An ANXA6 + Use of tumor-associated fibroblasts in lung cancer prognosis

Abstract

The present application belongs to the field of biomedicine, and particularly relates to the application of fibroblasts in lung cancer prognosis; and specifically discloses the use of ANXA6 gene in the preparation of a preparation for diagnosing bone metastasis in a subject with lung cancer in vitro and / or for predicting the tendency of forming bone metastasis in a subject with lung cancer in vitro, wherein when overexpression of the ANXA6 gene in CAF is detected, the subject is positively diagnosed with metastasis or has a greater tendency of forming metastasis.

Description

Technical Field

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

[0002] 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. However, CAFs lack specific markers for expression, making current identification methods unclear. CAFs are typically identified by jointly detecting several of the following proteins: alpha-smooth muscle actin (α-SMA), aspartic acid, collagen 11-al (COL11A1), fibroblast-activating protein (FAP), platelet-derived growth factor receptor (PDGFR)α / , fibroblast-specific protein 1 (FSP1), vimentin, and microfibrillar-associated protein 5 (MFAP5). Numerous studies have found that CAFs can secrete chemokines and growth factors such as hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMPs), fibroblast growth factor (FGF), and CXC motif chemokine ligand 12 (CXCL12).The growth factors and inflammatory factors produced by CAFs can regulate fibroblast activation in an autocrine manner, as well as regulate the behavior of tumor cells and other stromal cells in a paracrine manner, recruiting them to primary lesions or metastatic lesions, remodeling the ECM, constructing the TME, and ultimately promoting tumor cell proliferation, invasion, metastasis, and drug resistance. CAFs are the most abundant cell type in the TME, exhibiting strong heterogeneity in their origin, function, phenotype, and biomarkers. Although most literature reports that CAFs can promote tumor development, 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). Therefore, in order to accurately locate the pro-cancer CAF subsets and the anti-cancer CAF subsets, it is necessary to define these subsets through specific markers or secretions and to understand their functions and mechanisms.

[0003] 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. Summary of the Invention

[0004] This invention, in detecting tumor tissue from lung cancer patients, found that ANXA6 was expressed at low levels or not at all in lung cancer cells, but was overexpressed in CAF (catheter arterial fluid), thus identifying ANXA6. + CAF is associated with the prognosis of lung cancer. Based on this finding, this invention was completed.

[0005] In a first aspect, the present invention provides the use of the ANXA6 gene in the preparation of a formulation for the in vitro diagnosis of bone metastasis in subjects with lung cancer and / or for the in vitro prognosis of the tendency to form bone metastasis in subjects with lung cancer, wherein the use is such that when overexpression of the ANXA6 gene is detected in CAF, then the subject has a positive diagnosis of metastasis or a greater tendency to form metastasis.

[0006] Secondly, the present invention provides the use of the ANXA6 gene in the preparation of a formulation for predicting the prognostic effect of in vitro treatment of subjects with lung cancer, wherein the use is such that when overexpression of the ANXA6 gene is detected in CAF, the treatment effect of the subject is poor.

[0007] Thirdly, the present invention provides a formulation for diagnosing bone metastasis in subjects with lung cancer in vitro and / or for predicting the propensity for bone metastasis in subjects with lung cancer in vitro, the formulation containing a reagent for detecting the expression level of the ANXA6 gene in CAF cells of the subject.

[0008] Fourthly, the present invention provides a formulation for evaluating the prognostic effect of in vitro treatment of subjects with lung cancer, the formulation containing a reagent for detecting the expression level of the ANXA6 gene in CAF cells of the subject.

[0009] Fifthly, the present invention provides a method for evaluating the effectiveness of treatment for lung cancer patients, the method comprising the following steps:

[0010] S1. Collect tumor tissue from the subject;

[0011] S2. Isolate CAF cells from tumor tissue;

[0012] S3. Detection of ANXA6 gene expression level in CAF cells.

[0013] Furthermore, if the ANXA6 gene is overexpressed in CAF cells in the tissues of lung cancer patients who have received treatment, it indicates that the treatment is not effective. Attached Figure Description

[0014] Figure 1This study analyzed the expression of ANXA6 in lung cancer cells (CAF). (A: RT-PCR detection of ANXA6 mRNA expression in lung cancer tissues and normal lung tissues from 15 lung cancer patients; B: ANXA6 expression in lung cancer tissues; C: ANXA6 expression in normal lung tissues; D: CAF cultured and isolated from tumor tissues of lung cancer patients; E: Identification of biomarkers and detection of ANXA6 expression in cultured CAF.)

