Method for combined treatment of glioma by inducing exosome to exert remote isolation effect at high temperature

Abstract

The invention relates to the technical field of biological medicine, in particular to a method for inducing exosomes at high temperature to play a far-isolating effect for glioma combined therapy, which comprises the following steps: step 1, putting glioma cells in a high-temperature environment for stimulation for a period of time to promote the glioma cells to release the exosomes; step 2, separating and purifying the released exosome, so as to obtain H-Exos induced at high temperature; 3, the H-Exos is applied to far-isolating effect treatment of glioma to inhibit growth, migration and invasion of far tumor cells, according to the method for combined treatment of glioma by inducing exosomes at high temperature to play the far-isolating effect, a bilateral subcutaneous tumor-bearing mouse model is established to simulate the biological characteristics of diffuse growth of clinical brain tumor, and the biological characteristics of the far-isolating effect of the glioma can be simulated to achieve the far-isolating effect of the glioma to achieve the far-isolating effect of the glioma to achieve the far-isolating effect of the glioma to achieve the far-isolating effect of the glioma. MWA is adopted to ablate tumors on one side, then exosomes generated by tumor cells MHT are creatively combined and applied, and a basic support is provided for subsequently discussing a treatment strategy that thermal therapy and exosomes are combined as glioma.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically to a method for combined treatment of glioma by inducing exosomes to exert remote effects through high temperature. Background Art

[0002] Glioma is a common primary malignant tumor of the central nervous system with a high recurrence rate and mortality rate. [1] The principle of clinical treatment is to safely remove the tumor to the greatest extent possible, relieve clinical symptoms and prolong survival. Removing the tumor as much as possible and reducing the recurrence rate under the premise of less damage to normal brain tissue has become a difficult problem that needs to be solved in the treatment of malignant brain tumors, and new treatment methods are urgently needed.

[0003] Hyperthermia therapy is the use of different physical means to increase the temperature of tumor tissue and (or) the whole body, using high temperature to kill and its secondary effects to treat tumors. It has developed into the fifth tumor treatment method after surgery, radiotherapy, chemotherapy and biological therapy. [2] Among them, microwave ablation (MWA) has the advantages of low invasiveness and multiple targeted operations, and has achieved good therapeutic effects in the treatment of brain gliomas. However, the weak remote effect of MWA alone is not enough to reduce the recurrence rate of tumors. [3] Previous studies have found that different hyperthermia methods and different degrees of heat have different effects on cells. Temperatures around 43°C (Mild Hyperthermia, MHT) have been proven to be effective in tumor treatment. [4] , and significantly affects aerobic glycolysis in tumor tissues and the synthesis of nucleic acids and proteins in tumor cells [5,6] , increasing the release of exosomes [7] At the same time, studies have found that exosomes carry unique tissue- or cell-specific proteins and genetic material [8] , which can change the tumor microenvironment [9] , affecting the activity of surrounding cells

[10] This also provides a new idea to make up for the inadequacy of MWA in treating micrometastases and residual lesions.

[0004] However, there are few studies on the direct effects of exosomes released by glioma cells after thermal stimulation on surrounding cells. MWA is still a major challenge in clinical treatment in inhibiting the progression and metastasis of glioma.

[11] Therefore, in view of the above situation, it is urgent to develop a method for high temperature to induce exosomes to exert remote effects for combined treatment of gliomas in order to overcome the shortcomings in current practical applications. Summary of the invention

[0005] The purpose of the present invention is to provide a method for combined treatment of glioma by inducing exosomes to exert remote effects through high temperature, so as to solve the problems raised in the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The method of hyperthermia-induced exosomes exerting remote effects for combined treatment of gliomas comprises the following steps:

[0008] Step 1: placing the glioma cells in a high temperature environment for stimulation for a period of time to induce the glioma cells to release exosomes;

[0009] Step 2, isolating and purifying the released exosomes to obtain high temperature induced H-Exos;

[0010] Step 3: Apply the H-Exos to the abscopal effect treatment of glioma to inhibit the growth, migration and invasion of distant tumor cells.

[0011] As a further solution of the present invention: in step 1, the glioma cells are rat brain glioma cells C6, and the rat brain glioma cells C6 are cultured in a complete growth medium.

[0012] As a further solution of the present invention: take 1.0×10 7 The glioma cells were added into a serum-free culture medium and placed in a 43° C. incubator for continuous high temperature stimulation for 60 min.

[0013] As a further solution of the present invention: in step 2, the operation steps of isolating and purifying exosomes include ultracentrifugation, nanoparticle tracking analysis, electron microscopy observation and protein blotting analysis, and finally H-Exos are obtained.

[0014] As a further solution of the present invention: in step 3, the H-Exos are used for remote effect treatment of glioma via intravenous injection, local injection or other administration methods.

[0015] As a further scheme of the present invention: it also includes: evaluating the biological effects of H-Exos on glioma cells after high temperature stimulation, including the detection of cell proliferation, apoptosis, migration and invasion ability.

