An exosome with low E-cadherin expression, its preparation method and application

Abstract

This invention discloses an exosome with low E-cadherin expression, belonging to the field of tumor immunology. The cells with low E-cadherin expression are HCC827-CDH1. KD In addition to cell lines, this invention also discloses the preparation method and application of the above-mentioned exosomes. The exosomes of this invention are derived from tumor cells with low expression of E-cadherin, and the content of E-cadherin protein loaded in them is significantly reduced. Exosomes with reduced E-cadherin protein content can promote the expression of IFN-γ and Ki67 in CD8 cells, and enhance the cytotoxic function and proliferation ability of CD8 cells. These tumor cell-derived exosomes with reduced E-cadherin expression can be used as cancer vaccines to enhance the effect of tumor immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of tumor immunology, and more particularly to the preparation and application of exosomes expressing low levels of E-cadherin. Background Technology

[0002] In recent years, cancer vaccines based on tumor cell-derived exosomes (TDEs) have shown promising tumor immunotherapy effects in vitro and in animal models, but their performance in clinical trials has been less than satisfactory. This is because, on the one hand, TDEs possess parental tumor cell-associated antigens, giving them the potential to induce anti-tumor immunity; on the other hand, exosomes carry various immunosuppressive molecules such as PD-L1, thereby inhibiting anti-tumor immunity. Therefore, TDEs directly obtained from tumor cells cannot induce strong anti-tumor activity. Enhancing the tumor immunotherapy efficacy of TDEs is a pressing technical problem that needs to be solved in this field. Summary of the Invention

[0003] One of the objectives of this invention is to provide an exosome with low E-cadherin expression to solve the above-mentioned problems.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows: an exosome with low expression of E-cadherin, wherein the E-cadherin loading of the exosome is 9.0%-11.2% of that of conventional exosomes.

[0005] Compared with conventional exosomes, the exosomes described above exhibit significantly reduced expression of E-cadherin protein. The greater the downregulation of E-cadherin protein expression in the exosomes, the more pronounced the enhanced cytotoxicity and proliferative potential of CD8+ T cells. The conventional exosomes referred to are those secreted by HCC827 cells that normally express E-cadherin.

[0006] As a preferred technical solution: the cells expressing low levels of E-cadherin are HCC827-CDH1. KD Cell lines HCC827shEcad-1 and HCC827shEcad-2.

[0007] The cell lines HCC827shEcad-1 and HCC827shEcad-2 can stably express low levels of E-cadherin, thereby secreting exosomes containing large amounts of low-expressing E-cadherin.

[0008] As a preferred technical solution, the above-mentioned HCC827-CDH1 KDThe preparation method of cell lines HCC827shEcad-1 and HCC827shEcad-2 includes the following steps:

[0009] (1) Construction of PLKO.1-Puro-TRC-E-cadherin plasmid: The shRNA sequences 1 and 2 of E-cadherin were synthesized, and then the shRNA sequences of E-cadherin were loaded into the PLKO.1-Puro-TRC vector to obtain PLKO.1-Puro-TRC-E-cadherin plasmid 1 and PLKO.1-Puro-TRC-E-cadherin plasmid 2.

[0010] (2) Preparation of lentivirus: HEK293T cells were transfected with a mixture containing PLKO.1-Puro-TRC-E-cadherin plasmid 1 or plasmid 2, pCMV-dR8.91 and VSV-G plasmids respectively; HEK293T cells were cultured, the culture medium was collected, and the culture medium was processed to obtain lentiviruses packaged with E-cadherinshRNA-1 and E-cadherinshRNA-2.

[0011] (3)HCC827-CDH1 KD Cell line establishment: HCC827 cells were infected with a mixture of lentiviral suspensions containing E-cadherin shRNA-1 or E-cadherin shRNA-2, 1640 medium, and polybrene, respectively. Infected HCC827 cells were further cultured and selected using puromycin to obtain HCC827-CDH1 cells with low E-cadherin expression. KD Cell lines HCC827shEcad-1 and HCC827shEcad-2.