[0015] Figure 2 For ANXA6 + CAF promotes the malignant biological behavior of HCC827 cells;

[0016] A: Growth curves showing the proliferation of HCC827 cells and HCC827+CAF1 and HCC827+CAF2 cells; *p<0.05

[0017] B: Colony formation and gross morphology of HCC827 cells, HCC827+CAF1 cells, and HCC827+CAF2 cells;

[0018] C: Statistical graph of colony formation in HCC827 cells, HCC827+CAF1 cells, and HCC827+CAF2 cells; *p<0.05

[0019] D: Comparison of migration ability of HCC827 cells and HCC827+CAF1 and HCC827+CAF2 cells;

[0020] E: Comparison of the invasive ability of HCC827 cells and HCC827+CAF1 and HCC827+CAF2 cells;

[0021] F: Growth of subcutaneous xenografts in nude mice from HCC827 cells, CAF cells, and HCC827+CAF1 and HCC827+CAF2 cells; *p<0.05;

[0022] G: Gross image of subcutaneous xenograft tumors of HCC827 cells, CAF cells, and HCC827+CAF1 and HCC827+CAF2 cells in nude mice;

[0023] H: HE staining of subcutaneous xenograft tumor sections of HCC827 cells and HCC827+CAF1 and HCC827+CAF2 cells from nude mice (red arrow: tumor cells; black arrow: blood vessels);

[0024] I: Weight of subcutaneous xenograft tumors in nude mice containing HCC827 cells, CAF cells, and HCC827+CAF1 and HCC827+CAF2 cells; *p<0.05

[0025] Figure 3 For ANXA6 + Effects of CAF on the long-term effects of HCC827 cells.

[0026] A: Growth curves showing the proliferation of HCC827 and HCC827-3 cells; *p<0.05;

[0027] B: Comparison of migration and invasion abilities of HCC827 cells and HCC827-3 cells;

[0028] C: Growth of subcutaneous xenografts of HCC827 cells and HCC827-3 cells in nude mice; *p<0.05;

[0029] D: Comparative statistical chart of migration ability between HCC827 cells and HCC827-3 cells; *p<0.05;

[0030] E: Statistical comparison of the invasive ability of HCC827 cells and HCC827-3 cells; *p<0.05;

[0031] F: Gross image of subcutaneous xenograft tumors of HCC827 cells and HCC827-3 cells in nude mice;

[0032] G: Weight of subcutaneous xenograft tumors of HCC827 cells and HCC827-3 cells in nude mice; *p<0.05;

[0033] H: Comparison of HCC827 and HCC827-3 cell migration in NOD / SCID mice

[0034] Figure 4 The effect of altered ANXA6 expression on the malignant phenotype of HCC827 cells.

[0035] A: Verification of the effect of ANXA6 knockdown;

[0036] B: Effect of altered ANXA6 expression on HCC827 cell proliferation; *p<0.05;

[0037] C: Effects of altered ANXA6 expression on HCC827 cell migration and invasion;

[0038] D: Statistical chart comparing the effects of altered ANXA6 expression on the migration ability of HCC827 cells; *p<0.05;

[0039] E: Statistical graph comparing the effects of altered ANXA6 expression on the invasive ability of HCC827 cells; *p<0.05.

[0040] Figure 5 For ANXA6 + The impact of CAF on the survival time of lung cancer patients. Detailed Implementation

[0041] 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.

[0042] 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.

[0043] Example

[0044] 1. In all examples, the cell culture conditions were: 37°C, 5% CO2 static culture.

[0045] 2. The basal culture medium used in the examples is DMEM medium.

[0046] 3. The basal culture medium used in the examples was as follows: serum-free RPMI-1640 medium was mixed with fetal bovine serum to a concentration of 10%, penicillin to a concentration of 1%, and streptomycin to a concentration of 1%. The 0.2% BSA RPMI-1640 medium was prepared by mixing serum-free RPMI-1640 medium with bovine serum albumin (BSA) to a concentration of 0.2%, penicillin to a concentration of 1%, and streptomycin to a concentration of 1%.

[0047] 4.15 Lung Cancer Patients: Inclusion and Exclusion Criteria, Clinicopathological Parameters, and Follow-up Data

[0048] This study included patients newly diagnosed with non-small cell lung cancer at Beijing Chest Hospital, Capital Medical University. Patients with other tumors, those who had undergone any anti-tumor treatment, or those with incomplete clinicopathological or follow-up data were excluded. Cancerous and adjacent tissues were collected from the study subjects.

[0049] Example 1: Expression Analysis of ANXA6 in CAF

[0050] 1.1 RT-PCR detection of ANXA6 mRNA expression in lung cancer tissues and normal lung tissues from 15 lung cancer patients

[0051] 1. Tumor tissue and normal lung tissue were collected from 15 lung cancer patients, flash-frozen in liquid nitrogen, and stored at -80 degrees Celsius;

[0052] 2. Grind the tissue samples thoroughly with liquid nitrogen, add 1 ml of Trizol solution, mix well, and let stand at room temperature for 5 min to allow for complete lysis;

[0053] 3. Add 200 μl of chloroform, shake vigorously to mix for 30 seconds, and let stand at room temperature for 3-5 minutes.

[0054] 4. Centrifuge at 14000g for 15 min at 4℃. The RNA will be in the upper aqueous phase. Transfer it to another new RNase-free EP tube.

[0055] 5. Add an equal volume of isopropanol and mix thoroughly. Let stand at room temperature for 10 minutes.

