A lung cancer PDL1 immunotherapy-resistant cell line LLC-R and its application

Abstract

The present invention discloses a cell line LLC-R that is resistant to immunotherapy for PDL1 lung cancer and its application, which relates to the field of cell biology technology. The cell line LLC-R 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. The present invention provides a reliable experimental model for studying the resistance mechanism of lung cancer to PDL1 immunotherapy and related signaling pathways, enabling those skilled in the art to have a deeper understanding of the response of lung cancer cells to PDL1 immunotherapy, and helping to explore new biomarkers, research and develop new combination therapy strategies, improve treatment effects and reduce the occurrence of drug resistance. The present invention has important scientific and clinical significance for in-depth understanding of the resistance to immunotherapy for PDL1 lung cancer and improving the clinical treatment effects of patients.

Description

Technical Field

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

[0002] Primary lung cancer is the most common malignant tumor, with the highest morbidity and mortality rates. Non-small cell lung cancer accounts for approximately 85% of cases. The overall five-year survival rate for patients with advanced lung cancer is around 20%.

[0003] Currently, the main treatments for lung cancer include surgical resection, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. Programmed cell death ligand 1 (PDL1) is a type I transmembrane protein that inhibits T cell activation and maintains immune homeostasis by binding to programmed cell death protein 1 (PD-1). PDL1 inhibitors, as representatives of immune checkpoint inhibitors (ICIs), have become one of the mainstream treatments for 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 therapy is limited, and many patients are prone to resistance to immunotherapy due to innate or acquired factors, suffering from primary resistance and acquired resistance. Currently, these potential resistance mechanisms remain unknown, and new research methods and experimental techniques are urgently needed to further elucidate the resistance mechanisms of immunotherapy.

[0004] The lack of existing lung cancer cell lines resistant to PDL1 immunotherapy makes experiments difficult. Therefore, establishing a PDL1-resistant lung cancer cell line is crucial for identifying key molecules responsible for immunotherapy resistance, mapping the mechanisms of resistance, and providing a theoretical basis for treatment strategies following resistance. This is essential for guiding future precision lung cancer treatments and expanding response rates. Summary of the Invention

[0005] The present invention aims to provide a lung cancer cell line LLC-R that is resistant to immunotherapy targeting PDL1, and its use, to address the aforementioned problems of the prior art. This invention provides a reliable experimental model for studying the mechanisms of lung cancer resistance to PDL1 immunotherapy and related signaling pathways. This model enables those skilled in the art to gain a deeper understanding of the response of lung cancer cells to PDL1 immunotherapy, facilitates the discovery of new biomarkers, and facilitates the research and development of novel combination therapy strategies to improve treatment efficacy and reduce the incidence 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 animals are mice.

[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 mechanism of drug resistance in PDL1 immunotherapy.

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

[0015] This study has established a cell line, LLC-R, that is resistant to immunotherapy for PDL1 in lung cancer. This provides a reliable experimental model for studying the mechanisms of lung cancer resistance to PDL1 immunotherapy and related signaling pathways. This model will enable those skilled in the art to gain a deeper understanding of the response of lung cancer cells to PDL1 immunotherapy, facilitate the discovery of new biomarkers, and facilitate the research and development of novel combination therapy strategies to improve treatment efficacy and reduce the incidence of drug resistance. This study has important scientific and clinical significance for deepening understanding of PDL1 immunotherapy resistance in lung cancer and improving clinical treatment outcomes for 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 following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

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

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

[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 + The proportion change diagram of 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; F is the quantitative diagram of E;

[0020] Figure 4 The results of the in vitro proliferation and clone formation ability test of the lung cancer PDL1-resistant cell line LLC-R and the wild-type control cell line LLC are shown in Figure 1. A is a microscope image; B is a plate clone morphology image; C is a statistical graph of the clone number results;

[0021] Figure 5 Schematic diagram of commonly upregulated genes in two lung cancer cell lines resistant to PDL1;