[0012] The HCC827 cell lines HCC827shEcad-1 and HCC827shEcad-2, which were prepared by the above method, can continuously and stably express E-cadherin at low levels. The content of E-cadherin in the exosomes obtained is 9.0%-11.2% of that in conventional exosomes, and the stability is good.

[0013] More preferably, in step (1) above, the method for synthesizing the E-cadherin shRNA sequence is as follows: designing E-cadherin shRNA sequence 1 and sequence 2 and verifying them with NCBI; synthesizing them using the solid-phase phosphoramidite method; the E-cadherin shRNA sequences used are sequences with SEQ.ID NO:1 and SEQ.ID NO:2 respectively;

[0014] The sequence of SEQ.ID NO:1 is:

[0015] CCGGACCGATGACAACAAGCTCGAGCTTGTTGTCATTCTGATCGGTTTTTTG;

[0016] The sequence of SEQ.ID NO:2 is:

[0017] CCGGGAACGAGGCTAACGTCGTAATCTCGAGATTACGACGTTAGCCTCGTTCTTTTTG.

[0018] The lentiviral vector PLKO.1-Puro-TRC used in step (1) above contains AMP resistance, which facilitates subsequent screening for successfully transfected cells using puromycin. The PLKO.1-Puro-TRC vector has a 5' restriction site AgeI and a 3' restriction site EcoRI.

[0019] More preferably, in step (1), the method of loading E-cadherin shRNA sequence 1 or sequence 2 into the PLKO.1-Puro-TRC vector is as follows: after double digestion of E-cadherin shRNA sequence 1 or sequence 2 and PLKO.1-Puro-TRC vector with AgeI and EcoRI, they are ligated with T4 DNA ligase.

[0020] More preferably, in step (1), bacterial transformation is used to...

[0021] The PLKO.1-Puro-TRC-E-cadherin plasmids 1 and 2 were proliferated. Specifically, PLKO.1-Puro-TRC-E-cadherin plasmid 1 or plasmid 2 was transformed into Escherichia coli, and positive monoclonal colonies were screened for expansion culture to extract a large amount of PLKO.1-Puro-TRC-E-cadherin plasmid 1 and plasmid 2.

[0022] Further preferred, in step (2), when transfecting HEK293T cells, using antibiotic-free DMEM medium containing 20% ​​FBS to culture HEK293T cells can maintain the cells in a good growth state, thereby improving the transfection effect.

[0023] More preferably, in step (2), when transfecting HEK293T cells with the mixture, xtremeGeneHP transfection reagent is used for transfection; this transfection reagent can improve transfection efficiency.

[0024] Further preferably, in step (2), pCMV-dR8.91 and VSV-G are both lentiviral assembly plasmids; PLKO.1-Puro-TRC-E-cadherin is an E-cadherin shRNA encoding plasmid; preferably, the mass ratio of PLKO.1-Puro-TRC-E-cadherin plasmid 1 or plasmid 2, pCMV-dR8.91 and VSV-G in the mixture is 3:2.7:0.3; at this ratio, HEK293T cells have the best transfection efficiency and the highest viral titer.

[0025] Further preferred, the preparation method of the mixture in step (2) includes: mixing pCMV-dR8.91 and VSV-G according to the mass ratio, and then adding PLKO.1-Puro-TRC-E-cadherin plasmid 1 or plasmid 2 according to the mass ratio; adding 582ul serum-free opti-MEM and waiting for 5min; then adding 18ul xtremeGene HP transfection reagent and waiting for 15min; finally obtaining the mixture.

[0026] In a further preferred embodiment, the method for culturing transfected HEK 293T cells in step (2) includes: culturing the transfected HEK 293T cells for 10 hours, then culturing them in DMEM medium containing 20% ​​FBS for another 24 hours, and then collecting the medium.