[0056] 6. Centrifuge at 14000g for 10 min at 4℃, collect the RNA precipitate, and discard the supernatant.

[0057] 7. Wash twice with 75% ethanol (centrifuge at 12,000g for 5 min), air dry in a clean bench, and add an appropriate amount of DEPC water to dissolve the precipitate, depending on the amount of precipitate.

[0058] 8. Take 1 μl of RNA sample, dilute it 50 times, and measure the OD value on a nucleic acid protein detector. If the ratio of OD260 / OD280 is greater than 1, it indicates that the prepared RNA is relatively pure and free from protein contamination.

[0059] 9. Take 1 μl of RNA sample, perform 1% agarose gel electrophoresis at 80V for 20 min, stain with EB for 10 min, observe the bands and take pictures using a gel imaging system.

[0060] 10. Prepare solutions of RNA, dNTP, RNase inhibitor, and buffer in an RNase-free PCR tube. Incubate 20 μl of the above reaction solution at 30°C for 10 min, 42°C for 50 min, and 85°C for 10 min to complete cDNA synthesis.

[0061] 11. Vibrate evenly in a shaker or use a blower to mix evenly, then dispense 15ul into each of the 8 tubes.

[0062] 12. After diluting cDNA 1:20 with sterile pure water, the cDNA was sorted in a certain order and then subjected to RT-PCR to detect the results.

[0063] 1.2 Expression of ANXA6 in lung cancer tissues and normal lung tissues

[0064] The lung cancer tissues and normal lung tissues collected from lung cancer patients were stained using a double immunohistochemical staining method to observe their expression.

[0065] 1.4 Identification of biomarkers and detection of ANXA6 in CAF isolated and cultured from lung cancer patient tumor tissues.

[0066] 1. Collect tumor tissue from lung cancer patients and temporarily store it in DMEM culture medium.

[0067] 2. Place the tissue in a culture dish containing DMEM, cut the tissue into small pieces with scissors, and then transfer it to a centrifuge tube. Centrifuge at 1000 rpm for 5 min, discard the supernatant, add 5 ml of DMEM medium, mix well by pipetting, and centrifuge again.

[0068] 3. After centrifugation, discard the supernatant, add 3 ml of DMEM medium, mix well, transfer to a culture flask and spread it evenly to the bottom of the flask, place it in a cell culture incubator, and incubate at 37°C and 5% CO2 for 5 days.

[0069] 4. On the sixth day, CAF cells were observed adhering to the culture flask under a microscope. After changing the medium, the cells were allowed to continue growing for a period of time. CAF cells and lung tumor cells were observed to adhere to the culture flask and proliferate in large numbers.

[0070] 5. After the CAF cells have grown in large quantities, add trypsin digestion solution and digest at 37°C for about 5 minutes until the cells are completely digested into single cells. Then, add 1 ml of DMEM medium to neutralize the digestion solution, transfer to a centrifuge tube, centrifuge at 800 rpm for 5 minutes, discard the supernatant, add 3 ml of DMEM medium, transfer to a new cell culture flask, and wait for them to adhere and grow.

[0071] 6. The following day, two CAF cells were observed to adhere and grow under a microscope.

[0072] 7. Two CAF cells and lung tumor cells were seeded into 24-well plates and cultured statically at 37°C and 5% CO2 until the cell confluence reached 80%.

[0073] 8. Add the protease inhibitor to the RIPA lysis buffer and mix thoroughly.

[0074] 9. Remove the culture medium from the wells, wash the cells three times with physiological saline refrigerated at 4°C, and then discard the solution.

[0075] 10. Add 200 μl of RIPA lysis buffer to each well, then place in an ice bath for 15 minutes and vortex for 30 seconds every 5 minutes.

[0076] 11. Centrifuge at 13900 rpm and 4℃ for 15 minutes, collect the supernatant to obtain the cell protein product.

[0077] 12. Mix cell proteins and loading buffer at a ratio of 4:1 and incubate in a water bath at 100°C for 10 minutes. Place 20 μl of each sample into the well and perform electrophoresis at 80V for 30 minutes and then at 120V for 90 minutes.

[0078] 13. After electrophoresis, transfer the membrane at 65V in an ice bath for 3 hours, and then block it with 5% skim milk for 2 hours.

[0079] 14. Dilute the a-SMA, FAP-1 and ANXA6 antibodies at a ratio of 1:1000 and incubate overnight at 4°C.

[0080] 15. Wash the NC membrane three times with TBST solution for 10 minutes each time, then incubate with secondary antibody at room temperature for 4 hours, wash the NC membrane three times again, and expose it in an imaging system after using developing solution.

[0081] 16. After blocking for 30 minutes, GAPDH was diluted at a ratio of 1:4000 and incubated for 4 hours. The NC membrane was washed three times with TBST before imaging.

[0082] 1.5 Experimental Results

[0083] like Figure 1 A. According to RT-PCR results, the level of ANXA6 mRNA in lung cancer is higher than that in normal lung tissue.

[0084] like Figure 1 As shown in Figure B, ANXA6 (red portion) is mainly expressed in stromal cells in lung cancer tissue, but not in tumor cells (brown portion).

