Lung cancer immunotherapy acquired drug resistance cell line and application thereof

Abstract

The application discloses a lung cancer immunotherapy acquired drug resistance cell strain and application thereof in preparation of a lung cancer PD-1 inhibitor and / or PD-L1 inhibitor acquired drug resistance mouse model. The lung cancer immunotherapy acquired drug resistance cell strain is resistant to the PD-1 inhibitor and the PD-L1 inhibitor, can be used for constructing the lung cancer PD-1 inhibitor and / or PD-L1 inhibitor acquired drug resistance mouse model, and can be used for researching a PD-L1 and PD-1 immunotherapy drug resistance mechanism and simulating changes in a microenvironment in tumor tissue. The application further discloses the lung cancer PD-1 inhibitor and / or PD-L1 inhibitor acquired drug resistance mouse model and a construction method thereof.

Description

Technical Field

[0001] This invention belongs to the field of cell technology, specifically relating to acquired drug-resistant cell lines for lung cancer immunotherapy and their applications. Background Technology

[0002] Currently, lung cancer is the leading cause of cancer-related deaths worldwide. The latest data shows that in 2022, there were nearly 2.5 million new cancer cases globally, of which approximately 2 million were lung cancer cases; lung cancer caused approximately 1.8 million deaths, accounting for 18.7% of all cancer deaths.

[0003] Non-small cell lung cancer (NSCLC) accounts for 80%–85% of all lung cancer cases, and its treatment primarily includes surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. Among these treatments, immune checkpoint inhibitors (ICIs), represented by PD-1 / PD-L1 inhibitors and CTLA-4 inhibitors, have significantly improved patient survival and have become first-line treatments for advanced NSCLC. The most widely studied aspect is the link between PD-L1 molecules on the surface of tumor cells and PD-1 molecules on the surface of T cells, which can cause tumor immune escape. The function of PD-1 and PD-L1 inhibitors is to block this process and restore T-cell-mediated cytotoxicity. However, immunotherapy resistance (including primary and acquired resistance) has become a core issue limiting efficacy, with approximately 60% of initial responders eventually experiencing drug resistance and relapse.

[0004] Acquired resistance to immunotherapy in lung cancer is a major challenge in clinical practice. Its mechanisms are complex and multidimensional, involving intrinsic tumor cell characteristics (such as high expression of PD-L1 in tumor tissue), dynamic changes in the tumor microenvironment (such as the massive infiltration of immunosuppressive cells like tumor-associated macrophages (TAMs), immature dendritic cells (DCs), and regulatory T cells (Tregs), as well as immune system adaptation. Therefore, establishing models of immunotherapy resistance in lung cancer to investigate its potential resistance mechanisms is valuable for solving this problem. However, the methods for constructing models of acquired resistance to immunotherapy have the following drawbacks: (1) Limitation of clinical samples: They rely on patient-derived tumor xenograft (PDX) models, which are scarce and have high heterogeneity in resistance mechanisms; (2) Uniqueness of in vitro models: Traditional long-term drug shock methods for cell lines (such as paclitaxel pulsed administration) are difficult to simulate changes in the microenvironment within tumor tissues; (3) Insufficient drug resistance stability: Existing gene-edited drug-resistant cell lines lack dynamic drug resistance phenotypic evolution processes; (4) PD-1 inhibitors and PD-L1 inhibitors differ in the scope of pathway blockade and their impact on immune cell interactions. PD-L1 resistance is not necessarily related to PD-1 resistance. To achieve dual resistance to PD-1 and PD-L1, multiple inhibitors need to be used, and the preparation steps are cumbersome. Therefore, it is urgent to develop a lung cancer immunotherapy-acquired drug-resistant cell line that can simulate the clinical drug resistance process and has both stability and clinical relevance. Summary of the Invention

[0005] The purpose of this invention is to provide a lung cancer immunotherapy acquired resistance cell line that is resistant to both PD-L1 inhibitors and PD-1 inhibitors. This cell line can be used to construct mouse models of acquired resistance to PD-1 inhibitors and / or PD-L1 inhibitors, thereby solving the technical problems of scarce clinical samples and difficulty in simulating changes in the tumor microenvironment in existing technologies.