[0016] As a further scheme of the present invention, it also includes: after MWA treatment of glioma, applying the H-Exos in remote effect treatment to further inhibit the growth of distant tumor cells.

[0017] As a further scheme of the present invention: the H-Exos are administered at four time points, namely, day 3, day 14, day 21 and day 28 after MWA treatment.

[0018] A pharmaceutical composition for abscopal treatment of gliomas comprises the H-Exos described above and a pharmaceutically acceptable carrier.

[0019] As a further solution of the present invention: the pharmaceutical composition also includes a treatment plan for microwave ablation.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This application clarifies the direct biological effects of temperature around 43°C (MildHyperthermia, MHT) on cellular exosomes. By establishing a bilateral subcutaneous tumor-bearing mouse model to simulate the biological characteristics of diffuse growth of clinical brain tumors, MWA was used to ablate one side of the tumor, and then the exosomes (H-Exos) produced by MHT of tumor cells were innovatively combined to observe the effect of combined treatment on distant tumor growth, providing basic support for the subsequent in-depth study of hyperthermia combined with exosomes as a treatment strategy for gliomas, especially high-grade gliomas. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a roadmap for the remote effects of heat stress exosomes in the embodiments of the present invention.

[0023] Figure 2 This is a diagram of the exosome verification results in an embodiment of the present invention.

[0024] Among them, A is the specific protein WB result; B is the exosome NTA result; C is the exosome electron microscopy image.

[0025] Figure 3 This is a comparison of tumor sizes among three groups in an embodiment of the present invention.

[0026] Among them, A is the gross specimen of the tumor in the three groups of rat models; B is the maximum diameter of the tumor on the ablation side; C is the comparison of the tumor volume on the non-ablation side in the three groups.

[0027] Figure 4 It is a flow chart of ultracentrifugation in an embodiment of the present invention.

[0028] Figure 5 This is a diagram of MWA and drug administration time in bilateral tumor-bearing model mice in the examples of the present invention. DETAILED DESCRIPTION

[0029] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0031] The method provided by the embodiment of the present invention for combined treatment of glioma by inducing exosomes to exert a remote effect through high temperature includes the following steps:

[0032] Step 1: placing the glioma cells in a high temperature environment for stimulation for a period of time to induce the glioma cells to release exosomes;

[0033] Step 2, isolating and purifying the released exosomes to obtain high temperature induced exosomes (H-Exos);

[0034] Step 3: Apply the H-Exos to the abscopal effect treatment of glioma to inhibit the growth, migration and invasion of distant tumor cells.

[0035] This application clarifies the direct biological effects of temperature around 43°C (MildHyperthermia, MHT) on cellular exosomes. By establishing a bilateral subcutaneous tumor-bearing mouse model to simulate the biological characteristics of diffuse growth of clinical brain tumors, MWA was used to ablate one side of the tumor, and then the exosomes (H-Exos) produced by MHT of tumor cells were innovatively combined to observe the effect of combined treatment on distant tumor growth, providing basic support for the subsequent in-depth study of hyperthermia combined with exosomes as a treatment strategy for gliomas, especially high-grade gliomas. The research yielded the following:

[0036] 1. Increased exosome release after MHT

[0037] like Figure 2 As shown, the extracted H-Exos vesicles are round or oval, coated with a double membrane, and have a saucer-like structure. The results of the nanoparticle tracking instrument (NTA) analysis in the nanoparticle tracking analysis experiment showed that 98% of the exosomes had a diameter distribution ranging from 90 to 140nm, with a single peak at about 117nm, and no significant difference between the groups. The diameter of the exosomes observed in the TEM image was about 115nm, which was basically consistent with the measured value. The results of Western blot analysis showed that specific proteins of CD63, CD9 and TSG101 were positively expressed, that is, the extracted exosomes met the required standards. [14,15] , and can be used in subsequent experiments.

[0038] 2. Direct biological effects of H-Exos

[0039] (1) H-Exos enter C6 cells and cause death through apoptosis

[0040] After culturing CAMC-stained C6 cells with extracted PKH26-stained exosomes for 4 hours, confocal microscopy photos clearly showed that exosomes entered the cells and were distributed in the cytoplasm of C6 cells. The survival of C6 cells co-incubated with different concentrations of exosomes (20-200 μg / ml) after 24, 48 and 72 hours of incubation was observed. It is obvious that exosomes have induced C6 cell death. The half-maximal inhibitory concentration (IC50) after 48 hours of culture is about 80 μg / mL. Therefore, this study further set 10, 50 and 100 μg / ml groups for the next experiment with reference to the concentration of IC50. After 24, 48 and 72 hours of incubation, necrosis, late apoptosis, early apoptosis and living cells in the apoptosis flow cytometry experiment were represented by Q1, Q2, Q3 and Q4, respectively. The study showed that higher concentrations of exosomes led to more cell death in C6 cells, and more C6 cell death occurred with the extension of incubation time. Compared with necrosis (Q1), most cells died of apoptosis (Q2 and Q3) with increasing concentration and prolonged exposure time, and the increase was linear. In addition, the number of early apoptotic cells (Q3) increased with increasing concentration, and this phenomenon existed in different groups, which confirmed that at each time point, a part of the cells were in the early stage of apoptosis. It can be predicted that cell death caused by apoptosis will continue after 72 hours. As expected, the largest number of apoptotic cells was observed at the highest concentration and the longest exposure time, that is, after 72 hours.