[0085] like Figure 1 As shown in Figure C, ANXA6 is not expressed in normal lung tissue.

[0086] like Figure 1 As shown in D, observe its general shape and take a picture.

[0087] like Figure 1 The results showed that CAF1 and CAF2 expressed α-SMA, FAP-1, and ANXA6, confirming that they were tumor-associated fibroblasts with high ANXA6 expression. T1 and T2 showed no expression of α-SMA, FAP-1, and ANXA6, indicating that tumor cells hardly expressed ANXA6. CAF1 and CAF2 represent tumor-associated fibroblasts cultured from two lung cancer patients, while T1 and T2 represent tumor cells cultured from two lung cancer patients.

[0088] Example 2 ANXA6 + CAF promotes malignant phenotypes in HCC827 cells.

[0089] 2.1ANXA6 + Effects of CAF on HCC827 cell growth

[0090] 1. Add trypsin digestion solution to HCC827, HCC827+CAF1 and HCC827+CAF2 cells and digest at 37°C for about 5 minutes until the cells are completely digested into single cells. Neutralize with RPMI-1640 medium and transfer to centrifuge tubes. Centrifuge at 800 rpm for 5 minutes, resuspend in basal medium, and count cells using a Muse cell counter.

[0091] 2. Dilute according to the counting results, and seed the three types of cells into five 96-well plates at a density of 5000 cells per well.

[0092] 3. At the time points of 0 hours, 24 hours, 48 ​​hours, 72 hours and 96 hours after the cells were seeded into the wells, Cell Counting Kit-8 cell counting reagent was added to the wells in proportion. After incubating in a cell culture incubator for 1 hour, the OD value at 450 nm in each well was measured using a microplate reader.

[0093] 4. After organizing the measured OD values, use GraphPad Prism 8.0 software to plot them as a curve.

[0094] 2.2ANXA6 + Effect of CAF on colony-forming ability of HCC827 cells

[0095] 1. Add trypsin digestion solution to HCC827, HCC827+CAF1 and HCC827+CAF2 cells and digest at 37°C for about 5 minutes until the cells are completely digested into single cells. Neutralize with RPMI-1640 medium and transfer to centrifuge tubes. Centrifuge at 800 rpm for 5 minutes, resuspend in basal medium, and count cells using a Muse cell counter.

[0096] 2. Dilute according to the counting results, and finally dilute to 500 cells per 2 ml. After mixing, seed the cells into a 6-well plate and incubate at 37°C and 5% CO2. Observe after 7 to 9 days.

[0097] 3. Observe the cells in the well plate under a microscope. When visible colonies appear and the colonies do not merge with each other, stop the culture.

[0098] 4. Remove the culture medium from the well, wash the cells three times with physiological saline, add 1 ml of gentian violet staining solution containing 2% (v / v) methanol, and stain for 30 minutes.

[0099] 5. Rinse slowly with tap water to remove the gentian violet dye, then air dry and take photos.

[0100] 2.3ANXA6 + Effects of CAF on the migration and invasion abilities of HCC827 cells

[0101] 1. Replace the culture medium of HCC827, HCC827+CAF1, and HCC827+CAF2 cells with 0.2% BSA RPMI-1640 medium and incubate for 24 hours; CAF1 and CAF2 represent tumor-associated fibroblasts cultured from two patients.

[0102] 2. Add 600 ml of RPMI-1640 medium to the lower chamber of the Transwell plate and 100 μl of 0.2% BSARPMI-1640 medium to the upper chamber. Place the Transwell plate in a cell culture incubator and hydrate for 30 min.

[0103] 3. Add trypsin digestion solution to HCC827, HCC827+CAF1 and HCC827+CAF2 cells, digest at 37°C for about 5 minutes until the cells are completely digested into single cells. Neutralize with RPMI-1640 medium and transfer to centrifuge tubes. Centrifuge at 800 rpm for 5 minutes, resuspend in basal medium, and count cells using a Muse cell counter.

[0104] 4. Based on the counting results, the three types of cells were resuspended in 0.2% BSARPMI-1640. The cell concentration without base gel was 800,000 / ml, and the cell concentration with base gel was 2,000,000 / ml. The ratio of base gel to cell suspension was 1:5.

[0105] 5. Aspirate 100 μl of hydrated 0.2% BSA1640 medium from the Transwell chamber and add 100 μl of the above cell suspension to each well. Incubate at 37°C with 5% CO2.

[0106] 6. After 24 hours, remove the unmixed chambers, wash them twice with physiological saline, and then place them in gentian violet solution. After 30 minutes, wash off the gentian violet and observe the cells that have crossed the membrane and grown under a Leica microscope in bright field.

[0107] 7. After 48 hours, process the mixed gel chambers in the same way and observe the cells that have passed through the membrane and grown under bright field under a Leica microscope.

