Application of proton pump inhibitor

Abstract

The invention discloses a new application of a proton pump inhibitor. The proton pump inhibitor realizes treatment on related diseases and enhances the treatment effect by promoting endocytosis of extracellular vesicles. The proton pump inhibitor is used for enhancing anti-tumor immunity; the proton pump inhibitor is used for relieving pd-1 drug resistance in tumor treatment; the proton pump inhibitor is used for enhancing the drug delivery function of the extracellular vesicles; the proton pump inhibitor is used for removing extracellular vesicles. The invention finds that the proton pump inhibitor can enhance anti-tumor immunity, reduce drug resistance, enhance the delivery effect of extracellular vesicles on therapeutic drugs and reduce side effects of the drugs. The invention provides a pharmaceutical composition containing the proton pump inhibitor, and the pharmaceutical composition has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of a proton pump inhibitor. Background Art

[0002] Cancer is a disease that seriously endangers human health. Current treatments mainly include surgery, radiotherapy, and chemotherapy. However, these treatments have some limitations, such as large surgical trauma, radiation damage from radiotherapy, and toxic side effects from chemotherapy. More importantly, cancer cells will gradually develop resistance to chemotherapy drugs, resulting in decreased or failed treatment effects. Therefore, finding new treatment strategies and drugs is an urgent need for cancer treatment.

[0003] Extracellular vesicles (EVs) are a class of tiny membrane-structured particles secreted by cells, including exosomes, microvesicles, and apoptotic bodies, which can carry biological molecules such as proteins, nucleic acids, and lipids to communicate between cells. EVs play an important role in physiological and pathological processes and can serve as carriers of intercellular communication, delivering signal molecules such as proteins, nucleic acids, and lipids. The secretion and endocytosis of EVs is a dynamic equilibrium process that is regulated by multiple factors.

[0004] In recent years, EVs have attracted extensive attention in cancer diagnosis and treatment because they have the following advantages: (1) they can cross the blood-brain barrier and reach hard-to-reach sites; (2) they can evade clearance by the immune system and prolong circulation life; (3) they can selectively target specific cells or tissues to improve drug delivery efficiency; and (4) they can regulate the tumor microenvironment and inhibit tumor growth and metastasis.

[0005] However, EVs also have some problems, such as low content in the body and poor stability. In addition, the endocytosis of EVs is affected by many factors, such as pH, temperature, ion concentration, etc. Therefore, there is still a lack of a complete technology to use EVs to treat diseases such as cancer. Therefore, finding a technology and drug to improve the endocytosis efficiency and therapeutic effect of EVs is a major issue in the current field of cancer treatment. Summary of the invention

[0006] In order to solve the above problems, the present invention proposes a method for promoting the endocytosis of EVs by using proton pump inhibitors (PPIs), which can significantly improve the endocytosis efficiency and therapeutic effect of EVs. Among them, the proton pump is a type of protein that can transport protons from inside the cell to outside the cell, and participates in maintaining the pH balance inside and outside the cell. The expression level and activity of the proton pump on the plasma membrane affect the secretion and endocytosis of EVs. Proton pump inhibitors are a type of drug that can inhibit gastric acid secretion and are commonly used to treat peptic ulcers, gastroesophageal reflux, etc. However, the present invention has found that proton pump inhibitors also have anti-tumor activity, and further found that proton pump inhibitors increase the uptake of EVs by tumor cells, thereby enhancing the role of EVs in tumor treatment.

[0007] The present invention first studies and finds that the level of EVs in the supernatant of tumor cells is reduced under the action of proton pump inhibitors; then further examines the effect of PPIs on multivesicular bodies, and finds that PPIs have no effect on the degradation of multivesicular bodies, nor do they affect the fusion of MVBs and PMs, indicating that PPIs are unlikely to regulate the biogenesis of EVs; the present invention also proves through research that the reduction of supernatant EVs mediated by PPIs is due to the fact that PPIs promote the uptake of EVs by cells, and PPIs can promote the uptake of EVs by a variety of cells.