[0006] In a first aspect, the present invention provides an acquired drug-resistant cell line for lung cancer immunotherapy, the cell line being a mouse Lewis lung cancer cell-derived cell line.

[0007] The acquired drug-resistant cell line for lung cancer immunotherapy provided by this invention is the mouse Lewis lung cancer cell-derived cell line LLC-R, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No. 67576 and deposit date of December 30, 2025.

[0008] The LLC-R cell line developed in this invention, which is an acquired drug-resistant cell line for lung cancer immunotherapy, exhibits significantly different morphology from Lewis (LLC) cells in vitro. It demonstrates stronger proliferation, migration, and colony formation capabilities, and shows marked resistance to PD-1 and PD-L1 inhibitors. PD-L1 protein levels are significantly reduced; and it is associated with CD8+. + When T cells are co-cultured, CD8 inhibitors are suppressed. + T-cell killing. And tumors form more rapidly and grow faster in vivo; the resulting tumor microenvironment (TME) is rich in infiltration of a large number of immunosuppressive cells.

[0009] In a second aspect, the present invention provides the use of the above-described lung cancer immunotherapy-acquired resistance cell lines in the preparation of lung cancer PD-1 inhibitor and / or PD-L1 inhibitor-acquired resistance mouse models.

[0010] A third aspect of the present invention provides a method for constructing a mouse model of acquired resistance to PD-1 inhibitors and / or PD-L1 inhibitors in lung cancer, comprising the following steps: inoculating a lung cancer immunotherapy-acquired resistant cell line into the axilla of a mouse.

[0011] In some implementations, the inoculum size of acquired drug-resistant cell lines for lung cancer immunotherapy is 1 × 10⁻⁶. 6 ~2×10 6 Cells / mouse. Preferably, the inoculation amount is 1×10⁻⁶ cells / mouse. 6 cells / mouse.

[0012] In some implementations, the mice are C57BL / 6 mice.

[0013] A fourth aspect of the present invention provides a mouse model of acquired resistance to PD-1 inhibitors and / or PD-L1 inhibitors in lung cancer, constructed by the above-described construction method.

[0014] A fifth aspect of the present invention provides a method for preparing acquired drug-resistant cell lines for lung cancer immunotherapy, comprising the following steps: (1) Lewis cells were inoculated into the armpits of 6 to 9 mice. After tumor formation, PD-L1 inhibitors were administered. The tumor volume was measured every 3 days until the largest tumor approached the maximum tumor volume stipulated by animal ethics. The mice were then sacrificed, and the tumor tissue of the mouse with the largest tumor volume was taken. The tumor tissue was digested, resuspended and cultured in DMEM complete medium to obtain tumor cells. (2) Take the tumor cells obtained in step (1) and inoculate them into the armpits of 6 to 9 mice. Treat the mice according to the administration method and sampling conditions in step (1) to obtain tumor tissue and tumor cells. (3) Take the tumor cells obtained in step (2) and repeat the experiment twice in the same way as in step (2). The resulting tumor cells are the acquired drug-resistant cell lines for lung cancer immunotherapy.

[0015] In some implementations, the number of mice in steps (1) and (2) is 8.

[0016] In some embodiments, the PD-L1 inhibitor is administered once every 3 days at a dose of 8-12 mg / kg. Preferably, the PD-L1 inhibitor is administered once every 3 days, twice a week, at a dose of 10 mg / kg.

[0017] In some implementations, PD-L1 inhibitor therapy is administered via intraperitoneal injection.

[0018] In some implementations, the seeding density of Lewis cells is 1 × 10⁻⁶. 6 ~2×10 6 cells / mouse. Preferably, the seeding density of Lewis cells is 1 × 10⁻⁶. 6 cells / mouse.

[0019] In some implementations, the seeding amount of tumor cells is 5 × 10⁻⁶. 6 ~7×10 6 Tumor cells / mouse. Preferably, the inoculum size is 6 × 10⁶ cells / mouse. 6 cells / mouse.

