ANXA6 + Use of tumor-associated fibroblasts in treatment of egfr mutation-mediated diseases

Abstract

The application belongs to the field of biomedicine, and particularly relates to application of fibroblasts in disease treatment; and specifically discloses application of ANXA6 gene in preparation of a diagnostic preparation for treating EGFR mutation-mediated diseases, wherein when overexpression of the ANXA6 gene in CAF cells is detected, the EGFR mutation-mediated diseases cannot be treated by using EGFR targeted drugs; further, the EGFR targeted drugs are epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs).

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of fibroblasts in disease treatment. Background Technology

[0002] Human epidermal growth factor receptor (EGFR, ErbB-1, or HER1) is a member of the epidermal growth factor receptor (HER) family. This family includes HER1 (erbB1, EGFR), HER2 (erbB2, NEU), HER3 (erbB3), and HER4 (erbB4). EGFR is expressed on the surface of normal epithelial cells. High or abnormal expression of EGFR is found in many solid tumor cells, including head and neck cancer, breast cancer, bladder cancer, ovarian cancer, kidney cancer, colon cancer, and non-small cell lung cancer, especially lung cancer. The EGFR mutation rate in Asian lung cancer populations can reach 50% (Seshacharyulu P et al., Expert Opinion Ther Targets, 2012; 16:15–31).

[0003] EGFR is a popular target for targeted cancer therapy. Currently discovered EGFR tyrosine kinases (EGFR TKs) can catalyze the transfer of high-energy phosphate bonds from ATP to several tyrosine residues on EGFR, causing phosphorylation and activating downstream signaling pathways such as PI3K / Akt and Ras / MAPK. This promotes cell growth, proliferation, and metabolism, while inhibiting apoptosis. When EGFR is overexpressed or mutated in cells, programmed cell death is hindered, leading to uncontrolled cell growth regulation and the acquisition of unlimited proliferation and invasion capabilities, resulting in malignant tumor cells. EGFR TKIs inhibit tumor cell growth, accelerate tumor cell apoptosis, and suppress angiogenesis, tumor cell invasion, and metastasis by competing with ATP for intracellular tyrosine kinase binding sites, preventing autophosphorylation of intracellular tyrosine residues and inhibiting EGFR tyrosine kinase activation. The basic structure of small molecule EGFR TKIs is quinazolinamine. EGFR TKIs can be divided into: 1) Reversible TKIs, mainly gefitinib, erlotinib, and lapatinib; 2) Irreversible TKIs, mainly afatinib and dacomitinib. Currently, both reversible and irreversible EGFR TKIs show significant efficacy against EGFR-mutant non-small cell lung cancer in the short term, but ultimately all fail due to acquired resistance, without significantly prolonging patient survival. Several targeted epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are currently approved for the treatment of patients with EGFR-sensitive mutations, achieving better efficacy, significantly prolonging survival, and improving quality of life. Third-generation EGFR-TKIs, represented by osimertinib, have brought patients long-term survival of over 3 years, but resistance inevitably develops eventually. Some fourth-generation EGFR-TKIs have entered clinical trials; whether they will benefit patients requires further investigation.

[0004] The human annexin A6 (ANXA6) gene consists of 26 exons, approximately 6000 bp in length, and is located on chromosome 5q32-q34. It is a highly conserved Ca2+ gene. 2+ANXA6, a membrane-dependent protein mainly found in the plasma membrane and endosomal compartments, plays multiple roles in cell development and differentiation. Besides participating in processes such as cell proliferation, differentiation, inflammation, membrane repair, and viral infection, studies have shown that ANXA6 is closely associated with various tumors, and its functions differ across different tumor types. These functions are often related to alterations in the activity of signal transduction pathways such as Ras, Ras / MAPK, and FAK / PI3K, exhibiting a "bidirectional regulatory" characteristic. In melanoma, epithelial carcinoma, breast cancer, gastric cancer, prostate cancer, and chronic myeloid leukemia, it has a tumor-suppressive effect; its downregulation may promote the development of these cancers and enhance their invasion and metastasis. Furthermore, ANXA6 can promote breast cancer cell adhesion, motility, and invasion, as well as the progression of acute lymphoblastic leukemia, lymphoma adhesion, and secretory processes in myeloma cells. Simultaneously, its expression is upregulated in cervical cancer; the mechanism of this "bidirectional regulatory" is currently unclear. Patent CN111388683B reports the application of an ANXA6 expression inhibitor in the preparation of a drug for treating lung cancer. The study found that after ANXA6 expression was inhibited, the proliferation of lung cancer cells was significantly inhibited, the colony-forming ability of lung cancer cells was inhibited, the subcutaneous tumorigenic ability of lung cancer cells was significantly reduced, and the growth rate of tumors was significantly inhibited.