[0041] (2) H-Exos inhibit the migration and invasion of C6 cells

[0042] The results of the scratch test of H-Exos co-incubated with C6 cells at different concentrations (0, 10, 50 and 100 g / mL) were studied, as well as the quantitative results of the scratch test after 12, 24, 36 and 48 hours of culture. It is obvious that H-Exos restricts the migration behavior of cells, and the higher the concentration of H-Exos, the more obvious the effect (P < 0.05). The control group almost covered about 90% of the scratch area at 24-36 hours. In the experimental group, especially the 100 μg / mL group, about 70% of the scratch distance was still not filled by C6 cells after 48 hours. The study showed that H-Exos had a greater effect on cell migration behavior within 24 hours. As time went on, the scratch healing inhibition rate of all three concentration groups showed a significant decrease between 24 and 36 hours. Further, compared with the control group, from the effects of the three different concentrations, the greatest effect of H-Exos should occur between 12 and 24 hours, which is independent of the influence of H-Exos concentration.

[0043] (3) High temperature exposure changes the protein profile of EVs

[0044] To determine whether H-Exos have an effective protein load that affects surrounding cells, this study used mass spectrometry to analyze H-Exos separated and purified after high temperature and matched untreated cell Exos from the same batch as the control group. A total of 1083 differential proteins were identified between the two groups, 511 upregulated proteins, and 532 downregulated proteins. Comprehensive analysis showed that high temperature significantly regulated multiple proteins. We provide a comprehensive list of significantly regulated H-Exos proteins in Table 1 below. Using the STRING database and CytoScape software, the differential proteins between the H-Exos group and the Control group were subjected to PPI analysis and the Eccentricity algorithm to obtain the top ten key node proteins with the highest scores. Compared with Exos from untreated cells, in Exos derived from cells exposed to high temperature, proteins PARP1, STIP1, and BANF1 were significantly upregulated. At the same time, proteins such as MAPK1, FLNB, MAPK3, ALB, PXDN, AGRN, C1S, etc. were significantly downregulated on H-Exos.

[0045] (4) Analysis of therapeutic efficacy in rat subcutaneous glioma model

[0046] like Figure 3 As shown in the figure, in order to evaluate the effect of MWA combined with H-Exos on distant tumor cells, tumor cells were inoculated subcutaneously in the bilateral groins. After 6-7 days, bilateral groin subcutaneous tumors were formed, and the average maximum diameter of the tumor was about 1.4 cm, with no statistical difference between the groups (P>0.05). All rats had no obvious skin thermal damage after ablation, and only scars remained at the skin ablation site 1-2 weeks later. The ablation lesions were gradually absorbed and shrunk 2-3 weeks after ablation, and no obvious contrast agent was observed in ultrasound angiography. By the sixth week of observation, the tumor on the MWA-treated side had basically disappeared, and some thickened connective tissue remained. There was no significant statistical difference between the MWA group and the MWA+H-Exos group. For the subcutaneous tumor on the non-ablated side, there was a significant statistical difference in tumor volume between the MWA+H-Exos group, the MWA group and the control group. Compared with the control group, the tumor growth on the non-ablation side was inhibited in both the MWA group and the MWA+H-Exos group. There was no statistical difference in tumor volume between the MWA+H-Exos group and the MWA group at week 2, and there was a significant statistical difference between the two groups from week 3 to week 6 (P < 0.05). There was no significant statistical difference between the MWA group and the control group after week 3.

[0047] In step 1, the glioma cells are rat brain glioma cells C6, and the rat brain glioma cells C6 are cultured in a complete growth medium.

[0048] Take 1.0×10 7 The glioma cells were added into a serum-free culture medium and placed in a 43° C. incubator for continuous high temperature stimulation for 60 min.

[0049] In step 2, the steps of isolating and purifying exosomes include ultracentrifugation, nanoparticle tracking analysis, electron microscopy observation and Western blot analysis, and finally H-Exos are obtained.

[0050] In step 3, the H-Exos are used for remote effect treatment of glioma via intravenous injection, local injection or other administration methods.

[0051] It also includes: evaluating the biological effects of H-Exos on glioma cells after high temperature stimulation, including detection of cell proliferation, apoptosis, migration and invasion ability.

[0052] It also includes: after microwave ablation (MWA) treatment of glioma, applying the H-Exos in remote effect treatment to further inhibit the growth of distant tumor cells.