[0020] Regarding the inoculation site, this invention selected the axilla, which has a richer blood supply, as the inoculation site to ensure better tumorigenicity. After four cycles of tumor cell culture, the lung cancer immunotherapy-acquired drug-resistant cell line LLC-R was obtained, and it was found that this cell line also showed resistance to PD-1 inhibitors.

[0021] The beneficial effects of this invention are: (1) The lung cancer immunotherapy acquired drug resistance cell line of the present invention is prepared by in vivo administration. The applicant found that when Lewis cells are treated with PD-L1 inhibitors according to the construction method of the present invention, the resulting lung cancer immunotherapy acquired drug resistance cell line is not only resistant to PD-L1 inhibitors, but also resistant to PD-1 inhibitors. (2) The lung cancer immunotherapy acquired drug-resistant cell line of the present invention is implanted into animals to construct a drug-resistant model, which can be used to simulate the changes in the microenvironment in tumor tissue; the whole process does not rely on patient-derived tumor xenograft (PDX) model, and does not involve gene editing, thus solving the problem of insufficient drug resistance stability caused by gene editing; (3) The lung cancer immunotherapy acquired drug resistance cell line of the present invention can be used to study the drug resistance mechanism of PD-L1 and PD-1 immunotherapy, and can explore the target of immunotherapy resistance, which provides a potential target worth exploring for subsequent drug development or drug intervention. Attached Figure Description

[0022] Figure 1 A methodology and workflow for constructing an acquired resistance model for lung cancer immunotherapy; Figure 2 Tumor volume growth curves at each stage during the construction of a model for acquired resistance to immunotherapy in lung cancer; Figure 3 Microscopic morphological observation results of LLC and LLC-R cells; Figure 4 Results of proliferation assays for LLC and LLC-R cells; Figure 5 Results of clonogenic experiments for LLC and LLC-R cells; Figure 6 (A) is a graph showing the results of the migration ability assay for LLC and LLC-R cells; Figure 6 (B) represents the percentage of wound area for LLC and LLC-R cells; Figure 7 (A) is CD8 + T cell magnetic bead sorting results; (B) is CD8 + Microscopic morphological observation of T cells co-cultured with LLC cells or LLC-R cells; (C) represents CD8. + After co-culturing T cells with LLC or LLC-R cells, IC50 induced by αPD-L1 and αPD-1 intervention 50 Measurement results; Figure 8 Western blot results for PD-L1 expression levels in LLC and LLC-R cells; Figure 9 (A) Comparison of tumor size, tumor volume growth curve and tumor quality in an LLC cell xenograft animal model with and without PD-L1 inhibitor intervention; Figure 9 (B) Comparison of tumor size, tumor volume growth curve and tumor quality in the LLC-R cell xenograft animal model with and without PD-L1 inhibitor intervention; Figure 10 Flow cytometry analysis results of the proportions of macrophages, M1 macrophages, and M2 macrophages in tumor tissues of LLC and LLC-R cell xenograft animal models; Figure 11 CD8 in tumor tissues of LLC and LLC-R cell xenograft animal models+ Flow cytometry analysis results of the ratio of T cells to Treg cells; Figure 12 Flow cytometry analysis results of the percentage of mature dendritic cells in tumor tissues of LLC and LLC-R cell xenograft animal models. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to the accompanying drawings, but the embodiments of the present invention are not limited thereto. The raw materials and reagents involved in the following embodiments are all commercially available.

[0024] The following are embodiments of the present invention, the instruments, reagents and materials required for the experiment.

[0025] 1. Instruments Centrifuge tubes; centrifuges; shakers; electrophoresis equipment; transfer equipment; cell culture incubators; upright microscopes; flow cytometers.

[0026] 2. Reagents and Materials The reagents and materials are shown in Table 1.

[0027]

[0028] The PD-L1 inhibitor is Anti-mouse PD-L1 (B7-H1)-InVivo, sourced from Selleck; clone number: 10F.9G2; catalog number: A2115. The PD-1 inhibitor is Anti-mouse PD-1 (CD279)-InVivo, sourced from Selleck; clone number: RMP1-14; catalog number: A2122.