[0008] Subsequently, the present invention studied the mechanism of the above research and found that: PPIs treatment did not affect the membrane fusion of EVs, but increased macropinocytosis and PM localization of ATP6V1A (ATP6V1A is the name of a gene, encoding a protein complex called v-ATPases (proton pump), v-ATPases are located on the plasma membrane (PM), which can convert ATP into ADP and pump protons from the cell to the outside of the cell), indicating that PPIs increase EVs endocytosis by increasing macropinocytosis. Inhibition of vesicle protein- or clathrin-mediated endocytosis can moderately or slightly reduce EVs uptake by PEMs and 4T1 cells, but has no effect on EVs uptake by T cells. Inhibition of macropinocytosis inhibits the uptake of EVs by all cells. The increase of v-ATPases on the plasma membrane is a key step in PPIs-induced macropinocytosis.

[0009] Finally, the present invention verifies that PPIs-mediated EV endocytosis is carried out in a pH-dependent manner, and low pH induces PM accumulation of ATP6V1A. Finally, we found and verified that PPIs-mediated reduction of cytoplasmic pH promotes the dissociation of ATP6V1A from VM, thereby enhancing the assembly of PM v-ATPases.

[0010] In one aspect, the present invention provides a use of a proton pump inhibitor for preparing an anti-tumor preparation, wherein the proton pump inhibitor is selected from one or more of omeprazole, lansoprazole, esomeprazole, pantoprazole, rabeprazole, dacilprazole or a pharmaceutically acceptable salt thereof.

[0011] Furthermore, the proton pump inhibitor improves anti-tumor immunity by promoting the clearance of extracellular vesicles.

[0012] Furthermore, the proton pump inhibitors can lead to accelerated EV uptake by tumor cells, accelerate the arrival of the absorption platform and enhance the absorption intensity; PPIs-mediated inhibition of anti-tumor T-cell responses depends on macropinocytosis; PPIs enhance anti-tumor immunity by promoting the clearance of tumor-derived extracellular vesicles (TEVs).

[0013] On the other hand, the present invention provides a use of a proton pump inhibitor for preparing a preparation for reducing drug resistance to anti-PD-1 treatment, wherein the proton pump inhibitor is selected from one or more of omeprazole, lansoprazole, esomeprazole, pantoprazole, rabeprazole, dacilprazole or a pharmaceutically acceptable salt thereof.

[0014] Furthermore, the proton pump inhibitors attenuate resistance to anti-PD-1 therapy by clearing extracellular vesicles.

[0015] Furthermore, PPIs can prevent PD-L1+TEV-induced immunosuppression and eliminate resistance to anti-PD-1 therapy. The mechanism is to weaken resistance to anti-PD-1 therapy by eliminating immunosuppressive TEV.

[0016] On the other hand, the present invention provides a use of a proton pump inhibitor for preparing a preparation for promoting drug delivery, characterized in that the proton pump inhibitor is selected from one or more of omeprazole, lansoprazole, esomeprazole, pantoprazole, rabeprazole, dacilprazole or a pharmaceutically acceptable salt thereof.

[0017] Furthermore, the drug is delivered via extracellular vesicles as carriers; the proton pump inhibitor promotes the endocytosis of extracellular vesicles, thereby promoting the delivery of drugs by extracellular vesicles.

[0018] Specifically, PPIs can enhance the delivery effect of EVs on therapeutic drugs and have a synergistic effect on the downregulation of miR-155 levels in ALF liver induced by rbc-ev / miR-155-aso, significantly improving the therapeutic effect of RBC-EVs / miR-155-ASOs on ALF.

[0019] On the other hand, the present invention provides a use of a proton pump inhibitor for preparing a preparation for reducing the content of extracellular vesicles in an organism, characterized in that the proton pump inhibitor is selected from one or more of omeprazole, lansoprazole, esomeprazole, pantoprazole, rabeprazole, dacilprazole or a pharmaceutically acceptable salt thereof.