[0027] In step (2), the culture medium obtained is a virus-containing culture medium, which is obtained by centrifuging at 4°C and 2000g to remove cell debris.

[0028] In a further preferred embodiment, in step (3), HCC827 cells are revived, and when the cell density reaches 40%-60%, virus-containing culture medium is added and cultured for 24 hours.

[0029] More preferably, in step (3), the working concentration of polybrene is 10 mg / ml. At this concentration, the virus infection efficiency is the highest and the cytotoxicity is the lowest.

[0030] Further preferred, in step (3), puromycin is used to screen successfully transfected cells. The working concentration of puromycin is 2.5 ug / ml, and the treatment time is 48 h. Under these conditions, untransfected cells can be removed most effectively, and HCC827 cells HCC827shEcad-1 and HCC827shEcad-2 with stable low expression of E-cadherin can be obtained.

[0031] The second objective of this invention is to provide a method for preparing exosomes with low E-cadherin expression, the technical solution of which includes the following steps:

[0032] (1) Preparation of HCC827 cells HCC827shEcad-1 and HCC827shEcad-2 with low expression of E-cadherin;

[0033] (2) The HCC827 cells HCC827shEcad-1 and HCC827shEcad-2 with low expression of E-cadherin obtained in step (1) were cultured in 1640 medium without exosomes. After the culture was completed, the culture medium was collected.

[0034] (3) The culture medium collected in step (2) was subjected to gradient centrifugation to obtain exosomes exo-HCC827shEcad-1 and exo-HCC827shEcad-2 containing low expression of E-cadherin.

[0035] Preferably, in step (3), the method for separating and purifying the culture supernatant includes: a) centrifuging the culture medium at 4°C and 300g for 10 min, discarding dead cells and cell debris, and keeping the supernatant for later use; b) centrifuging the supernatant at 4°C and 2000g for 10 min, discarding dead cells and cell debris, and keeping the supernatant for later use; c) centrifuging the supernatant at 4°C and 10000g for 30 min, discarding large protein particles, and keeping the supernatant for later use; d) centrifuging the supernatant at 4°C and 100000g for 70 min, and taking the precipitate for later use; e) adding PBS to the precipitate to resuspend the precipitate, and centrifuging at 4°C and 100000g for 70 min, the precipitate being exosomes exo-HCC827shEcad-1 and exo-HCC827shEcad-2 with low expression of E-cadherin.

[0036] A third objective of this invention is to provide the application of the above-mentioned exosomes in the preparation of cancer vaccines for tumor immunotherapy.

[0037] The inventors of this application, through extensive experimentation, have provided a modified exosome with low E-cadherin expression. This exosome is derived from tumor cells exhibiting low E-cadherin expression. Experiments have demonstrated that the E-cadherin expression carried by this exosome enhances the cytotoxicity and proliferative potential of CD8+ T cells through interaction with them. This invention provides tumor cell-derived exosomes modified with reduced E-cadherin expression, which can serve as a cancer vaccine and enhance the efficacy of tumor immunotherapy.

[0038] Compared with the prior art, the advantages of the present invention are as follows: The exosomes of the present invention are derived from tumor cells with low expression of E-cadherin, and the content of E-cadherin protein loaded in them is significantly reduced. Exosomes with reduced E-cadherin protein content can promote the expression of IFN-γ and Ki67 in CD8 cells, enhance the cytotoxic function and proliferation ability of CD8 cells, and these tumor cell-derived exosomes with reduced E-cadherin expression can be used as cancer vaccines to enhance the effect of tumor immunotherapy. Attached Figure Description

[0039] Figure 1 The images show the identification results of the infected HCC827 cell lines in Example 1 and Comparative Example 1 ((A) is the Western blot result; (B) is the real-time quantitative PCR result).