[0029] 3. Cell culture methods Cells were cultured in DMEM complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution.

[0030] Example 1 This embodiment provides an acquired drug-resistant cell line for lung cancer immunotherapy, the preparation method of which includes the following steps: (1) Cells: Lewis cells (LLC); Animals: 55 female 6-8 week old C57BL / 6 mice.

[0031] (2) Subcutaneous xenograft model construction: Using a 1ml syringe, 1×10 6Eight parental LLC cells were injected into the right axilla of eight mice. Tumor formation was defined as tumors palpable and measurable by calipers on day 3 post-inoculation. PD-L1 inhibitors (10 mg / kg) were administered intraperitoneally every three days. Tumor volume was measured every three days. Mice were euthanized when the largest tumor approached the ethically permissible maximum tumor volume. Tumor tissue from the mice with the largest tumor volume was digested using the Reward Mouse Tumor Mild Enzymatic Digestion Kit. The tumor suspension was resuspended in DMEM complete medium and cultured overnight at 37°C in a 5% CO2 cell culture incubator. The supernatant was discarded the following day, and the culture dishes were washed twice with sterile PBS. The cells were then cultured again in DMEM complete medium. The resulting cells were named LLC-R-1.

[0032] (3) Construction of acquired drug-resistant cell lines for lung cancer immunotherapy: Culture a sufficient number of LLC-R-1 cells (approximately 6 × 10⁻⁶) 6 (100 cells), after digestion with trypsin to obtain LLC-R-1 tumor cell suspension, which was then inoculated into the right axilla of a new batch of 8 mice. This was the second cycle. The mice were treated according to the administration method and sampling conditions in step (2). After 4 cycles, the lung cancer immunotherapy acquired drug resistance cell line was finally obtained and named LLC-R.

[0033] The method and process for constructing a model of acquired resistance to immunotherapy in lung cancer are as follows: Figure 1 As shown, from Figure 1 It can be seen that after four cycles of construction, the acquired drug-resistant cell line LLC-R for lung cancer immunotherapy was obtained.

[0034] In this four-cycle culture, the cells obtained in the second cycle were named LLC-R-2, the cells obtained in the third cycle were named LLC-R-3, and the cells obtained in the fourth cycle were named LLC-R-4. The growth of cell nodule accumulation was observed in each cycle, and the cell nodule accumulation growth curves for each cycle are shown below. Figure 2 As shown. Figure 2 The labels "A", "B", "C", etc., in the smaller images refer to individual C57BL / 6 mice within each cycle. From Figure 2 It can be seen that the third cycle exhibits ideal conditions of rapid tumor proliferation and LLC-R-3 resistance to PD-L1 during its construction, but its stability is unknown.

[0035] It can be observed that from the first to the fourth cycle, the time for different individuals to reach the maximum tumor volume stipulated by animal ethics after drug administration was significantly shortened. At the same time, in the fourth cycle, an additional PD-1 inhibitor intervention group (3 mice were given PD-1 inhibitor 10 mg / kg intraperitoneal injection) and a PD-L1 inhibitor intervention group (6 mice were given PD-L1 inhibitor 10 mg / kg intraperitoneal injection) were set up. After being inoculated with PD-L1 inhibitor and PD-1 inhibitor in the manner of step (2), it was found that the growth rate of LLC-R-4 cell tumor volume was also significantly faster. This indicates that LLC-R-4 is significantly resistant to both PD-1 inhibitor and PD-L1 inhibitor, indicating that after 4 cycles, the acquired drug-resistant cell line LLC-R for lung cancer immunotherapy was finally obtained. In addition, Figure 2 The consistent growth trend of tumors in different individuals indicates that the drug resistance of the acquired drug-resistant cell line LLC-R in lung cancer immunotherapy is stable.

[0036] The following in vitro experiments were conducted on the acquired drug-resistant lung cancer immunotherapy cell line LLC-R obtained in Example 1.