[0020] Furthermore, the proton pump inhibitor increases the endocytosis of extracellular vesicles by increasing macropinocytosis, thereby reducing the extracellular vesicle content.

[0021] Furthermore, the proton pump inhibitors promote the assembly of PM v-ATPases by lowering pH, thereby increasing macropinocytosis.

[0022] In another aspect, the present invention provides a product for anti-tumor, or drug delivery, or reducing resistance to anti-PD-1 therapy, the product comprising a proton pump inhibitor.

[0023] Furthermore, the product for anti-tumor, drug delivery, or reducing resistance to anti-PD-1 therapy can be a combination of PPIs and one or more other drugs, and the PPIs can significantly increase the delivery efficiency of other drugs.

[0024] Furthermore, the other drugs may be anti-tumor drugs, and may be one or more of chemotherapeutic drugs, radiotherapeutic drugs, and biotherapeutic drugs; PPIs can increase the anti-tumor effect of anti-tumor drugs, and the two have a synergistic effect.

[0025] Furthermore, the other drugs may be anti-resistance drugs, such as docetaxel, verapamil, cyclosporine A, etc., or any one or a combination thereof; used to overcome the drug resistance of cancer cells to anti-tumor drugs and improve treatment efficiency.

[0026] Furthermore, the other drugs may be immunotherapy drugs, which may be any one or a combination of immune checkpoint inhibitors, cancer vaccines, cytokines, monoclonal antibodies, etc.; used to activate or enhance the body's immune system to identify and eliminate cancer cells.

[0027] Furthermore, the other drugs may be targeted drugs, which may be any one or a combination of tyrosine kinase inhibitors, protein kinase inhibitors, nuclear receptor modulators, epigenetic regulators, etc.; they are used to target specific molecules or signal pathways on the surface or inside cancer cells to achieve precision treatment.

[0028] Furthermore, the selection of other drugs includes but is not limited to the above-mentioned anti-tumor drugs, anti-resistance drugs, immunotherapy drugs, and targeted therapy drugs; in actual application, the drug combination can adjust the types and dosages of various drugs according to different disease types and patient conditions to achieve the best therapeutic effect. The drug combination has a synergistic effect and reduces adverse reactions and toxic side effects.

[0029] The beneficial effects of the present invention include:

[0030] 1. Improve the therapeutic effect of tumors: As a potential anticancer drug, proton pump inhibitors can improve anti-tumor immunity by promoting the endocytosis of extracellular vesicles. In addition, by encapsulating the drugs outside the tumor cells in the vesicles, the direct contact between the drugs and the cytoplasm can be reduced, thereby avoiding the resistance of tumor cells to the drugs and improving the therapeutic effect.

[0031] 2. Overcoming chemotherapy resistance: Some tumor cells are prone to drug resistance to chemotherapy drugs such as PD-1. Proton pump inhibitors can be used to promote the endocytosis of extracellular vesicles, which can encapsulate anti-resistance drugs inside the vesicles, thereby improving their ability to penetrate the cell membrane and overcome chemotherapy resistance.

[0032] 3. Improve drug loading efficiency: Using proton pump inhibitors to promote the endocytosis of extracellular vesicles can not only promote the penetration of drugs into cells, but also greatly improve the drug loading efficiency and avoid excessive use of drugs. This is very useful for some drugs that require high-dose treatment.

[0033] 4. Reduce the side effects of drugs: For some drugs with large side effects, by encapsulating them in vesicles, not only can the targeting of the drugs be improved, but also the impact of the drugs on healthy cells can be reduced, reducing unnecessary side effects.