[0005] A tumor is a complex structure composed of malignant tumor cells and a large number of non-tumor cells, which interact to form the tumor microenvironment (TME). The TME mainly consists of blood vessels surrounding the tumor, the extracellular matrix (ECM), and other non-tumor cells (including fibroblasts, adipocytes, vascular endothelial cells, and immune cells such as T lymphocytes, B lymphocytes, NK cells, and tumor-associated macrophages), as well as cytokines and exosomes secreted by these cells. Among these, cancer-associated fibroblasts (CAFs) are an important cellular component of the TME, with diverse origins, derived from intrinsic fibroblasts, bone marrow mesenchymal stem cells and hematopoietic stem cells, adipose stem cells, endothelial cells, and hepatocytes and stellate cells. CAFs are the most abundant cell type in the TME, exhibiting strong heterogeneity in their origin, function, phenotype, and biomarkers. Most literature reports that CAFs can promote tumor development and progression, but a small number of researchers have found that CAFs also play a role in inhibiting tumors. For example, Brechbuhl HM et al. found that CD146-positive CAFs in breast cancer enhance the tamoxifen sensitivity of breast cancer cells, and Patel et al. found that CAFs with low α-SMA expression in oral cancer can inhibit the self-renewal of stem cell-like tumor cells through bone morphogenetic protein 4 (BMP4). Summary of the Invention

[0006] Based on the research on human annexin A6 gene and tumor-associated fibroblasts (CAF) in this application, it was found that overexpression of human annexin A6 gene in CAF cells can promote the resistance of EGFR-mutant lung adenocarcinoma cells to targeted drugs, while decreased ANXA6 expression in CAF cells significantly reverses the resistance of EGFR-mutant lung adenocarcinoma cells to targeted drugs. Based on this, the present invention was completed.

[0007] In a first aspect, the present invention provides an application of the ANXA6 gene in the preparation of diagnostic agents for treating EGFR mutation-mediated diseases, wherein when overexpression of the ANXA6 gene is detected in CAF cells, EGFR mutation-mediated diseases cannot be treated with EGFR-targeted drugs.

[0008] Furthermore, the EGFR-targeting drug is an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKIs).

[0009] Furthermore, the EGFR-TKIs include, but are not limited to, gefitinib, erlotinib, icotinib, afatinib, dacomitinib, osimertinib (AZD9291), or vometinib.

[0010] Furthermore, the diseases mediated by the EGFR mutation include cancer.

[0011] Furthermore, the cancers mentioned include, but are not limited to, glioblastoma, head and neck cancer, pancreatic cancer, lung cancer, cancers of the nervous system, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, kidney cancer, retinal cancer, skin cancer, liver cancer, genitourinary cancer, and bladder cancer.

[0012] Furthermore, EGFR-mediated cancers are preferably lung adenocarcinoma, lung squamous cell carcinoma, or non-small cell lung cancer.

[0013] Secondly, the present invention provides a method for screening lung adenocarcinoma patients who are not suitable for EGFR-targeted therapy, the method comprising the following steps:

[0014] S1. Collect tumor tissue from the subject;

[0015] S2. Isolate CAF cells from tumor tissue;

[0016] S3. Detection of ANXA6 gene expression level in CAF cells.

[0017] S4. Population determination: The population is determined as not applicable based on whether there is overexpression of the ANXA6 gene in CAF cells.

[0018] Furthermore, the population that is not suitable for EGFR-targeted therapy refers to those with overexpression of the ANXA6 gene in CAF cells.