[0037] (1) In vitro experiments The cell morphology and migration ability of LLC cells and LLC-R cells were observed and photographed under a microscope. Crystal violet staining was used to explore their colony-forming ability. Mouse spleens were obtained, and CD8 negative selection magnetic beads were used to obtain CD8. + T cells; CD8 cells were prepared at a ratio of 20:1. + T cells and LLC, CD8 + T cells and LLC-R cells were co-cultured in 96-well plates, and CD8 cells were photographed under a microscope. + The killing ability of T cells against tumor cells was assessed. Intervention was initiated with PD-L1 inhibitors at concentrations of 80, 160, 240, 320, 400, 480, 560, and 640 μg / ml. CD8+ was photographed under a microscope. + The killing ability of T cells against tumor cells was assessed by detecting cell proliferation activity using the CCK-8 assay.

[0038] The specific analyses and their results are shown below.

[0039] 1. Morphological characteristics LLC cells and LLC-R cells were seeded into 96-well plates (5000 cells / well), and after 24 hours of adhesion, their morphological differences were observed and photographed under a microscope.

[0040] Microscopic observation results of LLC cells and LLC-R as follows: Figure 3 As shown. From Figure 3LLC cells were observed to be small, round or oval cells with diverse morphologies, mainly spindle-shaped or star-shaped, with some cells extending short pseudopodia. The cells were loosely connected and distributed in a scattered manner. In contrast, LLC-R cells were predominantly elongated spindle-shaped with clear, sharp boundaries. These cells extended numerous slender protrusions, tightly connecting with neighboring cells to form an interwoven network structure. The cells connected in sheets, forming a dense monolayer resembling "paving stones," exhibiting a swirling or concentric circle arrangement. This arrangement trend may endow LLC-R cells with stronger migration potential.

[0041] 2. Proliferative capacity Proliferative capacity is one of the most fundamental characteristics of cancer cells. Through an unlimited number of divisions, they eventually exceed the division limit (such as the Hayflick limit), leading to abnormal tissue proliferation. LLC cells and LLC-R cells were seeded into 96-well plates (1000 cells / well), and proliferation was detected using the CCK-8 assay after 24h, 48h, 72h, 96h, 120h, and 144h of culture. The results of the proliferation capacity assay are shown below. Figure 4 As shown.

[0042] from Figure 4 It can be seen that compared with LLC cells, the greater the increase in OD value of LLC-R, the stronger its cell proliferation activity is. Furthermore, LLC-R can reach its maximum tumor volume after 144 hours of culture after inoculation, indicating that LLC-R exhibits stronger proliferation capacity over time.

[0043] 3. Cloning ability Tumor clonogenic capacity refers to the ability of a single tumor cell to proliferate independently in vitro or in vivo and form cell colonies (clones). It reflects the self-renewal potential and tumorigenicity of tumor cells and is one of the core indicators for assessing the malignancy and metastatic potential of tumors.

[0044] LLC cells and LLC-R cells were seeded into 12-well plates (800 cells / well), and the medium was completely changed every 3 days. After 14 days, the cells were fixed with pre-cooled 4% paraformaldehyde and stained with crystal violet. After drying, the cells were observed and photographed. The staining results are shown below. Figure 5 As shown, from Figure 5 It can be seen that the staining area of ​​LLC-R is larger than that of LLC cells, indicating that LLC-R has a stronger clonogenic ability compared to LLC cells.

[0045] 4. Transferability Tumor migratory capacity is one of the core biological characteristics of malignant tumors, referring to the ability of cancer cells to penetrate tissue barriers, invade the circulatory system, and eventually colonize distant organs after detaching from the primary tumor. This ability is a major cause of death in cancer patients (approximately 90% of cancer deaths are related to metastasis).

[0046] LLC cells and LLC-R cells were seeded into 6-well plates (400,000 cells / well) and cultured for 24 hours. After adhesion, scratches were created in the 6-well plates using a 200 μL pipette tip. After washing with sterile PBS, the cells were observed and photographed under a microscope. After adding suitable culture medium, the cells were cultured overnight for 24 hours. After washing with sterile PBS, the cells were observed and photographed under a microscope.

