Application of macrophage-derived exosome in preparation of medicine for inhibiting microenvironment formation before pulmonary metastasis and preventing and treating tumor pulmonary metastasis

Abstract

The invention belongs to the field of biological medicines, and discloses application of a macrophage-derived exosome in preparation of medicines for inhibiting microenvironment formation before pulmonary metastasis and preventing and treating tumor pulmonary metastasis. The invention provides an application of a macrophage-derived exosome for inhibiting MTDH in preparation of a medicine for inhibiting microenvironment formation before tumor lung metastasis. The invention verifies that by inhibiting the macrophage-derived exosome Mac-shMTDH-Exo of MTDH, the integrity of vascular endothelial cells in a microenvironment before pulmonary metastasis can be protected, and the pulmonary metastasis of tumors can be obviously inhibited. The exosome of the scheme can be used as a biological preparation, inhibits formation of a microenvironment before tumor metastasis, and provides a new idea and possibility for lung metastasis inhibition diagnosis and clinical treatment of tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the use of macrophage-derived exosomes in the preparation of drugs for inhibiting the formation of a pre-metastatic microenvironment in the lung and preventing and treating tumor lung metastasis. Background Art

[0002] Metastasis is the main cause of clinical treatment failure and death in most cancer patients. Traditional anticancer treatments have numerous limitations, such as the toxic side effects of chemotherapy drugs and the multidrug resistance of tumor cells. Finding highly effective, low-toxic, and targeted anticancer treatment strategies has become a top priority.

[0003] The formation of a pre-metastatic microenvironment is a key step in tumor metastasis. Numerous studies have shown that the role of exosomes in the pre-metastatic microenvironment of cancer is gaining increasing attention. Exosomes, as a new type of nanoscale extracellular vesicles, have gradually become a highly promising drug carrier in anti-cancer therapy due to their unique biological properties in recent years, bringing new hope to cancer treatment. Exosomes as a method of tumor treatment have the following advantages: ① Compared with traditional research subjects, exosomes carry a large amount of important biological information such as nucleic acids and proteins; ② They have high stability; ③ Due to their endogenous nature and high biocompatibility, exosomes have relatively low cytotoxicity and immunogenicity; ④ Using exosomes to carry drugs can have a longer half-life in the circulation, etc.

[0004] Macrophages are the most numerous type of immune cell infiltrating tumor tissues. The oncogene MTDH is highly expressed in many tumors and is considered a key culprit in the spread or metastasis of cancer cells. The applicant has long studied the role of MTDH-positive macrophages in tumor development and progression. However, studies have yet to investigate whether macrophage MTDH, through the use of exosomes, regulates the formation of the premetastatic microenvironment and, in turn, induces distant tumor metastasis.

[0005] Our results highlight the importance of exosomes secreted by MTDH-suppressed macrophages in suppressing vascular leakage in lung tissue and the formation of lung metastases. These findings have implications for the development of new therapeutic strategies targeting vascular leakage in the pre-metastatic microenvironment. Summary of the Invention

[0006] Aiming at the problems existing in traditional tumor lung metastasis treatment, the present invention proposes the use of macrophage-derived exosomes in the preparation of drugs for inhibiting the formation of a microenvironment before lung metastasis and preventing and treating tumor lung metastasis.

[0007] In order to achieve the above object, the present invention is implemented by adopting the following technical solutions: The present invention proposes the use of macrophage-derived exosomes that inhibit MTDH in the preparation of a drug for inhibiting the formation of a pre-metastatic microenvironment in the lung.

[0008] A method for preparing macrophage exosomes comprising the following steps in sequence: S1: Preparation of THP-1 cell line with MTDH gene inhibition.

[0009] S2: Stimulate THP-1 cells with suppressed MTDH gene to differentiate into macrophages, and obtain macrophages Mac-shMTDH with suppressed MTDH gene.

[0010] S3: Stimulating the Mac-shMTDH cells to secrete exosomes.