[0040] Figure 2 Image showing the results of electron microscopy identification of exosomes: Morphological structure of exosomes observed under a transmission electron microscope;

[0041] Figure 3 Figure showing the results of exosome particle size identification: Exosome particle size distribution detected by nanoparticle tracking analysis (NTA);

[0042] Figure 4 Western blot results of exosomal protein markers and E-cadherin content ((A) Western blot results of exosomal markers; (B) Western blot results of exosomal E-cadherin content).

[0043] Figure 5 The effect of low-expression E-cadherin exosomes on the cytotoxicity of CD8+ T cells;

[0044] Figure 6 To investigate the effect of low-expression E-cadherin exosomes on the proliferation function of CD8+ T cells. Detailed Implementation

[0045] The invention will now be further described with reference to the accompanying drawings.

[0046] Example 1

[0047] The establishment of a tumor cell line with low E-cadherin expression includes the following steps:

[0048] (1) Construction of PLKO.1-Puro-TRC-E-cadherin plasmid: The shRNA sequences 1 and 2 of E-cadherin were synthesized, and then the shRNA sequences of E-cadherin were loaded into the PLKO.1-Puro-TRC vector to obtain PLKO.1-Puro-TRC-E-cadherin plasmid 1 and PLKO.1-Puro-TRC-E-cadherin plasmid 2.

[0049] The shRNA was synthesized using a solid-phase phosphoramidite method; the E-cadherin shRNA sequences 1 and 2 used were SEQ.ID NO:1 and SEQ.ID NO:2, respectively;

[0050] The sequence of SEQ.ID NO:1 is:

[0051] CCGGACCGATGACAACAAGCTCGAGCTTGTTGTCATTCTGATCGGTTTTTTG;

[0052] The sequence of SEQ.ID NO:2 is:

[0053] CCGGGAACGAGGCTAACGTCGTAATCTCGAGATTACGACGTTAGCCTCGTTCTTTTTG;

[0054] Among them, the lentiviral vector PLKO.1-Puro-TRC contains AMP resistance and has a 5' restriction site AgeI and a 3' restriction site EcoRI.

[0055] E-cadherin shRNA sequence 1 and sequence 2 and the PLKO.1-Puro-TRC vector were double-digested with AgeI and EcoRI, ligated with T4 DNA ligase, transformed into E. coli, and positive single colonies were screened and expanded to obtain a large number of PLKO.1-Puro-TRC-E-cadherin plasmids.

[0056] Cell preparation: HEK293T cells were revived and cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin and streptomycin at 37°C in a constant temperature incubator with 5% volCO2. After two passages, the cells were seeded into 10cm culture dishes. Lentiviral cells were packaged when the cells reached 45% confluence.

[0057] (2) Lentiviral preparation: Solution a was prepared by mixing a solution containing 3 μg pCMV-dR8.91 and 2.7 μg VSV-G; solutions b1 and b2 were prepared by adding 0.3 μg PLKO.1-Puro-TRC-E-cadherin plasmid 1 or plasmid 2 to solution a; solutions c1 and c2 were prepared by adding 582 μL silane-free opti-MEM to solutions b1 and b2 and waiting for 5 min; solutions d1 and d2 were prepared by adding 18 μl xtremeGene HP transfection reagent to solutions c1 and c2 and waiting for 15 min; solutions d1 and d2 were prepared by adding HEK to the above solution. In 293T cells, after culturing in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2 for 10 h, the culture medium was replaced with DMEM medium containing 20% ​​FBS and cultured for another 24 h. The culture medium was then collected. The supernatant obtained by centrifuging the culture medium at 4°C and 2000g to remove cell debris was the virus suspension.