[0108] 2.3ANXA6 + Effects of CAF on subcutaneous tumorigenesis of HCC827 cells in nude mice

[0109] 1. After culturing HCC827, CAF1, and CAF2 cells to 80%-90% confluence, trypsin digestion solution was added, and the cells were digested at 37°C for about 5 minutes. RPMI-1640 medium was then added, mixed, and centrifuged, discarding the supernatant. The cells were resuspended in RPMI-1640 medium, and counted using a Muse cell counter (CAF1 and CAF2 cells were both ANXA6 overexpressing cells).

[0110] 2. Based on the cell count results, five cell suspensions were prepared: HCC827, CAF1, CAF2, HCC827+CAF1, and HCC827+CAF2, with the HCC827 cell concentration being 1.5 × 10⁻⁶. 7 / ml, CAF1 and CAF2 cell concentrations were 0.5×10⁻⁶. 7 / ml, the concentrations of HCC827+CAF1 and HCC827+CAF2 cells were 2×10⁶. 7 / ml (including 1.5 × 10 HCC827 cells) 7 / ml and 0.5×10⁶ CAF1 or CAF2 cells 7 / ml)

[0111] 3. Prepare an appropriate number of BALB / c nude mice aged 4-6 weeks. After mixing the cell suspension, inject 100 μl of cell suspension into the armpit of the nude mice using a syringe. Inoculate 5-6 nude mice with each type of cell.

[0112] 5. Observe the tumor formation in the armpits of nude mice every 3 or 4 days and measure the size of the tumor.

[0113] 6. When the axillary tumor is close to 1000 mm in size 3 At that time, the mice were euthanized by cervical dislocation, and the tumor was removed, photographed, and weighed.

[0114] 7. Data analysis based on tumor growth volume and weight.

[0115] 8. Slices of tumor fragments from each group of nude mice were stained with hematoxylin and eosin (HE).

[0116] 2.4 Experimental Results

[0117] like Figure 2 A. The proliferation capacity of HCC827+CAF1+ and HCC827+CAF2 cells was stronger than that of HCC827 cells, and the difference was statistically significant (P<0.05), indicating that ANXA6 + CAF can promote the proliferation of HCC827 cells.

[0118] like Figure 2As shown in B, the number of colonies of HCC827+CAF1 and HCC827+CAF2 cells was greater than that of HCC827 cells, and the gross morphology of HCC827+CAF1 and HCC827+CAF2 cells was altered to some extent, with more obvious cell atypia and more dispersed growth.

[0119] like Figure 2 C. The number of cell colonies was counted and analyzed. The number of colonies of HCC827+CAF1 and HCC827+CAF2 cells was greater than that of HCC827 cells, and the difference was statistically significant (P<0.05), indicating that ANXA6 + CAF has a certain effect on the morphological changes of HCC827 cells and can promote the proliferation of HCC827 cells.

[0120] like Figure 2 In chambers D and E, both mixed and non-mixed gels showed that HCC827+CAF1 and HCC827+CAF2 cells crossed the membrane more frequently than HCC827 cells. Therefore, the migration and invasion abilities of HCC827+CAF1 and HCC827+CAF2 cells were stronger than those of HCC827 cells. (ANXA6) + CAF can promote the migration and invasion of HCC827 cells.

[0121] like Figure 2 H, HE staining showed ANXA6 + CAF promotes intratumoral angiogenesis and invasive growth of HCC827 cell transplantation, suggesting the presence of ANXA6. + CAF cells promote the malignant biological behavior of HCC827 cells.

[0122] like Figure 2 G shows that, within the same growth period, mice inoculated with CAF1 and CAF2 cells did not develop subcutaneous tumors, while mice inoculated with HCC827, HCC827+CAF1, and HCC827+CAF2 cells developed subcutaneous tumors.

[0123] like Figure 2 In mice inoculated with HCC827+CAF1 and HCC827+CAF2 cells, the subcutaneous tumor growth capacity and tumor weight were both stronger than those in mice inoculated with HCC827 cells.

[0124] This demonstrates that HCC827+CAF1 and HCC827+CAF2 cells exhibit stronger subcutaneous tumorigenicity than HCC827 cells, and the difference is statistically significant (P<0.05), indicating that ANXA6... + CAF can promote the subcutaneous tumorigenicity of HCC827 cells.

[0125] Example 3 ANXA6+ Effects of CAF on Long-Term Malignant Phenotype of HCC827 Cells

[0126] Cultured HCC827 cells and CAF cells (CAF cells are ANXA6 overexpressing 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 4-6 week old BALB / c nude mice were prepared, and after mixing the cell suspension, 2 million cells were injected into the axilla of each mouse using a syringe. After subcutaneous tumor formation, the mice were euthanized by cervical dislocation, and the tumor mass was dissected. The tumor mass was cut into small pieces with scissors, washed three times with physiological saline, added to DMEM medium, and transferred to a culture flask. The flask was then incubated for 5 days. After 5 days, tumor cells were observed adhering to the bottom of the culture flask under a microscope. These cells were digested, cultured, and named HCC827-3 cells.