[0019] Furthermore, the EGFR-targeted therapy includes, but is not limited to, drug treatment with gefitinib, erlotinib, icotinib, afatinib, dacomitinib, osimertinib (AZD9291), or vormetinib.

[0020] Thirdly, the present invention provides a formulation composition comprising an inhibitor that inhibits the expression of the ANXA6 gene in CAF cells, an EGFR-targeting agent, and pharmaceutically acceptable excipients.

[0021] Furthermore, the inhibitor also contains other active ingredients, which are active ingredients that enhance the expression of the ANXA6 gene in CAF cells and do not affect the efficacy of the EGFR-targeting agent.

[0022] Furthermore, the active ingredient may be an active ingredient that enhances the expression of the ANXA6 gene in CAF cells and also an active ingredient that improves the efficacy of EGFR-targeting agents.

[0023] Fourthly, the present invention provides the use of a formulation composition in the preparation of a medicament for the targeted treatment of EGFR mutation-mediated diseases.

[0024] Furthermore, the formulation composition contains an inhibitor that inhibits the expression of the ANXA6 gene in CAF cells, an EGFR-targeting agent, and pharmaceutically acceptable excipients.

[0025] Furthermore, the inhibitor also contains other active ingredients, which are active ingredients that enhance the expression of the ANXA6 gene in CAF cells and do not affect the efficacy of the EGFR-targeting agent.

[0026] Furthermore, the active ingredient may be an active ingredient that enhances the expression of the ANXA6 gene in CAF cells and also an active ingredient that improves the efficacy of EGFR-targeting agents.

[0027] Furthermore, the diseases mediated by the EGFR mutation include, but are not limited to, glioblastoma, head and neck cancer, pancreatic cancer, lung cancer, cancers of the nervous system, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, kidney cancer, retinal cancer, skin cancer, liver cancer, genitourinary cancer, and bladder cancer.

[0028] Furthermore, EGFR-mediated cancers are preferably lung adenocarcinoma, lung squamous cell carcinoma, or non-small cell lung cancer.

[0029] Furthermore, the EGFR-targeting agent is an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKIs).

[0030] Furthermore, the EGFR-TKIs include, but are not limited to, gefitinib, erlotinib, icotinib, afatinib, dacomitinib, osimertinib (AZD9291), or vometinib. Attached Figure Description

[0031] Figure 1 For ANXA6 + Sensitivity of lung cancer cells to osimertinib after treatment with CAF supernatant; A: ANXA6 + HCC827 cells were treated with CAF supernatant for 48 hours to assess the response of HCC827 cells to osimertinib; B: ANXA6 + HCC827 cells were treated with CAF supernatant for 72 hours to assess the response of HCC827 cells to osimertinib; C: ANXA6 +HCC827 cells were treated with CAF supernatant for 96 hours to assess the response of HCC827 cells to osimertinib; D: ANXA6 + PC-9 cells were treated with CAF supernatant for 48 hours to assess the response of PC-9 cells to osimertinib.

[0032] E: ANXA6 + PC-9 cells were treated with CAF supernatant for 72 hours to assess the response of PC-9 cells to osimertinib.

[0033] F: ANXA6 + PC-9 cells were treated with CAF supernatant for 96 hours to assess the response of PC-9 cells to osimertinib.

[0034] G: ANXA6 + H1975 cells were treated with CAF supernatant for 48 hours to assess the response of H1975 cells to osimertinib.

[0035] H: ANXA6 + H1975 cells were treated with CAF supernatant for 72 hours to assess the response of H1975 cells to osimertinib.

[0036] I: ANXA6 + H1975 cells were treated with CAF supernatant for 96 hours to assess the response of H1975 cells to osimertinib.

[0037] Figure 2 Sensitivity analysis of lung cancer cells to osimertinib after treatment with CAF supernatant containing ANXA6 knockdown;

[0038] A: The response of HCC827 cells to osimertinib after 96 hours of treatment with CAF supernatant containing ANXA6 knockdown.

[0039] B: PC-9 cells treated with CAF supernatant with ANXA6 knockdown for 96 hours, and the response of PC-9 cells to osimertinib; C: H1975 cells treated with CAF supernatant with ANXA6 knockdown for 96 hours, and the response of H1975 cells to osimertinib.