[0034] 5. Formation of combination drugs: Because proton pump inhibitors promote the endocytosis of extracellular vesicles and have wide applicability, they can be used in combination with a variety of drugs. Therefore, they can be used as a multifunctional drug treatment, providing more options for the treatment of various diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Flow cytometric analysis of CD9 percentage of EVs captured by anti-CD63-coated latex beads from supernatants of 4T1 cells treated with DMSO, Ome, or Rabe

[0036] Figure 2 BCA assay to measure protein content of EVs isolated from equal numbers of 4T1 cells treated with DMSO, Ome or Rabe

[0037] Figure 3Transmission electron microscopy to examine EV morphology in 4T1 cells treated with DMSO or Rabe

[0038] Figure 4 Nanoparticle tracking analysis of size distribution of EVs in 4T1 cells treated with DMSO or Rabe

[0039] Figure 5 Western blotting analysis of EVs in the supernatants of 4T1 cells treated with DMSO or Rabe and the same number of cells.

[0040] Figure 6 Confocal microscopy analysis of colocalization of CD63+MVBs and LAMP1+lysosomes in 4T1 cells treated with DMSO or Rabe

[0041] Figure 7 Confocal microscopy analysis of colocalization of HRS+MVBs and LAMP1+lysosomes in 4T1 cells treated with DMSO or Rabe

[0042] Figure 8 Confocal microscopy analysis of colocalization of EEA1+EEs and LAMP1+lysosomes in 4T1 cells treated with DMSO or Rabe

[0043] Fig. 9 Confocal microscopy analysis of colocalization of CD63+MVBs and LAMP1+lysosomes in 4T1 cells treated with DMSO or Rabe

[0044] Fig.10 TIRF microscopy analysis of the distribution of CD63+MVBs in 4T1 cells treated with DMSO or Rabe

[0045] Fig.11 Flow cytometric analysis of PKH26-labeled EVs in 4T1 cells treated with DMSO, Ome, or Rabe

[0046] Fig.12 Confocal microscopy analysis of PKH26-labeled EVs in 4T1 cells treated with DMSO, Ome, or Rabe

[0047] Fig.13 Real-time fluorescence microscopy analysis of PKH26-labeled EV uptake kinetics into DMSO- or Rabe-treated PEMs

[0048] Fig.14 Cre-Loxp system for monitoring the effects of PPIs on EV uptake in HEK293 cells

[0049] Fig.15Confocal microscopy analysis of red fluorescent protein (DsRed) and green fluorescent protein (eGFP)

[0050] Fig.16 Flow cytometric analysis of CD9 percentage of EVs captured by anti-CD63 coated latex beads from 4T1 cell supernatants treated with Rabe alone or in combination with cytochalasin D

[0051] Fig.17 Flow cytometric analysis of PKH26-labeled EVs in a variety of indicator cells treated with DMSO or Rabe, including mouse RAW264.7 macrophages, bone marrow-derived dendritic cells (BMDCs), and MC38 colon cancer cells; human THP-1 monocytic leukemia cells, A549 lung cancer cells, and HeLa cervical cancer cells

[0052] Fig.18 Confocal microscopy analysis of PKH26-labeled EV colocalization in phalloidin-labeled 4T1 cells treated with DMSO or Rabe

[0053] Fig.19 Flow cytometric analysis of PKH26-labeled EVs in 4T1 cells treated with Rabe alone or in combination with indomethacin

[0054] Fig. 20 Flow cytometric analysis of PKH26-labeled EVs in 4T1 cells treated with Rabe alone or in combination with chlorpromazine

[0055] Fig.21 Flow cytometric analysis of PKH26-labeled EVs obtained from 4T1 cells treated with Rabe alone or in combination with LY294002 or amiloride

[0056] Fig. 22 Flow cytometric analysis of PKH26-labeled EVs in 4T1 cells treated with DMSO or RABE in the absence or presence of ATP6V1A silencing

[0057] Fig.23 Western blotting analysis of ATP6V1A protein in 4T1 cells transfected with negative control (NC) or Atp6v1a siRNA

[0058] Fig.24 Flow cytometric analysis of PKH26-labeled EVs in 4T1 cells treated with Rabe alone or in combination with KM91104 or Baf-A1 (v-ATases inhibitor)

[0059] Fig.25Flow cytometric analysis of ATP6V1A on PM of 4T1 cells treated with DMSO or Rabe