[0127] 3.1 Growth curves of HCC827 and HCC827-3 cells

[0128] 1. Add trypsin digestion solution to HCC827 and HCC827-3 cells, digest at 37°C for about 5 minutes until the cells are completely digested into single cells. Neutralize with RPMI-1640 medium and transfer to centrifuge tubes. Centrifuge at 800 rpm for 5 minutes, resuspend in basal medium, and count cells using a Muse cell counter.

[0129] 2. Dilute according to the counting results, and seed the two types of cells into five 96-well plates at a density of 5000 cells per well.

[0130] 3. At the time points of 0 hours, 24 hours, 48 ​​hours, 72 hours and 96 hours after the cells were seeded into the wells, Cell Counting Kit-8 cell counting reagent was added to the wells in proportion. After incubating in a cell culture incubator for 1 hour, the OD value at 450 nm in each well was measured using a microplate reader.

[0131] 4. After organizing the measured OD values, use GraphPad Prism 8.0 software to plot them as a curve.

[0132] 3.2 Comparison of migration and invasion abilities between HCC827 and HCC827-3 cells

[0133] 1. Replace the culture medium of HCC827 cells and HCC827-3 cells with 0.2% BSARPMI-1640 medium and incubate for 24 hours.

[0134] 2. Add 600 ml of RPMI-1640 medium to the lower chamber of the Transwell plate and 100 μl of 0.2% BSARPMI-1640 medium to the upper chamber. Place the Transwell plate in a cell culture incubator and hydrate for 30 min.

[0135] 3. Add trypsin digestion solution to HCC827 cells and HCC827-3 cells, digest at 37°C for about 5 minutes until the cells are completely digested into single cells. Neutralize with RPMI-1640 medium and transfer to centrifuge tubes. Centrifuge at 800 rpm for 5 minutes, resuspend in basal medium, and count cells using a Muse cell counter.

[0136] 4. Based on the counting results, resuspend both types of cells in 0.2% BSARPMI-1640. The cell concentration without base gel was 800,000 / ml, and the cell concentration with base gel was 2,000,000 / ml. The ratio of base gel to cell suspension was 1:5.

[0137] 5. Aspirate 100 μl of hydrated 0.2% BSA1640 medium from the Transwell chamber and add 100 μl of the above cell suspension to each well. Incubate at 37°C with 5% CO2.

[0138] 6. After 24 hours, remove the unmixed chambers, wash them twice with physiological saline, and then place them in gentian violet solution. After 30 minutes, wash off the gentian violet and observe the cells that have crossed the membrane and grown.

[0139] 7. After 48 hours, process the mixed gel chambers in the same way and observe the cells that have crossed the membrane and grown.

[0140] 3.3 Growth of HCC827 and HCC827-3 cell subcutaneous xenografts in nude mice

[0141] 1. After culturing HCC827 and HCC827-3 cells to 80% to 90% confluence, add trypsin digestion solution and digest at 37°C for about 5 minutes. Add RPMI-1640 medium, mix well, centrifuge, and discard the supernatant. Resuspend in RPMI-1640 medium, and count cells using a Muse cell counter.

[0142] 2. Based on the counting results, dilute the cell suspension to 1×10⁻⁶. 7 / ml cells.

[0143] 3. Prepare an appropriate number of BALB / c nude mice aged 4-6 weeks. After mixing the cell suspension, inject the cells into the armpits of the nude mice using a syringe. Inject 2 million cells of cell suspension into each site, and inoculate 5-6 nude mice with each type of cell.

[0144] 4. Observe the tumor formation in the armpits of nude mice every 3 or 4 days and measure the size of the tumor.

[0145] 5. When the axillary tumor is close to 1000 mm in size 3 At that time, the mice were euthanized by cervical dislocation, and the tumor was removed, photographed, and weighed.

[0146] 3.4 Comparison of the metastatic ability of HCC827 cells and HCC827-3 cells

[0147] 1. Culture HCC827 cells and HCC827-3 cells to 80% to 90% confluence.

[0148] 2. Add trypsin digestion solution to the cells and digest at 37°C for about 5 minutes. Add RPMI-1640 medium to neutralize, mix well, centrifuge, and discard the supernatant. Resuspend in RPMI-1640 medium and count cells using a Muse cell counter.

[0149] 3. Wash the cells three times with physiological saline, dilute according to the counting results, and finally dilute with physiological saline to 1×10⁻⁶. 7 / ml cells.

[0150] 4. Prepare an appropriate number of NOD / SCID mice aged 4-6 weeks. After mixing the cell suspension, inject HCC827 cells and HCC827-3 cells into each mouse via the tail vein. Inject 0.2 ml (2 million) of cell suspension into each mouse, and inoculate 5 to 6 mice per group.

[0151] 5. Every 7 days, inject luciferase intraperitoneally at a dose of 150 mg / kg. After 15 minutes, observe the lung metastasis in mice using a small animal in vivo imaging system.

[0152] 3.5 Experimental Results

[0153] like Figure 3 A. The proliferation capacity of HCC827-3 cells was stronger than that of HCC827 cells, and the difference was statistically significant (P<0.05).

[0154] like Figure 3 B. In both mixed and unmixed chambers, the number of HCC827-3 cells that crossed the membrane was much greater than that of HCC827 cells.

