 + The proportion of immune cells also decreased ( Figure 3 This is consistent with the results of flow cytometry ( Figure 3 CF in the middle). The present invention speculates that various cells in these microenvironments regulate each other to form a complex network system that promotes rapid tumor progression. These changes and data further illustrate that the tumor cells constructed by the present invention have developed drug resistance, and anti-PDL1 immunotherapy induces a suppressive tumor immune microenvironment after drug resistance. Data are expressed as mean ± SE (n = 5 / group); * P<0.05, ** P<0.01, *** P<0.001.

[0083] 3. Detection of the in vitro proliferation and clone formation ability of the lung cancer PDL1-resistant cell line LLC-R

[0084] (1) Take the lung cancer cells LLC in the logarithmic growth phase and the lung cancer PDL1-resistant cell line LLC-R and observe them under an inverted microscope (see the cell morphology diagram). Figure 4 The cells were then collected by trypsin digestion and centrifugation, and the cell pellets were washed twice with sterile 1× PBS, centrifuged at 1000 rpm at room temperature for 5 min, the supernatant was removed, 2 mL of serum-free DMEM medium was added to resuspend the cell pellet, and the cells were counted using a cell counter.

[0085] (2) For the clone formation experiment, the concentration of LLC and LLC-R cells was adjusted to 8×10 2 1 mL of cell suspension was added to a six-well plate, supplemented with 800 μL of DMEM medium and 200 μL of fetal bovine serum, 5 replicate wells were set up for each well, and then placed in a 37°C, 5% CO2 incubator for 10 days.

[0086] (3) Staining and colony counting: Take the six-well plate out of the incubator at 37°C and 5% CO2, wash the cells twice with sterile 1×PBS, discard the supernatant, add 1mL 4% paraformaldehyde to fix for 20min, discard the supernatant, add 1mL 1% crystal violet staining solution to stain for 30min, discard the supernatant, and carefully wash the residual crystal violet dye with clean water. Take pictures and perform colony counting and statistical analysis. The results are shown in Figure 4 Medium B and C.

[0087] according to Figure 4 It can be seen that the in vitro proliferation and clone formation ability of the lung cancer PDL1-resistant cell line LLC-R is stronger than that of the wild-type control cell line LLC ( * P<0.05, ** P<0.01, *** P<0.001).

[0088] 4. Screening of common upregulated genes in two lung cancer PDL1-resistant cell lines

[0089] At the same time, the present invention also cultured another mouse lung cancer PDL1 resistant cell line CMT167-R, and analyzed the drug resistance mechanism of it and LLC-R. The intersection of the upregulated genes in LLC-R and CMT167-R was taken, and it was found that 106 genes were significantly upregulated in both resistant cells (see Figure 5 ).

[0090] 5. KEGG pathway enrichment analysis and GO function enrichment analysis of commonly upregulated genes in two lung cancer PDL1-resistant cell lines

[0091] The present invention uses the "DESeq2" R package to perform differential analysis on the expression data, and the differentially expressed genes are identified with FDR < 0.05 and log2FC absolute value > 1. The present invention uses the "ClusterProfiler" package and "Pathview" R package in R to perform KEGG pathway enrichment analysis on the differentially expressed genes (see Figure 6 ) and GO functional enrichment analysis (see Figure 7 ), and then relevant drawings were made after obtaining the results.

[0092] 6.JAK-STAT Signaling Pathway

[0093] Studies have reported that the JAK-STAT signaling pathway is associated with drug resistance, especially in certain cancers and autoimmune diseases. In tumors, abnormal activation of the JAK-STAT pathway is often associated with cell proliferation, survival, and immune escape mechanisms, which may lead to drug resistance of tumor cells to treatment. Therefore, the present invention further discloses the gene expression differences in the JAK-STAT Signaling Pathway pathway of the lung cancer PDL1-resistant cell line LLC-R of the present invention ( Figure 8 ).

[0094] 7. Comparison of immune-related signaling pathway enrichment

[0095] Using the "clusterProfiler" R package, the present invention compared the enrichment of bulk sequencing immune-related signaling pathways in the lung cancer PDL1-resistant cell line LLC-R and the control wild-type LLC tumor tissue. The changes in the immune response signals of tumor cells in the tumor microenvironment were shown in Figure 2. Fig. 9 Middle A; Changes in the activation pathways of immune responses are shown in Fig. 9 Middle B; Changes in lymphocyte-mediated immune pathways are shown in Fig. 9 Middle C; Changes in the granzyme-mediated programmed cell death signaling pathway are shown in Fig. 9 Middle D; Changes in leukocyte-mediated immune signaling pathways are shown in Fig. 9 (LLC-R vs LLC, * P<0.05, ** P<0.01, *** P<0.001).

[0096] 8. GO functional enrichment analysis of bulk sequencing of drug-resistant tumor tissues

[0097] In addition, the present invention also performed bulk sequencing GO functional enrichment analysis for the lung cancer PDL1-resistant cell line LLC-R and the control wild-type LLC tumor tissue. The results are shown in Fig.10 .

[0098] 9. Screening of significantly differentially expressed genes

[0099] Using R packages such as "EnhancedVolcano" and "readxl", the present invention performed differential gene analysis on the lung cancer PDL1-resistant cell line LLC-R and the control wild-type LLC cell line. Fig.11 The specific locations of significantly up-regulated and down-regulated differentially expressed genes in the volcano plot are shown.

[0100] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A lung cancer PDL1 immunotherapy-resistant cell line, characterized in that: The cell line is mouse (Mus musculus) lung cancer cell LLC-R, which was deposited in the China Center for Type Culture Collection on November 6, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: C2024374.

2. Use of the cell line as claimed in claim 1 in studying the mechanism of drug resistance in PDL1 immunotherapy.

3. Use of the cell line as claimed in claim 1 in preparing an animal model of lung cancer resistant to PDL1 immunotherapy.

4. A method for constructing an animal model of lung cancer PDL1 immunotherapy resistance, characterized in that: The method comprises the steps of inoculating the cell line according to claim 1 subcutaneously into an experimental animal to construct an animal model of lung cancer PDL1 immunotherapy resistance.

5. The construction method according to claim 4, characterized in that: The experimental animals are mice.

6. The construction method according to claim 4, characterized in that: The cell line is inoculated subcutaneously in the experimental animal in the form of a cell suspension.

7. Use of an animal model constructed according to the method according to any one of claims 4 to 6 in studying the mechanism of resistance to PDL1 immunotherapy.

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