[0053] The H-Exos were administered at four time points: 3 days, 14 days, 21 days and 28 days after MWA treatment.

[0054] A pharmaceutical composition for abscopal treatment of gliomas comprises the H-Exos described above and a pharmaceutically acceptable carrier.

[0055] The pharmaceutical composition also includes a treatment regimen for microwave ablation.

[0056] The present application realizes the application of using high temperature to induce glioma cells to release exosomes in the remote effect through the following steps, which are as follows:

[0057] 1. Cell culture

[0058] Cell passage was performed in a 10 cm standard culture dish, and rat glioma cells (C6) were cultured in complete growth medium containing F-12K medium (Servicebio, Cat#G4560, China) supplemented with 2.5% fetal bovine serum (FBS) (ExCell, Cat#: FSD500, Shanghai ExCell Biology) + 15% horse serum (HS) (Gibco, Thermo Fisher Scientific) + 1% double antibody. Cells were maintained at 37°C and 5% CO2.

[0059] 2. In vitro cell MHT stimulation

[0060] 1.0×10 7C6 cells were added to serum-free medium and placed in a 43°C incubator for about 60 min after the cells adhered to the wall. After MHT stimulation, the cells were transferred to 37°C and 5% CO2 and cultured for 24 h, after which the supernatant was harvested and clarified (300 g, 4°C for 10 min).

[0061] 3. Isolation and Characterization of Exosomes

[0062] (1) Exosome extraction

[0063] like Figure 4 As shown, exosomes were extracted by ultracentrifugation

[12] , the supernatant extracted above was centrifuged at 300g for 10min at 4°C to remove residual cells, and 2000g for 10min to remove cell debris. To remove apoptotic bodies, the supernatant was centrifuged again at 10000g for 15min at 4°C and then filtered with a 0.22μm filter. Finally, two ultracentrifugations were performed to separate exosomes (OptimaXE-100ultracentrifuge; BeckmanCoulter). In brief, centrifuge at 100000g for 90min at 4°C, resuspended with PBS, and ultracentrifuged again for 90min. The exosomes were then resuspended in 50ulPBS and stored at -80°C for subsequent experiments or storage.

[0064] (2)NTA

[0065] Take 4ul of the isolated and purified exosome PBS resuspension, dilute it 1000 times with PBS, and use the Nanoparticle tracking analysis (NTA) device ZetaView (Particle Metrix, Germany) to detect the particle size and concentration of the exosomes.

[0066] (3) Electron microscope

[0067] The electron microscope sample-carrying copper grid was covered on a 10 ul drop of exosome heavy suspension and allowed to stand at room temperature for 5 min. The excess sample liquid at the edge was removed with filter paper. The electron microscope sample-carrying copper grid was then covered on a drop of phosphotungstic acid negative staining liquid and allowed to stand at room temperature for 5 min. The excess liquid was removed with filter paper. After drying, the sample was observed and photographed under a H-7650 transmission electron microscope (Hitachi, Japan).

[0068] (4) Protein blotting

[0069] The stimulated cell supernatant was collected and, after ultracentrifugation, total protein was extracted from the cells using RIPA lysis buffer (Cat#C1053+, Applygen, China) supplemented with Phenylmethanesulfonylfluoride (PMSF, 1:100, Cat#ST505, Beyotime, China) and phosphatase inhibitors (1:100, Cat#G2007, Servicebio, China). Protein concentration was measured by BCA protein detection kit (Cat#23225, ThermoFisherScientific). Equal amounts of protein were separated by electrophoresis on a 10% polyacrylamide gel (Cat#PG112, EpiZymeBiotechnology, China), and then the protein was transferred to a PVDF membrane (Millipore, IPVH00010) with WB transfer buffer (Solarbio, D1005). Subsequently, Non-fat milk (5%) (BE6250; EASYBIO, Beijing, China) was used to block the nitrocellulose membranes for 2 at room temperature. Tris-buffered saline plus Tween-1 (TBST; Solarbio, T3) was incubated with antibodies TSG101 (1:1000, Cat#ab125011, Abcam), CD9 (1:1000, Cat#ab307085, Abcam), CD63 (1:1000, Cat#ab6102, Abcam), or β-actin (1:100, Cat#GB12001, Servicebio, China) at 4°C overnight. Afterwards, the membrane was incubated with HRP-conjugated goat anti-rabbit antibody (1:5000 dilution, bs-0295P-HRP; Bioss) or HRP-conjugated goat anti-Mouse antibody (1:5000 dilution, bs-0296P-HRP; Bioss) secondary antibody on a shaker at room temperature for 2 hours. PMID: 33111641. After washing 3 times with TBST, samples were detected with Enhanced Chemiluminescent (NC MBiotech, P10200) and visualized by using Amersham imager 600 (General Electric Company, USA).In some experiments, the previous primary and secondary antibodies were stripped from the PVDF membrane with stripping buffer (Cat#P0025, Beyotime, Shanghai, China) for 10 min. After reblocking, the membrane was re-incubated with another primary antibody at 4°C overnight. The following steps were the same as above.

