Method for up-regulating immune response of non-small cell lung cancer patient after osimertinib drug resistance and application

Abstract

The invention discloses an immune response up-regulation method after osimertinib drug resistance of a non-small cell lung cancer patient and application. The immune response up-regulation method comprises the following steps: 1, culturing PC9 and HCC827 cells to construct a drug-resistant strain; 2, protein extraction; 3, carrying out MTT experiment; 4, performing a cell clone formation experiment; 5, carrying out a cell transfection experiment; 6, RNA extraction and real-time quantitative PCR experiment; 7, selecting a sample and performing immunohistochemistry; 8, performing data analysis and statistics to confirm that a drug resistance mechanism depends on activation of a PI3K / AKT pathway; and 9, inhibiting the activation of an AKT pathway by using an AKT inhibitor, and regulating and controlling the expression of Etsl and PD-L1. According to the method for up-regulating the immunoreaction of the non-small cell lung cancer patient after the osimertinib resistance and the application, the immunotherapy curative effect of the non-small cell lung cancer patient after the osimertinib resistance of EGFR driven gene mutation and the PD-L1 expression up-regulation mechanism can be explored, the expression of PD-L1 is detected after Ets1 is interfered by using an AKT inhibitor, and the immunoreaction of the non-small cell lung cancer patient after the osimertinib resistance is detected. And the progression-free lifetime of the patient can be prolonged by the immune combination therapy.

Description

Technical Field

[0001] The present invention relates to the field of medical-related technologies, and specifically to a method and application for upregulating the immune response of patients with non-small cell lung cancer after osimertinib resistance. Background Art

[0002] Lung cancer is one of the most common malignant tumors in the world, among which the main pathological type is non-small cell lung cancer (NSCLC). Despite the continuous progress in anti-tumor treatment, lung cancer is still the main cause of cancer-related deaths in the world. Most patients have a poor prognosis, with an average five-year overall survival rate of about 10% to 20%. A large number of studies have shown that mutations in certain key genes initiate tumorigenesis and give lung cancer cells an invasive phenotype. Although nearly half of NSCLC patients are accompanied by epidermal growth factor receptor (EGFR) and KRAS mutations, there are also other rare mutations BRAF (5%), ALK (3%), MET (3%) and HER2 (3%). For patients with EGFR mutations, the main treatment method is currently targeted therapy, and immunotherapy is not effective for this group of people. However, whether it is the first, second, or even third generation tyrosine kinase inhibitors (TKIs), drug resistance is inevitable. The mechanism of drug resistance mostly depends on the EGFR pathway. Other mechanisms include bypass activation pathways, such as MET amplification, HER2 amplification, RET mutation, etc. In addition, some patients' drug resistance is due to epithelial-mesenchymal transition (EMT), small cell lung cancer (SCC) conversion, etc. 14. At present, the response strategy after drug resistance is mostly upgraded to third-generation TKIs, combined chemotherapy, and combined treatment plans containing anti-angiogenic therapy after second-generation sequencing;

[0003] With the development of medicine, immunotherapy has made significant breakthroughs in the treatment of various tumors. The expression of TMB and PD-L1 is related to the long-term benefits of immune checkpoint inhibitors (ICIs) in patients with NSCLC. For patients with negative driver genes, programmed death receptor 1 (PD-1) / programmed death ligand 1 (PD-L1) checkpoint inhibitors have the opportunity to bring sustained responses to patients. However, NSCLC with positive EGFR driver genes inhibits anti-tumor immunity by activating the PDI / PD-L1 pathway. The ATLANTICI phase clinical trial pointed out that in patients with EGFR / ALK mutations in NSCLC, more than 25% of tumor cells were positive for PD-L1 expression, and the objective response rate of durvalumab as a third-line or later treatment was only 12.2%. New clinical studies have shown that some patients with EGFR mutations can benefit from immunotherapy after using TKIs. However, there is still a lack of a suitable method to upregulate the immune response of NSCLC patients after osimertinib resistance;

[0004] Therefore, we propose a method and application for upregulating the immune response after osimertinib resistance in patients with non-small cell lung cancer in order to solve the problems raised above. Summary of the invention