[0040] Figure 3 For ANXA6 + CAF affects the response of EGFR-mutant lung adenocarcinoma cells to vormetinib.

[0041] Figure 4 For ANXA6 - CAF affects the response of EGFR-mutant lung adenocarcinoma cells to vormetinib.

[0042] Figure 5 For ANXA6+ The long-term effect of CAF on the response of metastatic lesions formed by EGFR-mutant lung adenocarcinoma cells to vortexinib. Detailed Implementation

[0043] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0044] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0045] Example

[0046] In all examples, the cell culture conditions were: 37°C, 5% CO2 static culture.

[0047] The basal culture media used in the examples were: 10% RPMI-1640 (take serum-free RPMI-1640 medium, add fetal bovine serum to a concentration of 10%, add penicillin to a concentration of 1%, add streptomycin to a concentration of 1%) and 10% DMEM (take serum-free DMEM medium, add fetal bovine serum to a concentration of 10%, add penicillin to a concentration of 1%, add streptomycin to a concentration of 1%).

[0048] Example 1 ANXA6 + Drug resistance in EGFR-mutated lung adenocarcinoma cells of CAF

[0049] 1. ANXA6 + CAF cells were placed in DMEM medium and incubated at 37°C in a 5% CO2 incubator. The cell culture supernatant was then collected.

[0050] 2. HCC827, PC-9 and H1975 cells were seeded in 10% RPMI-1640 medium. Cells that reached 90% confluence were passaged, and trypsin digestion solution was added. The cells were digested at 37°C for about 5 minutes until they were completely digested into single cells. Complete medium was added and the cells were mixed by pipetting. After mixing, the cells were counted using a Muse cell counter.

[0051] 3. HCC827, PC-9 and H1975 cells were seeded in 96-well plates, with 5000 cells per well. The cells were allowed to adhere and grow for 24 hours.

[0052] 4. Add ANXA6 +CAF cell culture supernatant was mixed with 10% RPMI-1640 medium at a 1:1 volume ratio to prepare osimertinib at different concentrations: 6 nM, 12 nM, 120 nM, 250 nM, 500 nM, 1200 nM, 6000 nM, 12000 nM, and 24000 nM. The supernatant in the 96-well plate was aspirated and discarded. 100 μl of osimertinib at different concentrations was added to each well and seeded into 96-well plates of HCC827 cells.

[0053] 5. Add ANXA6 + CAF cell culture supernatant was mixed with 10% RPMI-1640 medium at a 1:1 volume ratio to prepare osimertinib at different concentrations: 1 nM, 2 nM, 4 nM, 40 nM, 400 nM, 4000 nM, 6000 nM, 8000 nM, and 16000 nM. The supernatant in the 96-well plate was aspirated and discarded. 100 μl of osimertinib at different concentrations was added to each well of the 96-well plate seeded with PC-9 cells and H1975 cells.

[0054] 6. Place the 96-well plate into a cell culture incubator and measure the OD value of the cells after 48 hours, 72 hours and 96 hours of osimertinib treatment to obtain data.

[0055] 7. Draw a drug sensitivity curve of cells to osimertinib based on the data.

[0056] 8. Experimental Results:

[0057] like Figure 1 After 48 hours of treatment with osimertinib, the IC50 of HCC827 cells was 5.20 nM, and ANXA6... + The IC50 of CAF+HCC827 cells was 3469.2 nM, which was 667.15 times that of HCC827 cells; after 72 hours of osimertinib treatment, the IC50 of HCC827 cells was 5.20 nM, and ANXA6... + The IC50 of CAF+HCC827 cells was 2640.2 nM, which was 507.73 times that of HCC827 cells; after 96 hours of osimertinib treatment, the IC50 of HCC827 cells was 3.42 nM, and ANXA6... + The IC50 of CAF+HCC827 cells was 260.2 nM, which was 50.05 times that of HCC827 cells. These results suggest that ANXA6... + CAF leads to resistance in HCC827 cells to osimertinib.

[0058] In summary, compared with the three cell types themselves, ANXA6... +After treating HCC827, PC-9, and H1975 cells with CAF cell supernatant, ANXA6 + Three cell lines—HCC827, PC-9, and H1975—treated with CAF cell supernatant exhibited significant resistance to osimertinib.