[0011] The sequence of the MTDH gene is shown in the sequence listing as SEQ ID NO. 1. MTDH gene inhibition is achieved by RNA interference mediated by a lentiviral vector, and the RNA interference sequence is shown in SEQ ID NO: 2.

[0012] The present invention proposes the use of the above-mentioned macrophage exosomes in the preparation of a drug for inhibiting the permeability of vascular endothelial cells and the transvascular invasion ability of tumor cells.

[0013] Preferably, the THP-1 cell line culture method in step S1 is as follows: immerse the frozen THP-1 cell line in 37°C warm water and gently shake until thawed; add culture medium, centrifuge and discard the supernatant, and then culture at 37°C in a cell culture incubator containing 5% CO2 by volume; plate the cells at least 24 hours before transfection, and add a lentiviral suspension and polybrene that inhibits MTDH after the cell density reaches 70-80%; incubate at 37°C for 24 hours and then replace with fresh polybrene; centrifuge to remove the culture medium, add fresh culture medium again, and add puromycin for selection after 3-4 days; replace with fresh culture medium and puromycin every 2-3 days until a stable cell line is selected.

[0014] Compared with the prior art, the advantages and positive effects of the present invention are: This invention proposes the use of macrophage-derived exosomes that inhibit MTDH in the preparation of a drug for inhibiting the formation of a pre-metastatic microenvironment in the lung and preventing and treating tumor pulmonary metastasis. This invention demonstrates that MTDH-inhibiting macrophage-derived exosomes, Mac-shMTDH-Exo, can protect the integrity of vascular endothelial cells in the pre-metastatic microenvironment in the lung and significantly inhibit tumor pulmonary metastasis. This provides new insights and possibilities for the diagnosis and clinical treatment of tumor pulmonary metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the extraction and identification of macrophage-derived exosomes. Figure 1 A shows the successful construction of a macrophage cell line stably overexpressing or inhibiting MTDH. Figure 1B: The morphology and size of exosomes derived from macrophage MTDH were detected by transmission electron microscopy. Figure 1 C: The particle size of macrophage MTDH-derived exosomes was detected by nanoparticle tracking analysis (NTA). Figure 1 D: Western blot analysis of the expression of exosome markers derived from macrophage MTDH.

[0016] Figure 2 Exosomes secreted by macrophage MTDH increase lung metastasis of head and neck squamous cell carcinoma and breast cancer cells. Figure 2 A is the experimental plan for training macrophages to secrete exosomes MTDH and inoculating head and neck squamous cell carcinoma cells FaDu or breast cancer cells MDA-MB-231 cells. Figure 2 B shows representative images and HE staining of mouse lungs. Figure 2 C is Figure 2 Statistical chart of B. Figure 2 D is a representative image of mouse lungs. Figure 2 E is the HE staining image of mouse lung.

[0017] Figure 3 Exosomes secreted by macrophages MTDH promote increased vascular permeability and matrix degradation in lung tissue. Figure 3 AC is the result of vascular leakage; Figure 3 D shows the results of Masson staining.

[0018] Figure 4 It is the result of the transvascular invasion ability of tumor cells. Figure 4 A is the expression level and statistical analysis chart of marker molecules ZO-1 and VE-cadherin molecules. Figure 4 B is the result of endothelial migration experiment. Figure 4 C shows the results of angiogenesis experiment. Figure 4 D shows the results of microvascular invasion experiment. DETAILED DESCRIPTION

[0019] In order to more clearly understand the above-mentioned objects, features and advantages of the present invention, the present invention is further described below in conjunction with specific embodiments. It should be noted that, in the absence of conflict, the embodiments of the present application and the features therein can be combined with each other.

[0020] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways than those described herein. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0021] Example 1 1 Experimental Materials 1.1 Cell selection The human monocytic leukemia cell line THP-1, the human head and neck squamous cell carcinoma cell line FaDu, the human breast cancer cell line MDA-MB-231, and the human umbilical vein endothelial cells (HUVEC) used in this example were all purchased from Wuhan Punosai Life Science Technology Co., Ltd.