[0058] (3) Establishment of HCC827-CDH1KD cell lines HCC827shEcad-1 and exo-HCC827shEcad-2: HCC827 cells were revived and cultured in 1640 medium containing 10% fetal bovine serum and 1 wt% penicillin and streptomycin at 37°C in a constant temperature incubator with 5% vol CO2. After two passages, the cells were seeded in 10 cm culture dishes. When the cells reached 50% confluence, 1 ml of lentiviral suspension E-cadherin shRNA-1 or E-cadherin shRNA-2, 9 ml of 1640 medium, and 10 mg / ml of polybrene were added. After culturing 10 μl of the mixture for 24 h, the culture medium was changed, and 2.5 μg / ml of purine mold was added for selection. After 48 h, the culture medium was changed again and the successfully transfected cells were cultured to obtain HCC827 cells HCC827shEcad-1 and exo-HCC827shEcad-2 with stable low expression of E-cadherin.

[0059] Example 2

[0060] The preparation of exosomes with low E-cadherin expression includes the following steps:

[0061] HCC827shEcad-1 and exo-HCC827shEcad-2 cells, which expressed low levels of E-cadherin, were cultured in 1640 medium. When the cells reached 75% confluence, they were cultured for another 48 hours in 1640 medium containing 10 wt% exosome-free fetal bovine serum under 5% CO2 conditions. The culture supernatant was then collected. The collected culture supernatant was then separated and purified.

[0062] a. Centrifuge the culture medium at 4℃ and 300g for 10 minutes, discard dead cells and cell debris, and keep the supernatant for later use.

[0063] b. Centrifuge the supernatant at 4°C and 2000g for 10 minutes, discard dead cells and cell debris, and keep the supernatant for later use.

[0064] c. Centrifuge the supernatant at 4°C and 10,000g for 30 minutes, discard the large protein particles, and keep the supernatant for later use.

[0065] d. Centrifuge the supernatant at 4°C and 100,000g for 70 minutes, and collect the precipitate for later use;

[0066] e. Add PBS to the above precipitate to resuspend the precipitate, and centrifuge at 4℃ and 100,000g for 70 min. The precipitate is exo-HCC827shEcad-1 and exo-HCC827shEcad-2, which express low levels of E-cadherin.

[0067] Comparative Example 1

[0068] The difference between this comparative example and Examples 1 and 2 is that:

[0069] Cell preparation: HEK293T cells were revived and cultured in DMEM containing 10wt% fetal bovine serum and 1wt% penicillin and streptomycin at 37°C in a constant temperature incubator with 5% volCO2. After two passages, the cells were seeded in 10cm culture dishes. Lentiviral cells were packaged when the cells reached 45% confluence.

[0070] Lentiviral preparation: Solution a was prepared by mixing 3 μg pCMV-dR8.91 and 2.7 μg VSV-G; Solution b was prepared by adding 0.3 μg PLKO.1-Puro-TRC to Solution a; Solution c was prepared by adding 582 μl serum-free opti-MEM to Solution b and waiting for 5 min; Solution d was prepared by adding 18 μl xtremeGeneHP transfection reagent to Solution c and waiting for 15 min; Mixture d was added to HEK 293T cells and cultured in DMEM containing 10 wt% fetal bovine serum at 37°C and 5% volCO2 for 10 h, then cultured in DMEM containing 20 wt% FBS for another 24 h, and the culture medium was collected; The supernatant obtained by centrifuging the culture medium at 4°C and 2000g to remove cell debris was the viral suspension.

[0071] Establishment of the HCC827 scramble cell line: HCC827 cells were revived and cultured in a 37°C, 5% volCO2 incubator using 1640 medium containing 10 wt% fetal bovine serum and 1 wt% penicillin and streptomycin. After two passages, the cells were seeded into 10 cm culture dishes. When the cells reached 50% confluence, a mixture of 1 ml of the above lentivirus suspension, 9 ml of 1640 medium, and 10 μl of polybrene at a concentration of 10 mg / ml was added. After culturing for 24 h, the medium was changed, and 2.5 μg / ml of puromycin was added for selection. After 48 h, the medium was changed again and the successfully transfected cells were cultured to obtain the HCC827 scramble cell line.