[0059] Example 2: Effect of ANXA6 knockdown CAF cell supernatant on drug resistance in EGFR-mutant lung adenocarcinoma cells

[0060] 1. Using CRISPR-Cas9 technology in ANXA6 + After knocking down the ANXA6 protein gene in CAF cells, the cells were cultured in a cell culture incubator, and the cell culture supernatant was collected.

[0061] 2. HCC827, PC-9 and H1975 cells were seeded in 10% RPMI-1640 medium. Cells that reached 90% confluence were passaged, and trypsin digestion solution was added. The cells were digested at 37°C for about 5 minutes until they were completely digested into single cells. Complete medium was added and the cells were mixed by pipetting. After mixing, the cells were counted using a Muse cell counter.

[0062] 3. HCC827, PC-9 and H1975 cells were seeded in 96-well plates, with 5000 cells per well. The cells were allowed to adhere and grow for 24 hours.

[0063] 4. ANXA6 + CAF / ANXA6 - CAF (CAFANXA6sgRNA1, CAFANXA6sgRNA2, CAFANXA6sgRNA3) cell culture supernatant was mixed with 10% RPMI-1640 medium at a 1:1 volume ratio to prepare osimertinib at different concentrations: 1.5 nM, 3 nM, 30 nM, 150 nM, 300 nM, 600 nM, 1200 nM, 2500 nM, and 6000 nM. The supernatant in the 96-well plate was aspirated and discarded. 100 μl of osimertinib at each well was added to each well of the 96-well plate inoculated with HCC827 cells.

[0064] 5. Add ANXA6 + CAF / ANXA6 - CAF cell culture supernatant was mixed with 10% RPMI-1640 medium at a 1:1 volume ratio to prepare osimertinib at different concentrations: 1 nM, 2 nM, 4 nM, 40 nM, 400 nM, 4000 nM, 6000 nM, 8000 nM, and 16000 nM. The supernatant in the 96-well plate was aspirated and discarded. 100 μl of osimertinib at different concentrations was added to each well of the 96-well plate seeded with PC-9 cells and H1975 cells.

[0065] 6. Place the 96-well plate in a cell culture incubator and measure the OD value of the cells after 96 hours of treatment with osimertinib to obtain the data.

[0066] 7. Draw a drug sensitivity curve of cells to osimertinib based on the data.

[0067] like Figure 2 As shown, using ANXA6 + EGFR-mutant lung adenocarcinoma cells were treated with CAF supernatant and ANXA6 knockdown, followed by osimertinib treatment for 96 hours, and ANXA6 knockdown was achieved. + The IC50 values ​​for CAF+ HCC827 cells were 2610.2 nM, CAFANXA6sgRNA1+ HCC827 cells were 1.4 nM, CAFANXA6sgRNA2+ HCC827 cells were 1.3 nM, and CAFANXA6sgRNA3+ HCC827 cells were 3.1 nM. (ANXA6...) + The IC50 of HCC827 cells treated with CAF supernatant was 842-2007.85 times that of HCC827 cells treated with ANXA6 knockdown CAF supernatant.

[0068] ANXA6 + The IC50 values ​​for CAF+PC-9 cells were 405.2 nM, CAFANXA6sgRNA1+PC-9 cells were 16.1 nM, CAFANXA6sgRNA2+PC-9 cells were 310.2 nM, and CAFANXA6sgRNA3+PC-9 cells were 380.2 nM. (ANXA6...) + The IC50 of PC-9 cells treated with CAF supernatant was 1.07-25.17 times that of PC-9 cells treated with ANXA6 knockdown CAF supernatant.

[0069] ANXA6 + The IC50 values ​​for CAF+H1975 cells were 11021.1 nM, CAFANXA6sgRNA1+H1975 cells were 5968.2 nM, CAFANXA6sgRNA2+H1975 cells were 6498.2 nM, and CAFANXA6sgRNA3+H1975 cells were 7998.3 nM. (ANXA6...) + The IC50 of H1975 cells treated with CAF supernatant was 1.38–1.85 times that of H1975 cells treated with ANXA6 knockdown CAF supernatant.