[0047] Cell migration patterns of LLC cells and LLC-R cells, such as Figure 6 As shown in (A), the wound width of LLC-R cells was significantly smaller than that of LLC cells after 24 hours. The percentage of wound area for LLC cells and LLC-R cells is shown in Figure 1. Figure 6 As shown in (B), LLC-R cells exhibit stronger migration ability.

[0048] 5. CD8 + After co-culturing T cells with LLC or LLC-R cells, IC50 treated with aPD-L1 and aPD-1 50 Measurement IC 50 This is defined as the "half-maximal inhibitory concentration." This invention further investigated the IC50 of aPD-L1 and aPD-1 on LLC-R in vitro. 50 First, mouse spleens were obtained, and high-purity CD8+ was extracted from the spleens using magnetic bead sorting. + T cells ( Figure 7 (A) was then co-cultured with LLC cells and LLC-R cells at a ratio of 20:1 in 96-well plates and observed under a microscope after 48 hours of culture. Subsequently, the cells were treated with aPD-L1 (80, 160, 240, 320, 400, 480, 560, 640 μg / ml) and aPD-1 (100, 200, 300, 400, 500, 600, 700 μg / ml), and finally, the changes in cell proliferation activity were detected using the CCK-8 assay.

[0049] Microscopic observation results as follows Figure 7 As shown in (B). Microscopic observation revealed that after 48 hours of co-culturing, CD8... +T cells significantly attacked LLC cells, causing loss of cell integrity, but had no inhibitory effect on LLC-R cells. After the additional addition of a PD-L1 inhibitor, the IC50 of LLC cells decreased. 50 The concentration was 310.6 μg / mL, while the IC50 of LLC-R was... 50 The concentration was 1344 μg / mL, and its IC50 was... 50 It was 4.32 times that of LLC cells; after adding a PD-L1 inhibitor, the IC50 of LLC cells was... 50 The concentration was 467.2 μg / mL, and the IC50 of LLC-R was... 50 The concentration was 1820 μg / mL, and its IC50 was... 50 This is 3.89 times that of LLC cells. These results indicate that LLC-R cells have significantly reduced sensitivity to aPD-L1 and aPD-1, suggesting that the LLC-R cell line has developed significant resistance to aPD-L1 and aPD-1, which also provides a basis for the successful construction of immunotherapy resistance models.

[0050] Example 2 This embodiment provides a method for constructing a mouse model of acquired resistance to PD-1 inhibitors and / or PD-L1 inhibitors in lung cancer, including the following steps: 1×10 6 The lung cancer immunotherapy acquired drug-resistant cell line obtained in Example 1 was injected into the right axilla of a mouse.

[0051] The following in vivo experiments verified the successful construction of the acquired drug-resistant mouse model.

[0052] Six mice were selected from each group and injected with 1×10⁻⁶ mol / L of the solution into the right axilla of each mouse according to step (2) of Example 1. 6 LLC cells or LLC-R cells were administered intraperitoneally twice weekly with a PD-L1 inhibitor at 10 mg / kg per injection. Tumor volume was measured every 3 days until the largest tumor approached the maximum tumor volume stipulated by animal ethics, at which point the mice were euthanized. Tumor tissue was obtained from the mice. A portion was used for flow cytometry to investigate changes in relevant immune cells within the tumor. The remainder was used for subsequent qRT-PCR and Western blotting.

[0053] 1. PD-L1 molecular detection The expression of programmed death ligand 1 (PD-L1) on the surface of tumor cells is one of the core mechanisms of tumor immune escape, and its detection results are of great guiding significance for predicting the efficacy of immunotherapy, selecting treatment regimens, and assessing prognosis. After PD-L1 binds to PD-1 on the surface of T cells, it can inhibit T cell activation and subsequently lead to T cell exhaustion or apoptosis, allowing tumor cells to evade immune clearance. Western blotting assays were used to detect the expression level of PD-L1 in LLC and LLC-R cells or tumor tissues. The results are as follows... Figure 8 As shown.