[0005] To solve the problems raised in the above background technology. The present invention provides a method and application for upregulating immune response after osimertinib resistance in patients with non-small cell lung cancer, which can assist in guiding NSCLC patients to undergo combined immunotherapy, so that patients who develop resistance after targeted therapy can benefit from immunotherapy, and increase the treatment options for NSCLC patients.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for upregulating the immune response of patients with non-small cell lung cancer after osimertinib resistance, the method for upregulating the immune response comprising the following steps:

[0007] Step 1: Cultivate PC9 and HCC827 cells to construct drug-resistant strains;

[0008] Step 2: protein extraction;

[0009] Step 3: MTT assay;

[0010] Step 4: Cell clone formation experiment;

[0011] Step 5: Cell transfection experiment;

[0012] Step 6: RNA extraction and real-time quantitative PCR experiment;

[0013] Step 7: Sample selection and immunohistochemistry;

[0014] Step 8: Data analysis and statistics to confirm that the drug resistance mechanism depends on the activation of the PI3K / AKT pathway;

[0015] Step nine: Use AKT inhibitors to inhibit the activation of the AKT pathway and regulate the expression of Etsl and PD-L1.

[0016] As a preferred technical solution of the present invention, the construction of drug-resistant strains in step 1 includes the following steps:

[0017] Step 1.1: PC9 and HCC827 cells were cultured in complete medium containing 10% serum, 1640 medium and 1% double antibody (penicillin-streptomycin), under the conditions of 5% CO2 and 37°C;

[0018] Step 1.2: PC9-OR and HCC827-OR osimertinib-resistant cell lines were generated from PC9 and HCC827 cell lines respectively by induction with osimertinib;

[0019] Step 1.3: Induction started with 1 nM concentration of osimertinib. Osimertinib was added on the second day after cell passaging. The solution was changed every 2-3 days until 46 months later, when the IC50 of the resistant strain reached above 1 μM, the resistant cells were considered to be successfully induced.

[0020] As a preferred technical solution of the present invention, the protein extraction in step 2 comprises the following steps:

[0021] Step 2.1: Lyse the collected cells on ice using RIPA cell lysis buffer containing cocktail, sonicate several times and centrifuge, and detect protein concentration using the BCA method:

[0022] Step 2.2: Add 5× Loading Buffer and boil for 10 min, cool to room temperature and store at 20°C;

[0023] Step 2.3: Take 20 μg of sample for Western Blot experiment, block with TBS T containing 5% skim milk powder on a shaker for 2 h, incubate with primary antibody overnight, and wash the membrane 3 times with TBST, 5 min each time;

[0024] Step 2.4: Incubate with secondary antibody at room temperature for 2 h, wash the membrane with TBST and develop using ECL chemiluminescence.

[0025] As a preferred technical solution of the present invention, the MTT experiment in step 3 comprises the following steps:

[0026] Step 3.1: Weigh 0.1 g of MTT powder, dissolve it in 20 ml of ddH2O, filter it and store it in aliquots at -20°C away from light;

[0027] Step 3.2: PC9, PC9-OR, HCC827, and HCC827-OR cells were cultured and counted, 3000-5000 cells were plated per well, and osimertinib was added on the second day at concentrations of 0, 0.001, 0.01, 0.1, 1, and 10 μM;

[0028] Step 3.3: After 48 hours, replace the medium with complete medium containing 10% MTT. After 2 hours, remove the MTT-containing culture medium, add DMSO, shake for several minutes, detect the absorbance at 490 nm, and calculate the 48h ICs value.

[0029] As a preferred technical solution of the present invention, the cell clone formation experiment in step 4 is specifically as follows: PC9, PC9-OR, HCC827 and HCC827-OR cells are resuspended in complete culture medium and counted, 1000 cells / well are plated in a 6-well plate, and after adherence, the culture medium containing 200nM osimertinib is replaced, the medium is replaced every 3-5 days, and the culture is carried out for 10-14 days. The cells are washed 3 times with PBS, the cells are fixed with methanol for 15 minutes, and the cells are stained with Giemsa dye for 45 minutes, and then the cells are washed with water several times until the background is clean, and the cells are photographed and preserved after drying.