[0070] The results indicated that treatment of HCC827, PC-9, and H1975 cells with supernatant from CAF cells with reduced ANXA6 expression significantly reversed the resistance of these three cell types to osimertinib.

[0071] Example 3 ANXA6 + Relationship between CAF and the response of EGFR-mutant lung adenocarcinoma cells to voremerinib

[0072] 1. HCC827 cells and ANXA6 + After CAF culture reaches 80% confluence, trypsin digestion solution is added, and digestion is carried out at 37°C for about 5 minutes. Then, 10% RPMI-1640 medium is added, mixed well, centrifuged, and the supernatant is discarded. The cells are resuspended in 10% RPMI-1640 medium, and counted using a Muse cell counter.

[0073] 2. Based on the counting results, dilute the cell suspension to 1×10⁻⁶. 7 / ml, one group of cell suspensions consisted of HCC827 cells, and the other group of cell suspensions consisted of HCC827+CAF cells.

[0074] 3. Prepare an appropriate number of BALB / c nude mice with an age of 4 weeks. After mixing the cell suspension, use a syringe to inoculate the two groups of cells into the armpits of the nude mice. Inject 2 million cells of cell suspension into each site, and inoculate 6 nude mice with each type of cell.

[0075] 5. After tumors have formed in the axilla of nude mice, inject luciferase intraperitoneally at a dose of 150 mg / kg. After 15 minutes, observe the tumors in the axilla of nude mice using a small animal in vivo imaging system.

[0076] 6. When the tumor size is 400±32.97mm 3 Mice were treated with vometinib by gavage at a dose of 5 mg / kg, 0.1 ml / day / mouse, for 23 days.

[0077] 7. On days 5, 9, 12, 17 and 23 after administration, luciferase was injected intraperitoneally at a dose of 150 mg / kg. Fifteen minutes later, the changes in the axillary tumors of nude mice were observed using a small animal in vivo imaging system.

[0078] 8. Experimental Results

[0079] like Figure 3 As shown, ANXA6 + CAF and HCC827 cells were inoculated together into the axilla of nude mice. When the tumor size reached 400±32.97 mm... 3 After 23 days of intragastric treatment with vometinib, the HCC827 cell xenograft lesions shrank by 94±4%, and the HCC827+ANXA6 +The CAF cell xenograft tumor lesion shrank by 56±17.16%, suggesting that ANXA6 + CAF significantly increased the resistance of HCC827 cells to vortexinib.

[0080] Example 4: Knockdown of ANXA6 in CAF (ANXA6) - CAF (catheter oxidase) affects the response of EGFR-mutant lung adenocarcinoma cells to voremerinib.

[0081] 1. PC-9 cells, ANXA6 + CAF cells and ANXA6 - After CAF cells reached 80% confluence, trypsin digestion solution was added, and the cells were digested at 37°C for about 5 minutes. Then, 10% RPMI-1640 medium was added, mixed well, centrifuged, and the supernatant was discarded. The cells were resuspended in 10% RPMI-1640 medium and counted using a Muse cell counter.

[0082] 2. Based on the counting results, dilute the cell suspension to 1×10⁻⁶. 7 / ml, one group of cell suspensions is PC-9+ANXA6 + CAF cells, another group of cell suspensions consisted of PC-9 + ANXA6 - CAF cells.

[0083] 3. Prepare an appropriate number of BALB / c nude mice with an age of 4 weeks. After mixing the cell suspension, inject the cells into the armpits of the nude mice using a syringe. Inject 2 million cells of the cell suspension into each site, and inoculate 6 nude mice with each type of cell.

[0084] 5. After tumors have formed in the axilla of nude mice, inject luciferase intraperitoneally at a dose of 150 mg / kg. After 15 minutes, observe the tumors in the axilla of nude mice using a small animal in vivo imaging system.

[0085] 6. The tumor size is 390±28.61mm. 3 Mice were treated with vometinib by gavage at a dose of 5 mg / kg, 0.1 ml / day / mouse, for 29 days.