[0070] 4. Biological effects of H-Exos on C6 cells

[0071] (1) Exosome fluorescence labeling and in vitro cellular uptake

[0072] Exosomes were stained with the Pkh26 kit (Cat#D0030, SolarbioLifeScience, China). 500ul of diluent c was added to 50ul of extracted exosomes resuspended in PBS. 2ul of PKH26 was dissolved in another 500ul of solution c. The above solutions were mixed and incubated at room temperature for 5min. 1ml of 1% BSA was added to stop staining. In order to exclude unbound DiI residues, the solution was centrifuged at 100000g for 2h at 4°C. The supernatant was aspirated and resuspended in 50ul PBS for subsequent uptake experiments.

[0073] Cells were stained with CellTrackerBlueCMAC (Cat#HY-D1462). Labeled exosomes (10 μg) were incubated with C6 cells seeded on 24-well plates at 37°C for 3 hours. Cells were washed twice with PBS and fixed in 4% paraformaldehyde for 30 min at room temperature. The internalization of exosomes was observed and images were acquired using a Zeiss LSM710 confocal laser microscope.

[0074] (2) CCK-8 assay to detect cell proliferation

[0075] Cell Counting Kit-8 (CCK-8; Cat# BA00208, Bioss, China) was used to analyze and evaluate the cytotoxicity of isolated exosomes at different concentrations. C6 cells were collected in the exponential growth phase and three replicates of each group were inoculated into 96-well plates (5×10 3 , 100ul / well), and the edge wells of the 96-well plate were filled with 200μl sterile PBS. After cell attachment (confluence reached 70%–80% confluence), C6 cells were treated with different concentrations of EV (0, 0.5, 1, 5, 10, 50, 100μg / ml) and continued to be cultured in a standard incubator for 24h, 48h, and 72h. Then, 10μl CCK-8 was added to each well and incubated for 2 hours. The absorbance (A) value was recorded at a wavelength of 450nm using a microplate reader (Spectramax).

[0076] Cell inhibition rate (%) = [(Ac-As) / (Ac-Ab)] × 100%;

[0077] Wherein: As: absorbance of the experimental group; Ac: absorbance of the control group (containing cells, culture medium, CCK-8 solution, but no drugs); Ab: absorbance of the blank group.

[0078] (3) Cell apoptosis flow cytometry

[0079] Cell apoptosis was detected using Annexin V-APC / 7-AAD apoptosis kit (AT105, MultiSciences, China) according to the manufacturer's instructions. Briefly, 2 × 10 6 Cells were then resuspended in 500 μl of apoptosis positive control solution and the oviducts were placed on ice for 30 min. Resuspended in 1× binding buffer with annexin V-APC (5 μl / tube) and 7-AAD (10 μl / tube) and incubated in the dark for about 5 min. Finally, the cells were analyzed by flow cytometry.

[13] .

[0080] (4) Scratch assay to detect cell migration ability

[0081] To evaluate how the isolated exosomes affect the rate of C6 cell migration, a scratch assay was used. A total of 2.5 × 10 5 Cells / ml were incubated in a 6-well plate until 80% confluence. A straight line was then drawn in the middle of each well with a sterile 200 μl pipette tip. Culture medium with selected concentrations of exosomes (0, 10, 50, and 100 μg / ml) was added to the wells, and the cells were photographed at 0, 6, 12, 24, and 48 hours after scratching and the average migration distance was calculated. Cell migration rate = (mean initial intercellular distance - mean intercellular distance at time t) / mean initial intercellular distance.

[0082] (5) Transwell assay to detect cell invasion ability

[0083] To evaluate changes in cell invasion behavior after co-culture with exosomes, Transwell assays were performed. Briefly, BME (1:4, Cat#:3632-001-02, R&D Systems, Minneapolis, USA) was diluted with F-12K medium to coat the bottom of the Transwell upper chamber to form a matrix barrier for evaluating cell invasion. 5200 μL of C6 cell suspension was inoculated into the upper chamber of Transwell, H-Exos were added at different concentrations (0, 10, 50 and 100 μg / mL), and 650 μL of complete culture medium was added into the lower chamber of Transwell. After incubation at 37°C and 5% CO2 for 24 h, the cells in the upper chamber were gently wiped with a cotton swab, and the chamber was fixed with 4% paraformaldehyde (G1101, Servicebio, China) for 30 min. After rinsing with PBS, the cells were stained with 0.1% crystal violet (G1014; Servicebio, China), and rinsed with PBS three times to remove the crystal violet that was not bound to the cells. After air drying, the cells were photographed under an inverted phase contrast microscope (Nikon, ), and five fields of view under a 10x microscope were selected to observe the cells and counted to obtain the average value for quantification.