[0054] from Figure 8 It can be seen that the expression of PD-L1 protein in LLC-R cells is significantly decreased compared with LLC cells, while PD-L1 is highly expressed in LLC-R tumor tissue compared with LLC tumor tissue, which suggests an alteration in the tumor microenvironment.

[0055] 2. Validation of a mouse model of acquired drug resistance to lung cancer immunotherapy A mouse subcutaneous xenograft model was established by subcutaneously inoculating LLC and LLC-R cells into C57BL / 6 mice using in vivo experimental methods. Two groups were designated: a Con group and an aPD-L1 group. The aPD-L1 group received PD-L1 inhibitors every 3 days, while the Con group served as a control group without any drug treatment.

[0056] Tumor size, tumor volume changes, and tumor weight in LLC cell and LLC-R cell mouse subcutaneous xenograft models are shown in the following figures. Figure 9 As shown, PD-L1 inhibitor intervention in LLC xenograft mice significantly reduced tumor size, volume, and weight, while PD-L1 inhibitor intervention in LLC-R xenograft mice had no significant inhibitory effect on tumor size, volume, and weight. This suggests that LLC-R xenografts are highly resistant to PD-L1 inhibitor intervention and proliferate at a faster rate. These results indicate the successful establishment of an acquired resistance model for lung cancer immunotherapy.

[0057] Tumor microenvironment component detection Flow cytometry analysis was performed on macrophages (TAMs), T cells, and dendritic cells (DCs) in LLC tumor tissue and LLC-R tumor tissue obtained from a mouse model of acquired resistance to immunotherapy for lung cancer.

[0058] (1) Macrophages (TAMs) TAMs are macrophages that infiltrate tumor tissue. They are derived from blood monocytes and are the most abundant type of immune cells in the tumor microenvironment.

[0059] Flow cytometry analysis results of TAMs are as follows: Figure 10 As shown. From Figure 10 (A) It can be seen that compared to LLC tumor tissue, LLC-R tumor tissue contains a large number of TAMs, indicating significant macrophage infiltration in the tumor microenvironment, which is a marker of poor prognosis. From Figure 10 (B) It can be seen that the proportion of M1 macrophages is higher in the tumor tissue of LLC cells, while the proportion of M2 macrophages is higher in the tumor tissue of LLC-R. This indicates that the immune microenvironment in LLC-R tumors is dominated by M2 macrophages, suggesting that the tumor microenvironment presents a strong immunosuppressive state, which is conducive to tumor cells escaping the attack of the immune system.

[0060] (2) T cells T cells (T lymphocytes) are the core executors of adaptive immunity. They originate from hematopoietic stem cells in the bone marrow, differentiate and mature in the thymus, and exert their immune function by specifically recognizing and attacking pathogen-infected cells, cancer cells, and other abnormal cells. CD8+ is one such cell type. + T cells can directly kill tumor cells, while Treg cells utilize CD4+. + CD25 + It suppresses the immune response and maintains its own tolerance.

[0061] CD8 + Flow cytometry analysis results of T cells and Treg cells are as follows: Figure 11 As shown. From Figure 11 It can be seen that, compared with LLC cell tumor tissue, LLC-R tumor tissue has higher CD8 content. + The proportion of T cells was significantly reduced, while the proportion of Treg cells was significantly increased, suggesting that LLC-R tumor tissue contains fewer CD8 cells. + Infiltration of T cells and a greater number of Treg cells leads to immunosuppression. In addition, the extensive infiltration of TAMs makes LLC-R tumors even less responsive to immunotherapy.

[0062] (3) Dendritic cells Mature dendritic cells (DCs) are the most potent professional antigen-presenting cells (APCs), characterized by their ability to activate naïve T cells and exert cytotoxic effects. However, in their immature state, they possess an extremely strong antigen-phagocytic capacity, which may prevent tumor antigens from being further recognized by T cells, thus blocking the latter's mediated killing effect.

[0063] Flow cytometry analysis results of mature dendritic cells as follows Figure 12 As shown. From Figure 12 It can be seen that the proportion of mature dendritic cells in LLC tumor tissue is significantly higher than that in LLC-R tumor tissue. The content of mature dendritic cells in LLC-R tumor tissue is significantly reduced, indicating that it is more difficult for them to present tumor antigens to T cells to exert immune function.