[0072] HCC827 scramble exosomes acquisition: HCC827 scramble cells were cultured in 1640 medium. When the cells reached 75% confluence, they were cultured in 1640 medium containing 10 wt% exosome-free fetal bovine serum at 5% vol CO2 for 48 h. The culture supernatant was collected. The collected culture supernatant was then separated and purified: a) The culture medium was centrifuged at 4℃ and 300g for 10 min, and dead cells and cell debris were discarded, with the supernatant kept for later use; b) The supernatant was centrifuged at 4℃ and 2000g. a) Centrifuge at 4°C and 10000g for 10 min, discard dead cells and cell debris, and keep the supernatant for later use; b) Centrifuge the supernatant at 4°C and 10000g for 30 min, discard large protein particles, and keep the supernatant for later use; c) Centrifuge the supernatant at 4°C and 100000g for 70 min, and keep the precipitate for later use; e) Resuspend the precipitate in PBS, and centrifuge at 4°C and 100000g for 70 min, the precipitate is HCC827 scramble exosomes.

[0073] Experimental example:

[0074] 1. HCC827-CDH1 KD Identification of cell lines HCC827shEcad-1 and exo-HCC827shEcad-2:

[0075] The HCC827-CDH1 strain established in Example 1 was detected by Western blot and real-time quantitative PCR, respectively. KD The levels of E-cadherin protein and mRNA in cell lines HCC827shEcad-1 and exo-HCC827shEcad-2 and HCC827 scramble cells in Comparative Example 1.

[0076] The results showed that clear and dense E-cadherin protein bands were detected in HCC827 scramble cell lysates, while lighter and narrower E-cadherin protein bands were detected in HCC827shEcad-1 and HCC827shEcad-2 cell lysates of the same weight. Figure 1 A. Furthermore, the expression levels of E-cadherin mRNA in HCC827 scramble cells were 12.5 times and 20 times higher than those in HCC827shEcad-1 and HCC827shEcad-2 cells, respectively. Figure 1 B.

[0077] 2. Identification of exosomes:

[0078] Transmission electron microscopy revealed that the exosomes derived from the parent HCC827 cells were cystic structures. Figure 2 Nanosight nanoparticle size analysis showed that the exosome particle size distribution ranged from 30 to 100 nm. Figure 3 Western blot analysis showed that, compared to the cell group, the exosome group exhibited clearer and more intense bands for the exosome-positive marker proteins CD81 and CD63, while the exosome-negative marker protein Calnexin was extremely faint. Figure 4 A.

[0079] 3. Identification of exosomes with low E-cadherin expression:

[0080] Western blot analysis showed that the E-cadherin protein band in the HCC827 scramble exosomes obtained in Comparative Example 1 was clear and intense, while the E-cadherin protein bands in the HCC827shEcad-1 and HCC827shEcad-2 exosomes were lighter and narrower. Gel image analysis showed that the optical densities of the E-cadherin protein bands in the HCC827 scramble, HCC827shEcad-1, and HCC827shEcad-2 exosomes were 1268, 142, and 114, respectively. This means that the E-cadherin protein expression in the exosomes derived from HCC827shEcad-1 and HCC827shEcad-2 cells was 11.2% (142 / 1268 = 11.2%) and 9.0% (114 / 1268 = 9%), respectively, in the exosomes derived from HCC827 scramble cells. (See [link to article]). Figure 4 B.

[0081] 4. The experiment on the effects of exosomes with low E-cadherin expression on the proliferation and cytotoxicity of CD8+ T cells was conducted using the following steps:

[0082] Venous blood was drawn from healthy individuals, and peripheral blood mononuclear cells (PBMCs) were obtained using Ficoll separation solution according to the instructions; 1.5 x 10 6 One PBMC was seeded in a 24-well plate, and 5 μl of PBS, HCC827 scramble exosomes obtained in Comparative Example 1, HCC827shEcad-1 and HCC827shEcad-2 exosomes were added respectively. After co-culturing for 5 days, the expression of IFN-γ and Ki67 in CD8+ T cells was detected by flow cytometry.