[0084] 5. Mass spectrometry

[0085] Each protein sample was reduced with 10 mM DTT and 50 mM iodoacetamide in 0.1 M NH4HCO jinxing Alkylation for 30 min. Then, the sample was digested with 0.5 μg / μL trypsin at 37°C overnight. The next day, 0.1% formic acid was added to acidify the sample to terminate the digestion, and the peptide solution was collected. After repeated blowing, the sample was extracted by ZiptipC18 (Millipore, US) solid phase extraction to clean the salt in the sample, and the eluate was collected and separated by EASY-nLC liquid phase (A = 0.1% formic acid aqueous solution, B = 80% acetonitrile / 20% buffer A), low pH reversed phase C18 capillary chromatography (150μm×150mm, 1.9μm), with an effective elution gradient of 1%-35%, a total elution time of 90min, and a flow rate of 1.2μL / min. The peptide mixture was identified by OrbitrapExploris480 mass spectrometer analysis (ThermoFisherScientific). High sensitivity mode was used, and the scanning time was 90min. The first-level full scan resolution was 120,000, the scan range was 350-1200 m / z, and the dynamic gain control (AGC) was set to 3×10 6 , the maximum injection time was 50ms; the first 40 ions were screened after each primary scan and entered the collision cell for fragmentation, the collision energy was 30%, the secondary scan resolution was 30000, the charge state was screened (including precursors with charges from +2 to +7), and the dynamic elimination was 90s. The raw data files were processed using Thermo Scientific Proteome Discoverer 2.2, and the data were further visualized and analyzed using Perseus (vesion: 1.5.5.1) software.

[0086] 6. Bilateral subcutaneous tumor-bearing rat model

[0087] Rats were anesthetized with 20 mg / mL tribromoethanol working solution at 10 mL / kg intraperitoneally, and 1×10 7 C6 cells. When the maximum diameter of the tumor was ≥10 mm, one side of the tumor was treated with MWA. About 100 μg of H-Exos were injected through the tail vein on days 3, 7, 14, and 21 after ablation, and the three diameters of the tumor were measured by ultrasound or vernier caliper. The three diameters of the maximum section of the tumor, L (length), W (width), and H (height), were measured, and the tumor volume was calculated according to the calculation formula V = π / 6 × L × W × H. The bilateral subcutaneous tumor volume of the three groups was monitored regularly.

[0088] 7. Microwave ablation

[0089] like Figure 5 As shown, after the rats were anesthetized, the skin was disinfected. Under ultrasound guidance, an isolation zone was placed around the tumor, especially close to the skin, and saline-soaked gauze was covered to protect the skin around the tumor. The microwave ablation electrode needle (2450MHz, diameter = 1.6mm, length = 10cm, Nanjing Ruibo Medical Technology Co., Ltd.) was inserted into the tumor and the entire tumor was continuously ablated at a power of 5w. The MWA group only ablated unilateral tumors; in addition to MWA of unilateral tumors, the MWA+H-Exos group was given H-Exos at 4 time points after ablation to treat subcutaneous glioma model rats. Subsequently, unilateral ablation was performed randomly on inguinal tumors. To protect the surrounding tissues, an isolation zone was placed around the tumor, especially close to the skin, under ultrasound guidance before MWA.

[0090] 8. Ultrasound examination

[0091] Ultrasound examination was performed using a Canon I800 color ultrasound diagnostic instrument and an I18LX5 broadband probe, with a frequency of 18 MHz and a mechanical index of 0.09. The contrast agent was SonoVue for ultrasound contrast imaging. The imaging depth, sound pressure, and gain settings remained unchanged during the contrast imaging process. The rats were anesthetized and placed in a lateral position on an animal warming board. The contrast imaging mode was entered according to the preset contrast imaging conditions. The rat tail vein was disinfected, and 0.3-0.5 mL of SonoVue suspension was quickly injected. At the same time, the "Time" was turned on for timing, and dynamic observation was performed for 120 seconds and the images were stored.

[0092] In addition, the experimental data were statistically analyzed using SPSS27.0 software, the measurement data were expressed as x-±s, and the t test was used for comparison between groups. P<0.05 was considered statistically significant. All experiments were repeated three times.

[0093] In summary, glioma is a group of primary central nervous system malignant tumors originating from glial cells and is one of the most common and deadly types of brain tumors.

[16] Although in recent years, the treatment strategies of glioma and the genomic investigation of multicenter research alliances have made progress [17-20] However, due to its high heterogeneity, glioma has always been one of the most challenging diseases in oncology. Hyperthermia therapy is becoming increasingly important as a treatment option for cancer.

[21] , and has been validated in multiple cohort studies [22-24] Exosomes can be produced by most cell types and are approximately 40-160 nanometers in diameter.

[25] , a phospholipid bilayer structure [7] , releases the cell contents it carries through fusion of multivesicular bodies (MVBs) to the plasma membrane [26,27] , potentially playing a role in intercellular signaling [28,29] Previous studies have shown that exosomes released after cell stress may act as a signal transduction carrier to affect tumor cell activity.