[0022] 1.2 Mouse selection Balb / c nude mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., 4-5 weeks old, weighing 14-16 g, raised under specific pathogen-free (SPF) conditions) were used.

[0023] 1.3 Materials and Reagents Environmentally friendly transparent wax dipping and dewaxing liquid: YA0031, Beijing Solebow Company.

[0024] Anhydrous ethanol: 10009218, Sinopharm Group.

[0025] Eosin staining solution: E607321, Shanghai Sangon Biotechnology Co., Ltd.

[0026] Hematoxylin staining solution: A426825, Shanghai Sangon Biotechnology Co., Ltd.

[0027] Tris(hydroxymethyl)aminomethane (Tris-saturated phenol): A610195, Shanghai Sangon Biotechnology Co., Ltd.

[0028] Triton X-100: BS084, White Shark Biotechnology Co., Ltd.

[0029] TBST buffer: G0004, Shanghai Sangon Biotechnology Co., Ltd.

[0030] Sodium dodecyl sulfate (SDS): A600485, Shanghai Sangon Biotechnology Co., Ltd.

[0031] RPMI-1640 medium (PM150110), DMEM medium (PM150110), trypsin (PM150110), phosphate buffered saline (PBS) 1X (PB180327): Wuhan Punosai.

[0032] Phosphate buffered saline (PBS): BL302A, White Shark Biotechnology Co., Ltd.

[0033] Polybrene: HY-112735, MCE Company, USA.

[0034] Serum-free non-programmed cell freezing medium (protein-free): Dalian Meilun Company.

[0035] Fetal bovine serum (FBS): C04002, Shanghai Xiaopeng Company.

[0036] Exosome-free serum, C3801-0100, Shanghai Xiaopeng Company.

[0037] Serum-free non-programmed cell freezing medium (protein-free): MA0401, Dalian Meilun Company.

[0038] RIPA lysis buffer (strong): P0013B, Shanghai Beyotime Biotechnology Co., Ltd.

[0039] High-concentration gold-medal Matrigel: 0827245, Shanghai Nova Pharmaceuticals Biotechnology Co., Ltd. & Xiamen Model Biotechnology Co., Ltd.

[0040] Gold Matrigel: 0827045, Shanghai Nova Pharmaceuticals Biotechnology Co., Ltd. & Xiamen Model Biotechnology Co., Ltd.

[0041] 4% paraformaldehyde: HY-18739, White Shark Biotechnology Co., Ltd.

[0042] Triton X-100: BS084, White Shark Biotechnology Co., Ltd.

[0043] Neutral gum mounting medium: E675007, Sangon Biotech (Shanghai) Co., Ltd.

[0044] Phorbol 12-myristate 13-acetate (PMA): HY-18739, MCE Company, USA.

[0045] Puromycin aminonucleoside: HY-B1743, MCE, USA.

[0046] Polybrene: HY-112735, MCE Company, USA.

[0047] Crystal violet staining solution (0.1%): BL802A, White Shark Biotechnology Co., Ltd.

[0048] Transwell cell culture insert: for 24-well plates, 8.0 μm, 3422, Corning.

[0049] PVDF (polyvinylidene fluoride) membrane: LC2002, Thermo Fisher Scientific Inc.

[0050] Protease inhibitor cocktail (100×PIC): P6730; Beijing Solebow Technology Co., Ltd.

[0051] Protein phosphatase inhibitor cocktail: P1260; Beijing Solebao Technology Co., Ltd.

[0052] Skim milk: BS102, White Shark Biotechnology Co., Ltd.

[0053] Kit: SDS-PAGE gel preparation kit: BL508A, White Shark Biotechnology Co., Ltd., specifically including 30% Acr-Bis (29:1), 1M Tris-HCl pH 8.8, 10% SDS, APS (dry powder) and TEMED.