[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 function 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 in two PDL1-resistant lung cancer cell lines;

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

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

[0027] Figure 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents 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 described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be 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 the mouse lung cancer cell line LLC and C57BL / 6J mice to construct a subcutaneous tumor model, and isolates primary tumor cells from the anti-PDL1 antibody-resistant mouse tumors for culture and passage to obtain the 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, remove LLC cells from liquid nitrogen, and revive them in a 37°C water bath as soon as possible. Resuspend the cells and add them to 5 mL of complete medium. Centrifuge at 1000 rpm at room temperature for 5 min. Remove the supernatant, add 1 mL of DMEM complete medium, and gently pipette evenly. Transfer the cells to a 10 cm culture dish containing 9 mL of DMEM complete medium containing 10% fetal bovine serum and place them 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 passage method, resuspend the cells in sterile 1× PBS and count them, and adjust the cell concentration to 1×10 7The LLC cell suspension obtained 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 establish 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 as V = 0.5236ab. 2 Calculate tumor volume.

[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 injected with 200 μL / time, and the drug was administered once on the 1st, 4th, 7th, 10th, and 14th day, for a total of 5 times.

[0043] (6) The tumor volume of the mice was measured and calculated every 2 days. After 20 days of tumor bearing, the mouse with the largest tumor in the anti-PDL1 monoclonal antibody treatment group was considered to have anti-PDL1 resistance potential. The tumor tissue of the mouse was isolated under a sterile operating table and transferred to a 10 cm cell culture dish. The tissue was washed three times with sterile 1× PBS and the waste solution 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 cells were cultured in a 37°C, 5% CO2 incubator 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 until ready to use. Use a 1mL syringe to inoculate 100μL of the cell suspension subcutaneously into the back of C57BL / 6J mice to re-establish a subcutaneous tumor model in C57BL / 6J mice. Repeat the above screening process at least three times until the tumors in mice bearing the drug-resistant cell line LLC-R continue to grow rapidly after treatment with anti-PDL1 antibodies, and there is no significant difference compared to the control group.

[0046] (9) The largest tumor tissue in the anti-PDL1 antibody treatment group was isolated and washed twice with sterile 1× PBS. The waste liquid was discarded. After adding 1 mL of DMEM complete medium, the tumor tissue was cut into pieces of approximately 1 mm in size using sterile scissors. 3The cells were divided into small pieces and supplemented with DMEM complete medium to 10 mL. Penicillin / streptomycin was added and the cells were cultured and passaged in a 37°C, 5% CO2 incubator to obtain a stable PDL1-resistant lung cancer cell line 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, with the deposit address being Wuhan University, Wuhan, China, with the deposit number being 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) Subcutaneous tumor models were constructed in C57BL / 6J mice using the lung cancer cell line LLC and the obtained PDL1-resistant lung cancer cell line LLC-R, respectively. The construction method was as described in Example 1. Anti-PDL1 antibody treatment was performed and the changes in tumor volume were detected to verify the drug resistance effect of the PDL1-resistant lung cancer cell line LLC-R.

[0052] (2) The experiment was divided into four groups: the drug-resistant cell group LLC-R, the drug-resistant cell treatment group LLC-R-PDL1, the control cell group LLC, and the 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 volume was measured and photographed. 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 the drug-resistant cell line LLC-R bearing mice could still grow rapidly after being treated with anti-PDL1 antibodies, with no significant difference from the control group, indicating that the drug-resistant cell line LLC-R has 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, the culture medium is aspirated, leaving 0.1 mL, and the tumor tissue is minced.

[0061] (3) After mincing, 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 minutes. Shake and mix every ten minutes, and add 2 mL of 1640 buffer to each well to terminate the digestion.