[0086] like Figure 4 As shown, ANXA6 + CAF / ANXA6 knockdown CAF cells were inoculated together with PC-9 cells into the axilla of nude mice. Tumors were treated until the tumor size reached 390±28.61 mm. 3 After 29 days of treatment with vometinib, ANXA6 + CAF+PC-9 cell xenografts shrank by 23.6±13.3%, while ANXA6 knockdown CAF+PC-9 cell xenografts shrank by 65±9.16%, suggesting a correlation with ANXA6. +Compared with CAF, ANXA6 knockdown CAF significantly reversed the resistance of PC-9 cells to vormetinib.

[0087] Example 5 ANXA6 + The response of CAF to vortexinib in metastatic lesions of EGFR-mutant lung adenocarcinoma cells over a long period of time.

[0088] Cultured HCC827 cells and ANXA6 + CAF cells were digested into single cells using trypsin digestion solution, counted using a Muse counter, and mixed together at a 1:1 ratio. A corresponding number of 4-week-old BALB / c nude mice were prepared. After mixing the cell suspension, 2 million cells were injected into the axilla of each mouse using a syringe. Once subcutaneous tumors formed, the mice were euthanized by cervical dislocation, and the tumor masses were dissected. The tumor masses were cut into small pieces, washed three times with physiological saline, and then transferred to 10% DMEM medium and incubated in a culture flask. After the tumor cells grew to a certain number, they were digested and expanded for further culture, and named HCC827-3 cells.

[0089] Criteria for judging the shrinkage of metastatic lesions: Mice were injected intraperitoneally with luciferase substrate at a dose of 150 mg / kg. Ten minutes later, images were taken using a small animal imaging system to compare the intensity of fluorescence signals in the lesions before and after treatment and to calculate the changes in the lesions.

[0090] 1. HCC827 cells and HCC827-3 cells were seeded in 10% RPMI-1640 medium and cultured until 70% to 80% confluence.

[0091] 2. Add trypsin digestion solution and digest at 37°C for about 5 minutes, until the cells are completely digested into single cells. Add basal culture medium, mix well, centrifuge, and discard the supernatant. Resuspend in culture medium, and count cells using a Muse cell counter.

[0092] 3. Wash the cells three times with physiological saline, and resuspend the cells in physiological saline to a concentration of 1×10⁻⁶. 7 / ml.

[0093] 4. Prepare an appropriate number of NOD / SCID female mice, 4 weeks old.

[0094] 5. After the cell suspension was mixed, HCC827 / HCC827-3 cells were injected via the tail vein at a rate of 2 million per mouse, with each cell type inoculated into 5 mice.

[0095] 6. After 1.5 months, luciferase was injected intraperitoneally at a dose of 150 mg / kg. Fifteen minutes later, lung metastasis was observed in all mice using a small animal in vivo imaging system.

[0096] 7. Mice were treated with vometinib by gavage at a dose of 5 mg / kg, 0.1 ml / day / mouse.

[0097] 8. Luciferase was injected intraperitoneally at a dose of 150 mg / kg on days 10, 20, 30, 40, 51, 60, 80 and 90 after administration. The size of lung metastases in mice was observed using a small animal in vivo imaging system 15 minutes later.

[0098] To observe ANXA6 + The response of CAF to vortexinib in the formation of metastatic lesions in EGFR-mutant lung adenocarcinoma cells was investigated using long-term CAF treatment. Lung metastasis models were established using HCC827 and HCC827-3 cells, and vortexinib treatment was administered. After 90 days of treatment, metastatic lesions formed by HCC827 cells shrank by 70%-100%, and metastatic lesions formed by HCC827-3 cells shrank by 44.44%-70.83%. This indicates that ANXA6... + Long-term action of CAF on metastatic lesions formed by EGFR-mutant lung adenocarcinoma cells has shown resistance to vormetinib (see...). Figure 5 ).

Claims

1. The use of a reagent for detecting the expression of an ANXA6 gene in the preparation of a diagnostic preparation for treating EGFR mutation-mediated lung adenocarcinoma, wherein when overexpression of the ANXA6 gene in CAF cells is detected, the EGFR mutation-mediated lung adenocarcinoma cannot be treated with an EGFR-targeting drug; the EGFR-targeting drug is an epidermal growth factor receptor tyrosine kinase inhibitor, i.e., an EGFR-TKI; and the EGFR-TKI is osimertinib and foretinib.

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