[0054] Ultra-sensitive ECL chemiluminescence ready-to-use substrate (femtogram level): Wuhan Boster Bioengineering Co., Ltd.

[0055] Improved Sirius Red Staining Kit: G1472, Beijing Solebow Technology Co., Ltd., specifically including Sirius Red Staining Solution and Mayer's Hematoxylin Staining Solution.

[0056] MTDH antibody: CY7187, Shanghai Abiogen Biotechnology Co., Ltd.

[0057] β-Actin antibody: AF0003, Shanghai Beyotime Biotechnology Co., Ltd.

[0058] AF0006 antibody: AF0006, Shanghai Beyotime Biotechnology Co., Ltd.

[0059] CD63 antibody: CY5253, Shanghai Abiogen Biotechnology Co., Ltd.

[0060] CD9, CY5337, Shanghai Abiogen Biotechnology Co., Ltd.

[0061] TSG101 antibody: 28283-1-AP, Wuhan Tri-Taiwan Biotechnology Co., Ltd.

[0062] VE-cadherin: CY6599, Shanghai Abi Biotechnology Co., Ltd.

[0063] Occludin: CY5997, Shanghai Aibevi Biotechnology Co., Ltd.

[0064] HRP-conjugated secondary antibody (horseradish peroxidase-labeled goat anti-rabbit IgG (H+L)): A0352, Shanghai Beyotime Biotechnology Co., Ltd.

[0065] HRP-conjugated secondary antibody (horseradish peroxidase-labeled goat anti-mouse IgG (H+L)): A0350, Shanghai Beyotime Biotechnology Co., Ltd.

[0066] MTDH-overexpressing lentivirus, control lentivirus, and MTDH-inhibiting lentivirus were purchased from GeneCare. The MTDH gene sequence is shown in SEQ ID NO. 1, and its transcript number is NM_178812.4. The RNA interference-mediated lentiviral vector for MTDH inhibition is shown in SEQ ID NO. 2.

[0067] All other substances not otherwise specified were prepared using conventional reagents in the field of biology or chemistry, and the percentages of substances not otherwise specified refer to mass fractions.

[0068] 2. Experimental Methods 2.1 Cell recovery, culture, and cryopreservation Remove cryovials of THP-1, HUVEC, FaDu, and MDA-MB-231 cells from the liquid nitrogen container and immediately immerse in 37°C warm water. Gently shake the cryovials to rapidly thaw the cells. Add the cells to a centrifuge tube containing 10 times more culture medium (RPMI-1640 with 10% FBS for THP-1 macrophages; DMEM with 10% FBS for HUVEC, FaDu, and MDA-MB-231 cells) and centrifuge at 1,000 rpm for 5 minutes. Discard the supernatant and incubate the cells in a 5% CO2 (volume fraction) cell culture incubator at 37°C. Replace the culture medium the following day and continue culturing.

[0069] 2.2 Construction of stable cell lines by lentivirus infection of eukaryotic cells Prepare 10 5 Prepare a cell suspension at 100 μg / mL and plate 2 mL of the cell suspension in a 6-well plate. Keep the THP-1 cell density at approximately 70-80% during infection. The next day, remove 60 μL of viral suspension (1 x 108 TU / mL (transducing units per mL)) (MTDH-overexpressing lentivirus and control lentivirus, MTDH-inhibiting lentivirus, and control lentivirus) and 40 μL Polybrene (10 mg / mL). Incubate at 37°C. 24 hours after infection, replace with complete medium (RPMI-1640 with 10% FBS). Select with 0.2 μL of puromycin (10 mg / mL) starting 72 hours after transfection. Replace with fresh medium and 2 μg / mL puromycin every 2-3 days. Select for approximately two weeks to identify a stable cell line.

[0070] 2.3 Induction of macrophages 1 μL of phorbol ester PMA (1 mg / ml) was added to 10 mL of culture medium to induce the above stable cell lines for 48 h, then washed three times with 1XPBS and continued to culture with complete culture medium (RPMI-1640 + 10% FBS). The cells were induced to become MTDH-overexpressing macrophages (Mac-MTDH) and their control cells (Mac-vector), and MTDH-inhibited macrophages (Mac-shMTDH) and their control cells (Mac-shRNA).