[0064] The above results indicate that the acquired drug-resistant lung cancer immunotherapy cell line LLC-R of the present invention exhibits faster proliferation, stronger colony formation ability, and stronger migration ability in vitro compared to parental LLC cells, and inhibits CD8. +T cells kill it. Furthermore, a mouse subcutaneous xenograft model was established using LLC-R cells. It was observed that LLC-R xenografts were highly resistant to PD-1 and PD-L1 inhibitors and exhibited faster tumor growth. Within the tumor tissue, increased infiltration of TAMs and Treg cells, as well as CD8+, was observed in various cell subpopulations. + The reduction in T cells and mature dendritic cells provides a model construction method for further exploring acquired resistance to immunotherapy in lung cancer, which is beneficial for future refined drug research.

[0065] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A lung cancer immunotherapy-acquired drug-resistant cell line, characterized in that, The cell line in question is LLC-R, a mouse Lewis lung cancer cell-derived cell line, with accession number GDMCC No. 67576.

2. The application of the lung cancer immunotherapy-acquired resistance cell line according to claim 1 in the preparation of a mouse model of lung cancer with acquired resistance to PD-1 inhibitors and / or PD-L1 inhibitors.

3. A method for constructing a mouse model of acquired resistance to PD-1 inhibitors and / or PD-L1 inhibitors in lung cancer, characterized in that, The procedure includes the following steps: inoculating the lung cancer immunotherapy-acquired drug-resistant cell line according to claim 1 into the axilla of mice.

4. The method for constructing a mouse model of acquired resistance to PD-1 inhibitors and / or PD-L1 inhibitors in lung cancer according to claim 3, characterized in that, The inoculation density of the lung cancer immunotherapy acquired drug resistance cell line was 1×10⁻⁶. 6 ~2×10 6 cells / mouse.

5. The method for constructing a mouse model of acquired resistance to PD-1 inhibitors and / or PD-L1 inhibitors in lung cancer according to claim 3, characterized in that, The mice in question were C57BL / 6 mice.

6. A mouse model of acquired resistance to PD-1 inhibitors and / or PD-L1 inhibitors in lung cancer, characterized in that, It is constructed by the construction method of any one of claims 3-5.

7. The method for preparing the lung cancer immunotherapy acquired drug-resistant cell line according to claim 1, characterized in that, Includes the following steps: (1) Lewis cells were inoculated into the armpits of 6 to 9 mice. After tumor formation, PD-L1 inhibitors were administered. The tumor volume was measured every 3 days until the largest tumor approached the maximum tumor volume stipulated by animal ethics. The mice were then sacrificed, and the tumor tissue of the mouse with the largest tumor volume was taken. The tumor tissue was digested, resuspended and cultured in DMEM complete medium to obtain tumor cells. (2) Take the tumor cells obtained in step (1) and inoculate them into the armpits of 6 to 9 mice. Treat the mice according to the administration method and sampling conditions in step (1) to obtain tumor tissue and tumor cells. (3) Take the tumor cells obtained in step (2) and repeat the experiment twice in the same way as in step (2). The resulting tumor cells are the acquired drug-resistant cell lines for lung cancer immunotherapy.

8. The method for preparing acquired drug-resistant cell lines for lung cancer immunotherapy according to claim 7, characterized in that, The PD-L1 inhibitor is administered once every 3 days, at a dose of 8-12 mg / kg each time.

9. The method for preparing acquired drug-resistant cell lines for lung cancer immunotherapy according to claim 7, characterized in that, The PD-L1 inhibitor treatment is administered via intraperitoneal injection.

10. The method for preparing acquired drug-resistant cell lines for lung cancer immunotherapy according to claim 7, characterized in that, The inoculum size of the Lewis cells was 1 × 10⁻⁶. 6 ~2×10 6 cells / mouse; the seeding amount of the tumor cells was 5 × 10⁶ cells / mouse; 6 ~7×10 6 cells / mouse.

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