[10] Studies have shown that hyperthermia can increase the release of exosomes

[30] , and trigger anti-tumor immune response

[31] However, there are few studies on the direct effects of exosomes secreted by cells after heat stress on cells.

[0094] Normally, glial cells continuously release chemokines and other compounds that are favorable to the growth and proliferation of tumor cells. [32-35] After cell death, cell remnants containing chemokines are released, forming a positive feedback loop. In this study, we first extracted and isolated exosomes (H-Exos) from C6 cells stimulated at 43°C, and used protein blotting and TEM images to confirm that the collected vesicles were exosomes that met the characteristics of expert consensus. The number of exosomes excreted by cells after heat stimulation increased significantly, which is consistent with previous research results.

[30] , CAMC / PKH26 staining confirmed that the extracted exosomes could enter C6 cells. The results of CCK8 confirmed that the exosomes themselves significantly reduced the viability of C6 cells without any additional modification and only under heat stimulation, and this effect reached the highest level within the first 48 hours. This indicates that exosomes have caused rapid and consistent cell death in C6 cells. The main effect of H-Exos on inhibiting C6 cell migration occurred 12-24 hours after scratching, which also confirmed the rapid effect of exosomes on C6 cells. This is also consistent with the results of CCK8 and apoptosis flow cytometry, that is, the main effect on cell death was produced within 24 hours after co-culture. Transwell assays studied the invasive behavior of C6 cells cultured with H-Exos. Obviously, exosomes controlled the invasion of C6 cells within 24 hours. All the above findings indicate that H-Exos can limit the migration, proliferation and invasion of tumor cells, thereby preventing the growth of tumor numbers and reducing the chance of developing new tumors elsewhere. The reduction in the proliferation rate of the cells themselves will also lead to a reduction in their impact on the environment, especially when most of them have died through apoptosis, the microenvironment changes, and healthy cells can have a chance to return to normal. Although in all experiments, higher concentrations and durations can prolong the effect of exosomes on C6 cells, the rate of increase is different. Previous studies have shown that high temperatures can inhibit cell proliferation and kill some cells, and can also cause changes in cell morphology, intracellular sodium and calcium levels, and membrane potential, but there is no good correlation with cell mortality.

[36] , this study provides a new perspective on the mechanism of heat-induced cytotoxicity.

[0095] To further explore the potential role of H-Exos released after high temperature, we evaluated the proteome of GEV isolates by mass spectrometry. Proteomics can directly reflect the pathophysiological state of an organism. Comprehensive protein and functional analysis of exosomes can help us understand the mechanism of biological function. The proteome map showed that 1083 proteins in the H-Exos group were differentially expressed compared with the control group. Most of these proteins acted on the extracellular and Exos-related membrane structures, which were related to the formation, transportation and action of exosomes. It also showed that heat stimulation changed the tumor cell microenvironment through H-Exos. KEGG enrichment analysis enriched 59 signaling pathways, mainly involving cell metabolism, cell adhesion junctions, apoptosis and acute inflammatory response pathways. After analyzing the biological information of the biological role played by H-Exos from a macroscopic perspective, the study further screened the differential proteins. Using the Eccentricity algorithm in the protein interaction network, the top ten highest-scoring proteins included 7 downregulated proteins and 3 upregulated proteins. Among the down-regulated proteins, mitogen-activated protein kinase (MAPK) is a group of serine-threonine protein kinases that are activated when cells are stimulated by extracellular stimuli. It is an important transmission chain for signals to be transmitted from the cell surface to the nucleus. This signaling pathway is involved in a series of cell physiological activities such as cell growth, development, differentiation and apoptosis, and is the intersection of multiple signaling pathways. MAPK3 (ERK1) / MAPK1 (ERK2) is an important effector molecule in the pathway. The ERK1 / 2 signaling pathway plays a vital role in regulating cell proliferation, differentiation, apoptosis and migration, and can be activated by different extracellular stimuli such as cytokines, neurotransmitters and hormones.

[37] The RAS-RAF-MEK-ERK1 / 2 signaling pathway is the most widely studied signaling pathway, which directly or indirectly interacts with transcription factors and their regulatory factors to control cell survival, proliferation, and apoptosis.

[38] The significantly inhibited ERK1 / 2 protein pathway, together with the downregulation of proteins such as vascular endothelial growth factor (VEGF) and signal transducer and activator of transcription 3 (STAT3) in the differential protein list, all prove from the molecular mechanism level that the co-culture of C6 cells with H-Exos in the first section of the study inhibits cell migration and proliferation and increases cell apoptosis.