[0071] 2.4 Extraction of exosomes secreted by macrophages using ultracentrifugation MTDH-overexpressing and MTDH-inhibited macrophages were washed three times with 1X PBS, cultured in complete medium overnight, and then washed three times with PBS. Culture was continued in 1640 medium supplemented with 10% exosome-free serum, and the culture supernatant was collected after 48 hours. Exosomes were extracted using differential centrifugation, a classic exosome isolation method. The culture supernatant was collected and centrifuged at 12,000 g for 30 minutes. The exosomes were then collected by centrifugation at 110,000 g for 70 minutes. The supernatant was gently removed and PBS was added. After centrifugation at 110,000 g for 70 minutes, the cells were resuspended in PBS to obtain an exosome suspension.

[0072] 2.5 Exosome identification and transmission electron microscopy The sample was placed on a copper grid and counterstained with 20 μL of 3% phosphotungstic acid solution for 5 minutes. After blotting with filter paper, the sample was placed in the transmission electron microscope sample chamber for morphological observation and photography. Particle size determination: The parameters were zeroed with sterile PBS. After wiping dry, the exosome suspension was added to the sample chamber. The timer was set to 90 seconds. The liquid particle size was measured and repeated three times.

[0073] 2.6 Nanoparticle size analysis was performed by Shanghai Xiaopeng Company. The collected exosomes were diluted to 10 6 The exosomes were injected into the nanoparticle tracking analyzer using a 1 mL syringe; a laser beam passed through the exosome particles outside the sample chamber, and the particles were visualized using a microscope equipped with a camera to capture the Brownian motion of the exosomes. Finally, the concentration and hydrodynamic diameter were calculated based on their motion using equations.

[0074] 2.7 Identification of the expression of exosome surface markers CD9 and CD63 by Western blot Add cell lysis buffer (Biyuntian), add 500 μL per 10 cm culture dish and about 100 μL per six-well plate, mix thoroughly, and place on ice to lyse the cells for 30 minutes. After adding 2× loading buffer, denature the protein sample by boiling at 100°C for 5 minutes, then load directly or store at -20°C.

[0075] Western blot. Gel preparation: Top stacking gel (5% density, pH 6.8); bottom separating gel (8-12% density, pH 8.8); Sample loading: 20-50 μg protein; Electrophoresis: Constant voltage 80 V for stacking gel, 120 V for separating gel; Transfer: Activate PVDF membrane (Millipore) with methanol. Loading sequence: negative electrode / (black) sponge - filter paper - gel - PVDF membrane - filter paper - sponge (white) / positive electrode. Avoid air bubbles between layers. Transfer from negative electrode to positive electrode. Incubate on ice at a constant voltage of 150 V for 3 hours. Blocking: After transfer, remove the PVDF membrane and block with blocking solution (5% nonfat dry milk in 1× TBST) at room temperature with shaking for 1 hour. Primary antibody: Prepare the antibody according to the antibody manufacturer's instructions and incubate overnight at 4°C. Wash the membrane three times with 1× TBST (5 min each time); prepare the antibody (typically at a 1:10,000 concentration, diluted in blocking buffer) and incubate at room temperature for 1 hour. Wash the membrane three times with 1× TBST (15 min each time). Expose: Prepare reagent A and reagent B from the kit (A:B = 1:1) immediately before use and store in dark. Aspirate and apply developer solution to the membrane, ensuring that it completely penetrates the membrane. Allow the developer solution to react for 2 minutes before acquiring images using a fully automated chemiluminescence / fluorescence image analysis system (Tanon 5200 Multi).