[0155] like Figure 3 As shown in D and E, when the number of cells crossing the membrane was counted, the migration and invasion abilities of HCC827-3 cells were stronger than those of HCC827 cells, and the difference was statistically significant (P<0.05).

[0156] like Figure 3As can be seen from F, during the same growth period, mice inoculated with HCC827 cells did not develop subcutaneous tumors, while mice inoculated with HCC827-3 cells did develop subcutaneous tumors.

[0157] like Figure 3 C and G: No tumors formed under the skin in mice inoculated with HCC827 cells. The subcutaneous tumors in mice inoculated with HCC827-3 cells increased in size over time. The average weight of the subcutaneous tumors in mice inoculated with HCC827-3 cells was greater than that in mice inoculated with HCC827 cells.

[0158] This shows that HCC827-3 cells have a stronger subcutaneous tumorigenicity than HCC827 cells, and the difference is statistically significant (P<0.05).

[0159] like Figure 3 H, one mouse inoculated with HCC827-3 cells developed bone metastasis, and the lung metastasis was larger than that of the mouse inoculated with HCC827 cells, indicating that the metastatic ability of HCC827-3 cells is stronger than that of HCC827 cells.

[0160] Example 4: Effects of ANXA6-altered CAF on the malignant phenotype of HCC827 cells

[0161] 4.1 Knock down ANXA6 in CAF and verify using Western Blot

[0162] 1. Seed CAF cells into 24-well plates. When the cells have reached 80%-90% confluence and are in good condition, prepare to infect the target cells.

[0163] 2. Use a pipette to remove the culture medium from the wells, add the calculated lentivirus solution, virus infection enhancement solution and culture medium respectively, and place the plate in a 37°C, 5% CO2 incubator for overnight incubation.

[0164] 3. 24 hours after infection, aspirate the culture medium containing lentivirus, add basal culture medium back into the wells, continue culturing, and observe whether there are any abnormalities in the cell state.

[0165] 4. Observe the infection efficiency of lentiviral particles under a fluorescence microscope. If the infection efficiency is appropriate, conduct drug screening.

[0166] 5. Seed CAF cells and infected cells separately into 24-well containers. Add 1 μg / ml puromycin to the basal medium, mix well, and then add to the wells for screening. After the CAF cells die, collect the infected cells and culture them.

[0167] 6. CAF-NC, CAF-ANXA6sgRNA1, CAF-ANXA6sgRNA2 and CAF-ANXA6sgRNA3 (cells with ANXA6 knockdown) were seeded into 24-well plates and allowed to grow to 80% confluence.

[0168] 8. Add the protease inhibitor to the RIPA lysis buffer and mix thoroughly.

[0169] 9. Remove the culture medium from the wells, wash the cells three times with physiological saline refrigerated at 4°C, and then discard the solution.

[0170] 10. Add 200 μl of RIPA lysis buffer to each well, then place in an ice bath for 15 minutes and vortex for 30 seconds every 5 minutes.

[0171] 11. Centrifuge at 13900 rpm and 4℃ for 15 minutes, collect the supernatant to obtain the cell protein product.

[0172] 12. Mix cell proteins and loading buffer at a ratio of 4:1 and incubate in a water bath at 100°C for 10 minutes. Place 20 μl of each sample into the well and perform electrophoresis at 80V for 30 minutes and then at 120V for 90 minutes.

[0173] 13. After electrophoresis, transfer the membrane at 65V in an ice bath for 3 hours, and then block it with 5% skim milk for 2 hours.

[0174] 14. Dilute the ANXA6 antibody at a ratio of 1:1000 and incubate overnight at 4°C.

[0175] 15. Wash the NC membrane three times with TBST solution for 10 minutes each time, then incubate with secondary antibody at room temperature for 4 hours, wash the NC membrane three times again, and expose it in an imaging system after using developing solution.

[0176] 16. After sealing for 30 minutes, dilute GAPDH at a ratio of 1:4000 and incubate for 4 hours. Wash the NC membrane three times with TBST, and then expose it in an imager using a developing solution.

[0177] 4.2 Effects of CAF with altered ANXA6 expression on HCC827 cell growth

[0178] 1. Trypsin digestion solution was added to four types of cells: HCC827+CAF (CAF is ANXA6 overexpressing cell), HCC827+ANXA6sgRNA1, HCC827+ANXA6sgRNA2, and HCC827+ANXA6sgRNA3. The cells were digested at 37°C for about 5 minutes until they were completely digested into single cells. After neutralization with RPMI-1640 medium, the cells were transferred to centrifuge tubes and centrifuged at 800 rpm for 5 minutes. The cells were then resuspended in basal medium and counted using a Muse cell counter.

[0179] 2. Dilute according to the counting results, and seed the four types of cells into five 96-well plates at a density of 5000 cells per well.

[0180] 3. At the time points of 0 hours, 24 hours, 48 ​​hours, 72 hours and 96 hours after the cells were seeded into the wells, Cell Counting Kit-8 cell counting reagent was added to the wells in proportion. After incubating in a cell culture incubator for 1 hour, the OD value at 450 nm in each well was measured using a microplate reader.