[0030] As a preferred technical solution of the present invention, the cell transfection experiment in step 5 is specifically to culture the cells to the logarithmic growth phase, wait until the degree of confluence reaches about 75%, replace the medium with 1640 complete culture medium without dual antibodies, and then use jetPRIMEpolyplus transfection reagent to transfect si-NC or si-Ets1, and collect the cells after 48 hours for subsequent experiments.

[0031] As a preferred technical solution of the present invention, the RNA extraction and real-time quantitative PCR experiment in step 6 includes the following steps:

[0032] Step 6.1: Extract total RNA from PC9-OR and HCC827-OR cells using TRIzol reagent;

[0033] Step 6.2: Reverse transcribe RNA into cDNA using HiScript III RT SuperMix for qPCR (+gDNAwiper) reverse transcription kit;

[0034] Step 6.3: Perform qRT-PCR using HiScript II One Step qRT-PCR SYBR Green Kit;

[0035] Step 6.4: Use 2 ΔΔCt The relative expression level of the target gene was analyzed by

[0036] As a preferred technical solution of the present invention, the selection of samples and immunohistochemistry in step 7 include the following steps:

[0037] Step 7.1: Select 5 NSCLC patients with EGFR classic mutations detected by genetic testing. After these patients have been treated with osimertinib targeted anti-tumor therapy for a period of time, the efficacy is evaluated according to the Recist 1.1 standard to determine disease progression;

[0038] Step 7.2: Select NSCLC patients who receive targeted anti-tumor therapy with osimertinib. After a period of standardized treatment, evaluate the efficacy according to the Recist 1.1 standard to determine disease progression, perform a second biopsy, and select paraffin sections before and after drug resistance for immunohistochemistry experiments;

[0039] Step 7.3: bake the paraffin sections at 65°C for 30 min, dewax with xylene and hydrate with different concentrations of ethanol, perform antigen repair with pH 9.0 EDTA antigen repair solution at high temperature and high pressure for 10 min, cool to room temperature, and wash with PBS three times, 5 min each time;

[0040] Step 7.4: Incubate with endogenous peroxidase blocker at room temperature for 30 minutes to remove endogenous peroxidase; block with 5% goat serum at room temperature for 30 minutes, incubate with primary antibody in a 4°C refrigerator overnight, incubate with secondary antibody at room temperature for 1 hour, stain with DAB (Dako) for 3 minutes, counterstain with hematoxylin for 2 minutes, and finally dehydrate and seal the slides;

[0041] Step 7.5: After drying by ventilation, observe under a microscope, take photos and count.

[0042] As a preferred technical solution of the present invention, the data analysis and statistics in step eight specifically include drawing statistical graphs using GraphPad Prism 8.0, and using SPSS 25.0 to statistically analyze the experimental results. The quantitative results are tested using Student's t test, and a P value < 0.05 is considered to be statistically significant. Each group of experiments is repeated at least 3 times.

[0043] The present invention provides the following technical solution: an application of a method for up-regulating the immune response of patients with non-small cell lung cancer after osimertinib resistance, wherein the application of the method for up-regulating the immune response assists in guiding NSCLC patients to undergo combined immune therapy.

[0044] Compared with the prior art, the beneficial effects of the present invention are: it can explore the efficacy of immunotherapy and the mechanism of upregulation of PD-L1 expression after osimertinib resistance in patients with non-small cell lung cancer with EGFR driver gene mutations; in the resistant strains, adding AKT inhibitors can interfere with Ets1 and then detect the expression of PD-L1, and interfere with the expression of Ets1 on the basis of inhibiting the AKT pathway, so that patients with osimertinib-resistant non-small cell lung cancer can switch to combined immunotherapy to prolong the patient's progression-free survival. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0046] Figure 1 Schematic diagram of AKT pathway activation in PC9-OR and HCC827-0R resistant strains of the present invention;

[0047] Figure 2 Schematic diagram of the expression of Ets1 and PD-L1 after inhibiting the activity of AKT in the PC9-OR and HCC827-0R resistant strains of the present invention. DETAILED DESCRIPTION

[0048] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] The present invention provides the following technical solution: a method for upregulating immune response in patients with non-small cell lung cancer after osimertinib resistance, comprising the following steps:

[0050] 1.1 Cell culture and construction of drug-resistant strains

[0051] Both PC9 and HCC827 cells were cultured in complete medium containing 10% serum 1640 and 1% double antibody (penicillin-streptomycin) at 5% CO2 and 37°C.