[0076] 2.8 Exosome acclimation experiment and nude mouse lung metastasis experiment Four-week-old nude mice were injected with exosomes secreted by macrophage MTDH through the tail vein (10 μg / 200 μL / time, three times a week, for a total of 9 times). Three weeks later, 1.5×10 6 / 150μL DMEM / each or 1X10 MDA-MB-231 cells 6 After 2-3 months, nude mice were sacrificed by cervical dislocation, and lung tissue was removed and fixed with 4% paraformaldehyde for 24 hours. Dehydration, clearing, wax infiltration, embedding, and sectioning were performed as per the routine procedures. Sections were then stained with H&E as follows.

[0077] 2.9 HE staining Dewaxed sections were placed in hematoxylin stain for 6 minutes. Rinse with running water for 5 minutes. Stain with alcohol eosin stain for 2-3 minutes. Dehydrate the stained sections in pure alcohol for 1 minute, then clear them with environmentally friendly wax dewaxing and clearing solution for 5 minutes. Add a drop of gum to the cleared sections and cover with a coverslip for sealing. ① Dewax to water: 5 minutes in xylene, cycle three times, 1 minute in absolute ethanol, 1 minute in 95% ethanol, 1 minute in 75% ethanol, and rinse with tap water for a few seconds.

[0078] 2.10 Sirius red staining Dewax paraffin sections to dehydrate: Place sections in environmentally friendly paraffin immersion and dewaxing clearing solution 1 for 20 minutes, then in environmentally friendly paraffin immersion and dewaxing clearing solution for 20 minutes, then in anhydrous ethanol for 5 minutes, then in 95% ethanol for 5 minutes, then in 75% ethanol for 1 minute. Rinse in tap water for 10 seconds. Stain sections with picrosirius red: Stain sections in picrosirius red solution for 8 minutes, then dehydrate in two or three cylinders of anhydrous ethanol. Dehydrated sections: Place sections in clean environmentally friendly paraffin immersion and dewaxing clearing solution for 5 minutes, then mount sections with neutral gum. Microscopic examination and image acquisition and analysis: Interpretation of results: Collagen fibers appear red, and the background appears yellow under light microscopy.

[0079] 2.10 Transwell cell migration and invasion assays HUVEC cells co-cultured with exosomes (Mac-shMTDH-Exo and Mac-shRNA-Exo) for 48 h were digested to prepare cell suspension. 200 μL of cell suspension (5×10 5 800 μL of complete medium containing 10% FBS was added to the upper chamber of the chamber, and the 24-well plate (lower chamber) was added. The 24-well plate was placed in an incubator and cultured for 24 h. The chamber was removed, the liquid in the upper chamber was aspirated, and the cells were fixed with 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet for 15 min. The cells were immediately transferred to 1× PBS, rinsed until no excess dye was present, and inverted to air dry. The cells were photographed under an inverted microscope, and the counts in five randomly selected high-power fields were averaged for statistical analysis.

[0080] 2.11 Tube Formation Experiment HUVEC cells (20X10 4 2 μg exosomes were added to the cells) to observe the effect of MTDH-inhibiting macrophage-derived exosomes on the angiogenesis ability of human umbilical vein endothelial cells (HUVEC). 4 HUVEC cells were seeded onto Matrigel and cultured in an incubator for 6 hours. The tube-forming ability of HUVEC cells on Matrigel was observed and photographed using an inverted microscope. Statistical analysis was performed using GraphPad Prism 8. Means between the two groups were compared using an unpaired t-test. Data are expressed as mean ± SEM.