[0181] 4. After organizing the measured OD values, use GraphPad Prism 8.0 software to plot them as a curve.

[0182] 4.3 Effects of CAF with altered ANXA6 expression on the migration and invasion abilities of HCC827 cells

[0183] 1. Replace the culture medium of the four cell types HCC827+CAF, HCC827+ANXA6sgRNA1, HCC827+ANXA6sgRNA2 and HCC827+ANXA6sgRNA3 with 0.2% BSARPMI-1640 medium and incubate for 24 hours.

[0184] 2. Add 600 ml of RPMI-1640 medium to the lower chamber of the Transwell plate and 100 μl of 0.2% BSARPMI-1640 medium to the upper chamber. Place the Transwell plate in a cell culture incubator and hydrate for 30 min.

[0185] 3. Add trypsin digestion solution to four types of cells: HCC827+CAF, HCC827+ANXA6sgRNA1, HCC827+ANXA6sgRNA2, and HCC827+ANXA6sgRNA3. Digest at 37°C for about 5 minutes until the cells are completely digested into single cells. Neutralize with RPMI-1640 medium and transfer to centrifuge tubes. Centrifuge at 800 rpm for 5 minutes. Resuspend in basal medium and count cells using a Muse cell counter.

[0186] 4. Based on the counting results, the four cell types were resuspended in 0.2% BSARPMI-1640. The cell concentration without base gel was 800,000 / ml, and the cell concentration with base gel was 2,000,000 / ml. The ratio of base gel to cell suspension was 1:5.

[0187] 5. Aspirate 100 μl of hydrated 0.2% BSA1640 medium from the Transwell chamber and add 100 μl of the above cell suspension to each well. Incubate at 37°C with 5% CO2.

[0188] 6. After 24 hours, remove the unmixed chambers, wash them twice with physiological saline, and then place them in gentian violet solution. After 30 minutes, wash off the gentian violet and observe the cells that have crossed the membrane and grown under a Leica microscope in bright field.

[0189] 7. After 48 hours, process the mixed gel chambers in the same way and observe the cells that have passed through the membrane and grown under bright field under a Leica microscope.

[0190] 4.4 Experimental Results

[0191] like Figure 4 The results showed that ANXA6 was normally expressed in CAF-NC cells, while the expression levels of ANXA6 in CAF-ANXA6sgRNA1, CAF-ANXA6sgRNA2, and CAF-ANXA6sgRNA3 cells were extremely low, demonstrating that knocking down the three targets of ANXA6 was effective.

[0192] like Figure 4 B. The proliferation capacity of HCC827+CAF cells was stronger than that of HCC827+ANXA6sgRNA3 cells, and the difference was statistically significant (P<0.05), indicating that altering ANXA6 expression in CAF cells affects the proliferation capacity of HCC827 cells.

[0193] like Figure 4 C. In both mixed and unmixed chambers, the number of HCC827+CAF cells that crossed the membrane was much greater than that of the three cell types: HCC827+ANXA6sgRNA1, HCC827+ANXA6sgRNA2, and HCC827+ANXA6sgRNA3.

[0194] like Figure 4 As can be seen from D, when the number of cells crossing the membrane was counted, the migration and invasion abilities of HCC827+CAF cells were stronger than those of HCC827+ANXA6sgRNA1, HCC827+ANXA6sgRNA2 and HCC827+ANXA6sgRNA3 cells, and the differences were statistically significant (P<0.05).

[0195] Example 5 ANXA6+ Prognostic analysis of CAF cells in lung cancer

[0196] 5.1ANXA6 + The impact of CAF on survival time in lung cancer patients

[0197] Based on the immunohistochemical results, the expression of ANXA6 in lung cancer tissue and normal tissue was quantitatively analyzed using Image Pro Plus software, and the data were obtained.

[0198] 5.2 Based on immunohistochemical data, the impact of ANXA6 in CAF on the survival of lung cancer patients was analyzed using Statistical Product Service Solutions software.

[0199] like Figure 5 As shown, lung cancer patients with high ANXA6 expression in CAF had significantly shorter survival than those with low or no ANXA6 expression in CAF, with a statistically significant difference between the two groups (p<0.05). High ANXA6 expression in CAF can serve as a marker for poor prognosis in lung cancer patients.

Claims

1. Use of a reagent for detecting the expression of ANXA6 gene in the preparation of a preparation for in vitro diagnosing the tendency of bone metastasis in a subject with lung cancer, said use being that when overexpression of ANXA6 gene in CAF is detected, then the subject has a greater tendency to form bone metastasis; said lung cancer is non-small cell lung cancer.

2. Use of a reagent for detecting the expression of ANXA6 gene in the preparation of a preparation for in vitro prognosis analysis of the effect after treatment of a subject with lung cancer, said use being that when overexpression of ANXA6 gene in CAF is detected, then the treatment of the subject is ineffective; said lung cancer is non-small cell lung cancer.

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