[0052] PC9-0R and HCC827-OR osimertinib-resistant cell lines were induced by osimertinib from PC9 and HCC827 cell lines, respectively.

[0053] Induction started with 1 nM concentration of osimertinib. Osimertinib was added on the second day after cell passaging, and the medium was changed every 2-3 days. After 46 months, the induction of resistant cells was considered successful when the IC50 of the resistant strain reached above 1 μM.

[0054] 1.2 Protein extraction and protein electrophoresis

[0055] The collected cells were lysed on ice using RIPA cell lysis buffer containing cocktail, ultrasonically lysed several times and then centrifuged. The protein concentration was detected by the BCA method. After adding 5× Loading Buffer, the cells were boiled for 10 minutes, cooled at room temperature and stored at 20°C.

[0056] Take 20 μg of sample for Western Blot experiment, block with TBST containing 5% skimmed milk powder on a shaker for 2 h, incubate with primary antibody overnight (P-EGFR, CST, 1:1000; EGFR, CST, 1:1000; P-AKT, CST, 1:1000 AKT, CST, 1:1000; E1, CST, 1:1000; PD-L1, Abcam, 1:1000; GAP DH, proteintech, 1:5000, B-actin, Proteintech, 1:5000), wash the membrane with TBST 3 times, 5 min each time.

[0057] The secondary antibody (CST, 1:2000) was incubated at room temperature for 2 h, and the membrane was washed with TBST and developed using ECL chemiluminescence.

[0058] 1.3 MTT assay

[0059] Weigh 0.1 g of MTT powder, dissolve MTT in 20 ml of ddH2O, filter and aliquot, and store at -20°C away from light.

[0060] PC9, PC9-OR, HCC827, and HCC827-OR cells were cultured and counted, 3000-5000 cells were plated per well, and osimertinib was added the next day at concentrations of 0, 0.001, 0.01, 0.1, 1, and 10 μM;

[0061] After 48 hours, the medium was replaced with complete medium containing 10% MTT. After 2 hours, the MTT-containing culture medium was removed, DMSO was added, and the cells were shaken for several minutes. The absorbance at 490 nm was detected, and the 48h ICo value was calculated.

[0062] 1.4 Cell clone formation experiment

[0063] PC9, PC9-OR, HCC827 and HCC827-OR cells were resuspended in complete culture medium and counted, and 1000 cells / well were plated in a 6-well plate. After adhesion, the culture medium was replaced with 200 nM osimertinib. The medium was changed every 3-5 days and cultured for 10-14 days. The cells were washed 3 times with PBS, fixed with methanol for 15 min, stained with Giemsa dye for 45 min, and then washed with water several times until the background was clean. After drying, the cells were photographed and preserved.

[0064] 1.5 Cell transfection experiment

[0065] The cells were cultured to the logarithmic growth phase, and when the confluence reached about 75%, the medium was replaced with 1640 complete medium without dual antibodies. Then, si-NC or si-Ets1 (interference sequence: 5'GGAAUUACUCACUGAUAAA-3', 3'-CCUUAAUGAGUGACUA UUU-5') was transfected using jetPRIME polyplus transfection reagent. After 48 hours, the cells were collected for subsequent experiments.

[0066] 1.6 RNA extraction and real-time quantitative PCR (qRT-PCR) experiments

[0067] Total RNA was extracted from PC9-OR and HCC827-OR cells using TRIzol reagent. RNA was reverse transcribed into cDNA using Hi Script III RTSuperMix for qPCR (+gDNA wiper) reverse transcription kit (Novagen). Subsequently, qRT-PCR was performed using HiScript II One Step qRT-PCR SYBR Green Kit (Novagen). The relative expression of the target gene was analyzed using the 24 method.