[0081] 2.12 Transendothelial invasion assay Lentiviral vector-mediated green fluorescent protein (GFP, sequence as shown in SEQ ID NO: 3) was used to label the head and neck squamous cell carcinoma cell line FaDu cells. 200 μL of green fluorescent labeled FaDu cell suspension (5×10 5 ) were added to the chambers, and 800 μL of complete medium containing 10% FBS was added to the 24-well plate (lower chamber). The 24-well plate was placed in an incubator and cultured for 48 hours. Transwell chambers were prepared, and human umbilical vein endothelial cell (HUVEC) suspension was added and cultured in an incubator. After 24 hours, the cells were washed three times with 1XPBS, and exosomes (Mac-shMTDH-Exo and Mac-shRNA-Exo were added and cultured for 48 hours; 20X10 4 cells plus 2 μg of exosomes) were co-incubated for 48 h; 48 h later, head and neck squamous cell carcinoma cells labeled with green fluorescent protein were inoculated: complete medium containing 10% FBS was added to the lower chamber of the 24-well plate as a chemoattractant, and tumor cell suspension (1×10 5 cells / mL, 200 μL was added to each well); 24 h later, the Transwell chamber was removed, washed with PBS, and the endothelial cells and non-invaded tumor cells in the upper chamber were gently wiped off with a cotton swab. 4% paraformaldehyde fixative was added to clean wells of a 24-well plate, and the chamber was fixed for 30 min. The fixative was discarded, and the chamber was washed with PBS. The head and neck squamous cell carcinoma cells that migrated to the lower chamber were observed under a fluorescence microscope.

[0082] 2.13 Statistical Analysis Experimental data are presented as mean ± standard error of the mean (SEM). For comparisons between two groups, statistical significance was determined using a two-sided Student's t-test. P values ​​less than 0.05 (P < 0.05) were considered statistically significant.

[0083] 3 Results 3.1 Figure 1 The extraction and characterization of macrophage-derived exosomes. Using a lentiviral packaging system to infect THP-1 cells, THP-1 cell lines stably overexpressing or suppressing MTDH were successfully constructed. These cells were then induced with PMA (100 ng / ml) for 48 hours to become macrophages. Quantitative Western blotting (Figure 1A) revealed significant overexpression or knockdown of MTDH compared to the respective control cells. These results demonstrate the successful construction of cell lines stably overexpressing MTDH, providing an experimental basis for studying whether macrophage MTDH, through exosomes, plays a role in regulating distant cancer metastasis.

[0084] First, the exosomes secreted by macrophage MTDH were isolated by ultracentrifugation and identified by electron microscopy and NTA. The morphology of the exosomes was preliminarily determined to be cup-shaped or saucer-shaped by electron microscopy, and the particle size of the exosomes was about 30-120nm (Figure 1B). The particle size can be further detected by NTA ( Figure 1 C). Western blot confirmed the expression of exosome-specific proteins (Figure 1D). After ultracentrifugation, high-quality exosomes were obtained in this example based on the results of transmission electron microscopy (TEM), NTA, and Western blot analysis for subsequent animal experiments and co-incubation experiments.

[0085] Figure 2 Macrophage MTDH-derived exosomes increase lung metastasis of head and neck squamous cell carcinoma and breast cancer cells.

[0086] To investigate the role of exosomes derived from macrophages overexpressing MTDH in distant cancer metastasis, mice were intravenously injected with exosomes secreted by macrophage MTDH via the tail vein. It was found that exosomes secreted by macrophage MTDH significantly promoted the lung metastasis of FaDu or MDA-MB-231 cells. Figure 3 A shows the experimental plan for training macrophages to secrete exosomes secreted by MTDH and inoculating head and neck squamous cell carcinoma cells FaDu or breast cancer cells MDA-MB-231. Exosomes derived from macrophages overexpressing MTDH and control cells were injected through the tail vein. After 3 weeks of training, head and neck squamous cell carcinoma cells were inoculated. After 2 months, the mice were sacrificed, and the lungs were imaged and stained with H&E ( Figure 2 A). The results showed that HE staining results showed that the number of metastatic foci in the lung tissue of mice in the exosome-treated group secreted by macrophages MTDH was significantly higher than that in the control group ( Figure 2 B), and the difference was statistically significant ( Figure 2 C). Compared with the control group, exosomes secreted by macrophage MTDH significantly increased the size and number of lung metastases of breast cancer cells ( Figure 2 D), and HE staining results showed that the number of metastatic foci in the lung tissue of mice in the exosome-treated group secreting macrophage MTDH was significantly higher than that in the control group ( Figure 2 E).