[0096] In order to observe the effect of MWA combined with H-Exos on residual glioma cells after surgery, especially tumor cells that infiltrate and grow at a distance, this study established a subcutaneous bilateral tumor-bearing mouse model to simulate and observe the anti-tumor effect of diffusely growing tumors. This section of the study found that compared with the control group, both the MWA group and the MWA+H-Exos group could inhibit the growth of tumors on the untreated side; compared with the use of MWA alone, the MWA+H-Exos group could significantly inhibit the growth of tumors on the non-ablated side. At the same time, there was no significant difference between the MWA+H-Exos group and the MWA group before the second week, but there was a significant difference between the two from the third to the sixth week. The MWA group showed slow growth after the third week, and there was no significant difference between the control group. This shows that MWA can exert a short-term biological effect to inhibit the growth of homologous tumors. After the use of H-Exos, the biological effect of MWA was amplified, and it exerted a sustained anti-tumor effect on homologous tumor cells.

[39] This in vivo animal experiment confirms our findings in the cell experiment in Chapter 2, which is a supplement to the MWA remote effect mechanism. It is also a reflection of the efficient targeting and specific killing of tumor cells by H-Exos on C6 cells.

[0097] In the central nervous system, exosomes can easily cross the BBB

[40] , and because they are not living cells, they can be extracted and stored for future use more conveniently. This also provides us with a new idea for long-term treatment after infusion after in vitro expansion of exosomes. In addition, they have a lower incidence of adverse events [41,42] . This ideal effect also suggests that we can use exosomes themselves as drugs or use them as targeted drug carriers to deliver tumor cell-inhibiting chemicals to tumor cells. Although this study fully characterized the biological functions of H-Exos, it also has limitations. First, we did not verify its exact mechanism in this part of the study. At the same time, our main research purpose was to explore the anti-tumor effect of MWA combined with H-Exos. Therefore, there was no separate H-Exos group in this study. In the future, the research team will conduct in-depth research on the mechanism of action of H-Exos in affecting tumor cells in order to develop more effective treatments.

[0098] Therefore, cell exosomes have been used in a variety of tumor treatment studies, such as as delivery vehicles for transporting a variety of drugs to treat gliomas, etc. However, the inherent ability of exosomes from the same cell under stress has been overlooked in many cases. According to the results of this study, MHT-stimulated exosomes play an anti-tumor role by reducing the proliferation, migration and invasion of glial cells, which also explains the remote effect in the MWA process from another perspective and provides a new idea for preventing glioma recurrence.

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[0142] Table 1. List of some differentially expressed proteins in H-Exos / Control

[0143]

[0144]

[0145]

[0146] It should be noted that in the present invention, it should be understood that although this specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that those skilled in the art can understand.

Claims

1. A method for combined treatment of glioma by inducing exosomes to exert a remote effect through high temperature, characterized in that: The following steps are involved: Step 1: placing the glioma cells in a high temperature environment for stimulation for a period of time to induce the glioma cells to release exosomes; Step 2, isolating and purifying the released exosomes to obtain high temperature induced H-Exos; Step 3: Apply the H-Exos to the abscopal effect treatment of glioma to inhibit the growth, migration and invasion of distant tumor cells.

2. The method for combined treatment of glioma by inducing exosomes to exert abscopal effect by hyperthermia according to claim 1, characterized in that: In step 1, the glioma cells are rat brain glioma cells C6, and the rat brain glioma cells C6 are cultured in a complete growth medium.

3. The method for combined treatment of glioma by inducing exosomes to exert abscopal effect by high temperature according to claim 2, characterized in that: Take 1.0×10 7 The glioma cells were added into a serum-free culture medium and placed in a 43° C. incubator for continuous high temperature stimulation for 60 min.

4. The method for combined treatment of glioma by inducing exosomes to exert abscopal effect by hyperthermia according to claim 1, characterized in that: In step 2, the steps of isolating and purifying exosomes include ultracentrifugation, nanoparticle tracking analysis, electron microscopy observation and Western blot analysis, and finally H-Exos are obtained.

5. The method for combined treatment of glioma by inducing exosomes to exert abscopal effect by hyperthermia according to claim 1, characterized in that: In step 3, the H-Exos are used for remote effect treatment of glioma via intravenous injection, local injection or other administration methods.

6. The method for combined treatment of glioma by inducing exosomes to exert abscopal effect by hyperthermia according to claim 1, characterized in that: Also includes: The biological effects of H-Exos on glioma cells were evaluated after hyperthermia stimulation, including detection of cell proliferation, apoptosis, migration and invasion ability.

7. The method for combined treatment of glioma by inducing hyperthermia to exert remote effects of exosomes according to claim 1, characterized in that: Also includes: After MWA treatment of glioma, the H-Exos were applied in abscopal therapy to further inhibit the growth of distant tumor cells.

8. The method for combined treatment of glioma by inducing hyperthermia to exert remote effects of exosomes according to claim 7, characterized in that: The H-Exos were administered at four time points: 3 days, 14 days, 21 days and 28 days after MWA treatment.

9. A pharmaceutical composition for abscopal treatment of glioma, characterized in that: Comprising the H-Exos according to claim 1 and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition for abscopal treatment of glioma according to claim 9, characterized in that: The pharmaceutical composition also includes a treatment regimen for microwave ablation.

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