[0068] The qRT-PCR primer sequences are: Ets 1 forward: 5'-GATAGTTGTGATCGCCTCACC-3', Ets1 reverse: 5'-GTCCTCTGAGTCGAAGCTGTC-3'; PD-L1 for ward: 5'-TGGCATTTGCTGAACGCATTT-3', PD-L1 reverse: 5' TGCAGCCAGGT CTAATTGTTTT-3', β-actin forward: 5'ACGCCAACACAGTGCTGTCTG-3', β-actin reverse: 5'-GGCCGGACTCGTCATACTCC-3'.

[0069] 1.7 Selection of clinical samples and immunohistochemistry

[0070] Five NSCLC patients with classic EGFR mutations indicated by genetic testing were selected. After these patients used osimertinib for targeted anti-tumor treatment for a period of time, the efficacy was evaluated according to the Recist1.1 standard and the disease was judged to have progressed. The patients' gender, age, treatment regimen, etc. were collected to analyze the effective time of combined immunotherapy after the patients became resistant to osimertinib.

[0071] NSCLC patients who received targeted anti-tumor therapy with osimertinib were selected. After a period of standardized treatment, the efficacy was evaluated according to the Recist1.1 standard. Disease progression was judged and a second biopsy was performed. Paraffin sections before and after drug resistance were selected for immunohistochemistry experiments.

[0072] The paraffin sections were baked at 65°C for 30 min, dewaxed with xylene and hydrated with ethanol of different concentrations, and antigen repaired with pH 9.0 EDTA antigen repair solution at high temperature and high pressure for 10 min. After cooling at room temperature, they were washed three times with PBS for 5 min each time.

[0073] Next, the cells were incubated with an endogenous peroxidase blocker at room temperature for 30 min to remove endogenous peroxidase; the cells were blocked with 5% goat serum at room temperature for 30 min, incubated with primary antibodies (PD-L1 antibody, Abcam, 1:100; Ets1 antibody, Abcam, 1:100) in a 4°C refrigerator overnight, incubated with secondary antibodies (Maixin) at room temperature for 1 h, stained with DAB (Dako) for 3 min, counterstained with hematoxylin for 2 min, and finally dehydrated and sealed.

[0074] After drying by ventilation, the cells were observed under a microscope, photographed and counted.

[0075] 1.8 Data Analysis and Statistics

[0076] GraphPad Prism 8.0 was used to draw statistical graphs, and SPSS 25.0 was used to analyze the experimental results. The quantitative results were tested using Student's t test, and P values ​​< 0.05 were considered statistically significant. Each group of experiments was repeated at least 3 times.

[0077] In order to explore the drug resistance mechanism of the resistant strains, proteins of two pairs of parental strains and resistant strains were extracted for Western Blot experiments. The results showed that the expression of P-EGFR in PC9-OR and HCC827-0R resistant strains was lower than that in the parental strains, while the expression of P-AKT in the resistant strains was higher than that in the parental strains ( Figure 1 ).

[0078] Therefore, it is believed that the drug resistance mechanism of PC9-OR and HCC827-0R may depend on the activation of the PI3K / AKT pathway.

[0079] The above experimental results have found that P-AKT is activated in drug-resistant strains. Previous studies have also shown that the AKT pathway can upregulate the expression of PD-L1. In order to further explore whether the increased expression of PD-L1 and Ets1 in drug-resistant strains is due to the activation of the AKT pathway, two AKT inhibitors (Dactolisib and LY294002) were used to detect whether the expression of Ets1 and PD-L1 is affected by them.

[0080] PC9-OR and HCC827-0R drug-resistant cells were treated with 100nM Dactolisib or 50uM LY294002, respectively. After 24h, the cells were harvested and proteins were extracted for Western Blot experiments. The results showed that after the addition of Dactolisib and LY294002, the expression of P-AKT was significantly reduced compared with the control group, indicating that AKT inhibitors can effectively inhibit the activation of the AKT pathway.

[0081] In addition, we also found that the expression of Ets1 and PD-L1 was downregulated ( Figure 2 ), indicating that the AKT pathway can regulate the expression of Ets1 and PD-L1.