[0087] Figure 3 Exosomes derived from macrophages that overexpress MTDH promote increased vascular permeability and matrix degradation in lung tissue. To investigate the specific effects of exosomes secreted by macrophages on the lungs in vivo, exosomes secreted by macrophages were injected into nude mice via tail vein injection. Figure 3As shown in A, mice were treated with 10 μg of macrophage-secreted exosomes every 3 days for 3 weeks and then evaluated. The results showed that HE staining showed that there was very obvious blood cell exudate in the lung tissue of mice treated with macrophage-secreted exosomes ( Figure 3 A, B, C). Masson staining results showed that compared with the control group, fibrosis in the lung tissue of mice treated with exosomes secreted by macrophages was significantly reduced ( Figure 3 D).

[0088] Figure 4 Macrophage-derived exosomes that inhibit MTDH significantly protect HUVEC integrity and inhibit microvascular cancer cell invasion. To determine whether Mac-shMTDH-Exo protects endothelial cell integrity, human umbilical vein endothelial cells (HUVECs) were treated with Mac-shMTDH-Exo and Mac-shRNA-Exo. HUVECs were harvested after 48 hours of co-incubation. Western blot analysis was used to assess occludin and VE-cadherin protein expression, which indicate endothelial cell integrity. Figure 4 As can be seen in A, Mac-shMTDH-Exo protected the integrity of HUVEC cells compared with the control group. Figure 4 B. The results of endothelial migration assay showed that the migration ability of HUVEC in the co-culture of Mac-shMTDH-Exo was significantly reduced compared with the control group. Figure 4 As shown in C, the tube-forming ability of HUVECs co-cultured with Mac-shMTDH-Exo was significantly reduced compared with the control group. Figure 4 As can be seen in D, the invasive ability of HNSCC cells co-cultured with HUVECs stimulated with Mac-shMTDH-Exo was significantly reduced compared with that of FaDu (labeled with green fluorescence) in comparison with the control group.

[0089] The above description is merely a preferred embodiment of the present invention and does not constitute any other form of limitation to the present invention. Any person skilled in the art may utilize the technical contents disclosed above to change or modify them into equivalent embodiments with equivalent changes for application in other fields. However, any simple modification, equivalent change, and modification of the above embodiments made in accordance with the technical essence of the present invention without departing from the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A use of macrophage exosomes in the preparation of a drug for inhibiting the formation of a pre-metastatic microenvironment in the lung, characterized in that: The method for preparing macrophage exosomes comprises the following steps performed in sequence: S1: Preparation of THP-1 cell line with MTDH gene inhibition; S2: Stimulate THP-1 cells with suppressed MTDH gene to differentiate into macrophages, and obtain macrophages Mac-shMTDH with suppressed MTDH gene; S3: stimulating the Mac-shMTDH cells to secrete exosomes; The sequence of the MTDH gene is shown in SEQ ID NO. 1 in the sequence listing; lentiviral vector-mediated RNA interference is used to inhibit the MTDH gene, and the sequence of the RNA is shown in SEQ ID NO:

2.

2. Use of the macrophage exosomes according to claim 1 in the preparation of a drug for inhibiting the permeability of vascular endothelial cells and the transvascular invasion ability of tumor cells.

3. The application according to claim 1, characterized in that The THP-1 cell line culture method in step S1 is as follows: immerse the frozen THP-1 cell line in 37°C warm water and gently shake until thawed; add culture medium, centrifuge and discard the supernatant, and then culture in a cell culture incubator containing 5% CO2 at 37°C; plate the cells at least 24 hours before transfection, add a lentiviral suspension that inhibits MTDH and polybrene after the cell density reaches 70-80%; incubate at 37°C for 24 hours and then replace with fresh polybrene; centrifuge to remove the culture medium, add fresh culture medium again, and add puromycin for selection after 3-4 days; replace with fresh culture medium and puromycin every 2-3 days until a stable cell line is selected.

Images