[0083] The results showed that the expression of IFN-γ and Ki67 in CD8+ T cells in the HCC827shEcad-1 and HCC827shEcad-2 groups was significantly higher than that in the HCC827 scramble group. Figure 5 and Figure 6 .

[0084] The above description merely illustrates specific embodiments of the present invention and should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. An exosome expressing low levels of E-cadherin, characterized in that: The exosomes were prepared using the following steps: (1) Preparation of HCC827 cells with low expression of E-cadherin; the HCC827 cells are HCC827-CDH1 KD Cell lines HCC827shEcad-1 and HCC827shEcad-2; HCC827-CDH1 KD The preparation methods for cell lines HCC827shEcad-1 and HCC827shEcad-2 include the following steps: 1) Construction of PLKO.1-Puro-TRC-E-cadherin plasmid: E-cadherin shRNA sequences 1 and 2 were synthesized, and then the E-cadherin shRNA sequences were loaded into the PLKO.1-Puro-TRC vector to obtain PLKO.1-Puro-TRC-E-cadherin plasmid 1 and PLKO.1-Puro-TRC-E-cadherin plasmid 2. The method for synthesizing the E-cadherin shRNA sequence was as follows: the E-cadherin shRNA sequence was designed and verified by NCBI; it was synthesized using the solid-phase phosphoramidite method; the E-cadherin shRNA sequences used were SEQ ID NO:1 and SEQ ID NO:2, respectively. 2) Preparation of lentivirus: HEK293T cells were transfected with a mixture containing plasmid 1 or 2 of PLKO.1-Puro-TRC-E-cadherin, pCMVdR8.91 and VSV-G plasmids respectively; HEK293T cells were cultured, the culture medium was collected, and the culture medium was processed to obtain lentiviruses packaged with E-cadherin shRNA-1 and E-cadherin shRNA-2; 3) HCC827-CDH1 KD Cell line establishment: HCC827 cells were infected with a mixture of lentiviral suspensions containing E-cadherin shRNA-1 or E-cadherin shRNA-2, 1640 medium, and polybrene, respectively. Infected HCC827 cells were further cultured and selected using puromycin to obtain HCC827-CDH1 cells with low E-cadherin expression. KD Cell lines HCC827shEcad-1 and HCC827shEcad-2; (2) The HCC827 cells with low E-cadherin expression obtained in step (1) were cultured in 1640 medium without exosomes. After the culture was completed, the culture medium was collected. (3) The culture medium collected in step (2) is subjected to gradient centrifugation to obtain exosomes containing low expression of E-cadherin; the E-cadherin loading of the exosomes is 9.0% or 11.2% of that of conventional exosomes.

2. The exosomes according to claim 1, characterized in that, In step 2), HEK293T cells were cultured in antibiotic-free DMEM medium containing 20% ​​FBS during transfection.

3. The exosomes according to claim 2, characterized in that, In step 2), pCMV-dR8.91 and VSV-G are both lentiviral assembly plasmids; PLKO.1-Puro-TRC-E-cadherin plasmid 1 and plasmid 2 are E-cadherin shRNA-1 and E-cadherin shRNA-2 encoding plasmids; the mass ratio of PLKO.1-Puro-TRC-E-cadherin plasmid 1 or plasmid 2, pCMV-dR8.91 and VSV-G in the mixture is 3:2.7:0.

3.

4. The exosomes according to claim 3, characterized in that, In step 3), HCC827 cells were revived. When the cell density reached 40%-60%, virus-containing culture medium was added and cultured for 24 hours.

5. The exosome according to claim 4, characterized in that, In step 3), the working concentration of polybrene is 10 mg / ml.

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