[0082] To explore whether NSCLC patients with EGFR mutations in the real world can benefit from immunotherapy after becoming resistant to osimertinib, five patients with lung adenocarcinoma with EGFR mutations were selected and switched to a combination therapy regimen containing immunotherapy as a follow-up treatment after progression on osimertinib (Table 1).

[0083] It was found that one patient achieved an extremely long progression-free survival (PFS) and was still able to control the disease until the statistical cutoff. The patient's PFS with second-line use of osimertinib was 25.2 months. After becoming drug-resistant, he continued to be treated with atezolizumab combined with endostatin and chemotherapy, and obtained clinical benefits for more than 17 months. CT images showed that the lesions had been significantly reduced.

[0084] The average PFS of the other four patients was 5.35 m, suggesting that in the real world, combined immunotherapy can be used as a clinical option after EGFR-TKIs resistance.

[0085] Table 1 Patients who received immunotherapy after becoming resistant to osimertinib

[0086]

[0087] Finally, it should be noted that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for upregulating immune response in patients with non-small cell lung cancer who have become resistant to osimertinib, characterized in that: The immune response upregulation method comprises the following steps: Step 1: Cultivate PC9 and HCC827 cells to construct drug-resistant strains; Step 2: protein extraction; Step 3: MTT assay; Step 4: Cell clone formation experiment; Step 5: Cell transfection experiment; Step 6: RNA extraction and real-time quantitative PCR experiment; Step 7: Sample selection and immunohistochemistry; Step 8: Data analysis and statistics to confirm that the drug resistance mechanism depends on the activation of the PI3K / AKT pathway; Step nine: Use AKT inhibitors to inhibit the activation of the AKT pathway and regulate the expression of Etsl and PD-L1.

2. The method for upregulating immune response in patients with non-small cell lung cancer after osimertinib resistance according to claim 1, characterized in that: The construction of drug-resistant strains in step 1 includes the following steps: Step 1.1: PC9 and HCC827 cells were cultured in complete medium containing 10% serum, 1640 medium and 1% double antibody (penicillin-streptomycin), under the conditions of 5% CO2 and 37°C; Step 1.2: PC9-OR and HCC827-OR osimertinib-resistant cell lines were generated from PC9 and HCC827 cell lines respectively by induction with osimertinib; Step 1.3: Induction started with 1 nM concentration of osimertinib. Osimertinib was added on the second day after cell passaging. The solution was changed every 2-3 days until 46 months later, when the IC50 of the resistant strain reached above 1 μM, the resistant cells were considered to be successfully induced.

3. The method for upregulating immune response in patients with non-small cell lung cancer after osimertinib resistance according to claim 1, characterized in that: The protein extraction in step 2 comprises the following steps: Step 2.1: Lyse the collected cells on ice using RIPA cell lysis buffer containing cocktail, sonicate for several times and centrifuge, and detect protein concentration using the BCA method: Step 2.2: Add 5× Loading Buffer and boil for 10 min, cool to room temperature and store at 20°C; Step 2.3: Take 20 μg of sample for Western Blot experiment, block with TBS T containing 5% skim milk powder on a shaker for 2 h, incubate with primary antibody overnight, and wash the membrane 3 times with TBST, 5 min each time; Step 2.4: Incubate with secondary antibody at room temperature for 2 h, wash the membrane with TBST and develop using ECL chemiluminescence.

4. The method for upregulating immune response in patients with non-small cell lung cancer after osimertinib resistance according to claim 1, characterized in that: The MTT experiment in step 3 includes the following steps: Step 3.1: Weigh 0.1 g of MTT powder, dissolve it in 20 ml of ddH2O, filter it and store it in aliquots at -20°C away from light; Step 3.2: PC9, PC9-OR, HCC827, and HCC827-OR cells were cultured and counted, 3000-5000 cells were plated per well, and osimertinib was added on the second day at concentrations of 0, 0.001, 0.01, 0.1, 1, and 10 μM; Step 3.3: After 48 hours, replace the medium with complete medium containing 10% MTT. After 2 hours, remove the MTT-containing culture medium, add DMSO, shake for several minutes, detect the absorbance at 490 nm, and calculate the 48h ICs value.