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

[0063] (4) After digestion, remove the tube and place it on ice. Use a pipette to filter the liquid containing 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 of 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) Resuspend the cells in 1 mL of 2% PBS buffer (adjust the amount of buffer according to the cell volume) and transfer to an EP tube. Centrifuge at 4000 rpm for 4 min and discard the waste liquid. Mark 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, 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 minutes. Prepare the total solution in advance and protect 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. The single cell suspension obtained in step (6) was centrifuged at 1500 rpm for 5 min, then resuspended in 125 μL of 2% 1640 culture medium and incubated 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) per 1 mL of 1640 medium. Incubate at 37°C, 5% CO₂ for 4 hours. Mix three times during incubation.

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

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

[0077] e. Wash twice with 300 μL of 2% PBS buffer, centrifuge at 1500 rpm for 5 min at 4°C, discard the supernatant, and incubate with 200 μL of 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 antibodies in 1× permeabilization buffer and stain each sample with 60–100 μL of the mixture. Incubate at 4°C in the dark for 1 hour.

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

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

[0082] In this part of the study, the present invention compared the differences in the immune microenvironment between non-small cell lung cancer LLC tumor tissue and anti-PDL1 immunotherapy-resistant non-small cell lung cancer tumor tissue using an animal model of anti-PDL1 immunotherapy resistance. 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 subsets in tumor tissue 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 subsets, 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 hypothesizes that the various cells within these microenvironments regulate each other, forming a complex network system that promotes rapid tumor progression. These changes and data further demonstrate that the tumor cells constructed in this invention have developed drug resistance and that anti-PDL1 immunotherapy induces a suppressive tumor immune microenvironment after drug resistance. Data are presented 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 PDL1-resistant lung cancer 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 respectively and observe them under an inverted microscope (see the cell morphology diagram). Figure 4 The cells were then trypsinized and collected by centrifugation. The cell pellets were washed twice with sterile 1× PBS and centrifuged at 1000 rpm for 5 min at room temperature. The supernatant was removed and 2 mL of serum-free DMEM medium was added to resuspend the cell pellet. 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, and 5 replicates were set up for each well. The plate was then placed in a 37°C, 5% CO2 incubator for 10 days.

[0086] (3) Staining and colony counting: Remove the six-well plate from the incubator at 37°C and 5% CO2, wash the cells twice with sterile 1×PBS, discard the supernatant, add 1 mL of 4% paraformaldehyde and fix for 20 min, discard the supernatant, add 1 mL of 1% crystal violet staining solution and stain for 30 min, discard the supernatant, and carefully wash away the residual crystal violet dye with clean water. Take pictures and perform colony counting and statistical analysis. The results are shown in Figure 4 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 its resistance mechanism with LLC-R. The intersection of the upregulated genes in LLC-R and CMT167-R was found to have 106 genes that were significantly upregulated in both resistant cell lines (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 tumor cell resistance to treatment. Therefore, the present invention further discloses the gene expression differences in the JAK-STAT Signaling Pathway pathway in the PDL1-resistant lung cancer 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 immune-related signaling pathways in bulk sequencing of PDL1-resistant lung cancer cell line LLC-R and wild-type LLC tumor tissues. The changes in the immune response signals of tumor cells in the tumor microenvironment were shown. Figure 9 Middle A; Changes in the activation pathways of the immune response are shown in Figure 9 Middle B; Changes in lymphocyte-mediated immune pathways are seen Figure 9 Middle C; Changes in the granzyme-mediated programmed cell death signaling pathway are shown in Figure 9 Middle D; Changes in leukocyte-mediated immune signaling pathways are shown in Figure 9 (LLC-R vs LLC, * P<0.05, ** P<0.01, *** P<0.001).

[0096] 8. GO Function Enrichment Analysis of Bulk Sequencing of Drug-Resistant Tumor Tissues

[0097] In addition, the present invention also performed bulk sequencing GO function 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 Figure 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. Figure 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 merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined 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, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: C2024374.

2. Use of the cell line according to claim 1 in studying the mechanism of resistance to PDL1 immunotherapy.

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

4. A method for constructing an animal model of lung cancer resistant to PDL1 immunotherapy, 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 resistant to immunotherapy of lung cancer PDL1.

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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