5. The method for upregulating immune response in patients with non-small cell lung cancer after osimertinib resistance according to claim 1, characterized in that: The cell clone formation experiment in step 4 is specifically as follows: PC9, PC9-OR, HCC827 and HCC827-OR cells are resuspended in complete culture medium and counted, 1000 cells / well are plated in a 6-well plate, and after adherence, the culture medium containing 200nM osimertinib is replaced, the medium is replaced every 3-5 days, and the culture is carried out for 10-14 days. The cells are washed 3 times with PBS, the cells are fixed with methanol for 15 minutes, and the cells are stained with Giemsa dye for 45 minutes, and then the cells are washed with water several times until the background is clean, and the cells are photographed and stored after drying.

6. The method for upregulating immune response in patients with non-small cell lung cancer after osimertinib resistance according to claim 1, characterized in that: The cell transfection experiment in step 5 specifically involves culturing cells to the logarithmic growth phase, and when the confluency reaches about 75%, replacing the medium with 1640 complete medium without dual antibodies, and then transfecting si-NC or si-Ets1 using jetPRIME polyplus transfection reagent. After 48 hours, the cells are collected for subsequent experiments.

7. The method for upregulating immune response in patients with non-small cell lung cancer after osimertinib resistance according to claim 1, characterized in that: The RNA extraction and real-time quantitative PCR experiment in step 6 includes the following steps: Step 6.1: Extract total RNA from PC9-OR and HCC827-OR cells using TRIzol reagent; Step 6.2: Reverse transcribe RNA into cDNA using HiScript III RT SuperMix for qPCR (+gDNAwiper) reverse transcription kit; Step 6.3: Perform qRT-PCR using HiScript II One Step qRT-PCR SYBR Green Kit; Step 6.4: Use 2 ΔΔCt The relative expression level of the target gene was analyzed by 8. The method for upregulating immune response in patients with non-small cell lung cancer after osimertinib resistance according to claim 1, characterized in that: The step 7 of selecting samples and immunohistochemistry comprises the following steps: Step 7.1: Select 5 NSCLC patients with EGFR classic mutations detected by genetic testing. After these patients have been treated with osimertinib targeted anti-tumor therapy for a period of time, the efficacy is evaluated according to the Recist 1.1 standard to determine disease progression; Step 7.2: Select NSCLC patients who receive targeted anti-tumor therapy with osimertinib. After a period of standardized treatment, evaluate the efficacy according to the Recist 1.1 standard to determine disease progression, perform a second biopsy, and select paraffin sections before and after drug resistance for immunohistochemistry experiments; Step 7.3: bake the paraffin sections at 65°C for 30 min, dewax with xylene and hydrate with different concentrations of ethanol, perform antigen repair with pH 9.0 EDTA antigen repair solution at high temperature and high pressure for 10 min, cool to room temperature, and wash with PBS three times, 5 min each time; Step 7.4: Incubate with endogenous peroxidase blocker at room temperature for 30 minutes to remove endogenous peroxidase; block with 5% goat serum at room temperature for 30 minutes, incubate with primary antibody in a 4°C refrigerator overnight, incubate with secondary antibody at room temperature for 1 hour, stain with DAB (Dako) for 3 minutes, counterstain with hematoxylin for 2 minutes, and finally dehydrate and seal the slides; Step 7.5: After drying by ventilation, observe under a microscope, take photos and count.

9. The method for upregulating immune response in patients with non-small cell lung cancer after osimertinib resistance according to claim 1, characterized in that: The data analysis and statistics in step eight specifically include drawing statistical graphs using Graph Pad Prism 8.0, and using SPSS 25.0 to statistically analyze the experimental results. The quantitative results were tested using Student's t test, and a P value < 0.05 was considered statistically significant. Each group of experiments was repeated at least 3 times.

10. An application of the method for upregulating immune response in patients with non-small cell lung cancer after osimertinib resistance as claimed in claim 1, characterized in that: The application of the immune response upregulation method assists in guiding NSCL C patients to undergo combined immune therapy.

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