[0060] Fig.26 Confocal microscopy analysis of ATP6V1A localization in phalloidin-labeled 4T1 cells treated with DMSO or Rabe

[0061] Fig. 27 Flow cytometric analysis of PKH26-labeled EVs in indicator cells treated with DMSO, Baf-A1, indomethacin, or chlorpromazine

[0062] Fig.28 Flow cytometric analysis of PKH26-labeled EVs in 4T1 cells treated with DMSO or Rabe or DMSO+MeβCD or Rabe+MeβCD

[0063] Fig.29 Flow cytometric analysis of PKH26-labeled EVs in 4T1 cells treated with DMSO or Rabe or DMSO + azathioprine or Rabe + azathioprine

[0064] Fig.30 Western blotting analysis of p-PKA and PKA protein in 4T1 cells treated with DMSO or Rabe for the indicated time

[0065] Fig.31 Flow cytometric analysis of 4T1 cells treated with H89 alone or in combination with PKH26-labeled EV

[0066] Fig.32 Western blotting analysis of SLC4A7 expression

[0067] Fig.33 Flow cytometric analysis of PKH26-labeled EVs in 4T1 cells transfected with NC or SLC4A7 siRNA

[0068] Fig.34 Confocal microscopy analysis of pH-sensitive fluorescent probe BCECF-AM indicating pH value in 4T1 cells treated with DMSO or Rabe

[0069] Fig.35 Flow cytometric analysis of EVs labeled with pkh26, 4T1 cells cultured in normal pH (7.2) medium

[0070] Fig.36 Flow cytometric analysis of PKH26-labeled EVs in 4T1 cells cultured in low pH (6.4) medium

[0071] Fig.37Flow cytometric analysis of EVs labeled with pkh26 in 4T1 cells treated with Rabe alone or in combination with 2,4-dinitrophenol (2,4-DNP)

[0072] Fig.38 Flow cytometric analysis of EVs labeled with pkh26 in 4T1 cells treated with Rabe alone or in combination with 2,4-dinitrophenol (2,4-DNP)

[0073] Fig.39 Confocal microscopy analysis of A TP6V1A and LAMP1+ lysosomes in 4T1 cells

[0074] Fig.40 Confocal microscopy analysis of ATP6V1A localization in phalloidin-labeled 4T1 cells

[0075] Fig.41 Confocal microscopy analysis of ATP6V1A and LAMP1+ lysosomal colitis in HeLa cells (left) or ATP6V1A localization in phalloidin-labeled HeLa cells (right) transfected without or with ATP6V0A2 plasmid and without (left) or with (right) cholesterol

[0076] Fig.42 Confocal microscopy analysis of the localization of ATP6V1A in phalloidin-labeled HeLa cells (left) or the co-aggregation of ATP6V1A and LAMP1+ lysosomes in HeLa cells transfected or not with ATP6V1A in medium at normal pH (7.2) (left) or high pH (7.6) (right)

[0077] Fig.43 Western blotting confirmed the overexpression of ATP6V0A2 and ATP6V0A4 in HeLa cells

[0078] Fig.44 Western blotting to verify knockdown of ATP6V0A2 and ATP6V0A4 in HeLa cells

[0079] Fig.45 Confocal microscopy analysis of the localization of ATP6V1A in phalloidin-labeled HeLa cells, transfected with Atp6v0a2 siRNA, and without cholesterol

[0080] Fig.46 Confocal microscopy analysis of the localization of ATP6V1A in phalloidin-labeled HeLa cells, transfected with Atp6v0a4 siRNA, and containing cholesterol

[0081] Fig.47Confocal microscopy analysis of ATP6V1A and LAMP1+ lysosomal colocalization in HeLa cells transfected with Atp6v0a2 siRNA and cultured in high pH (7.6) medium

[0082] Fig.48 Confocal microscopy analysis of ATP6V1A and LAMP1+ lysosomal colocalization in HeLa cells transfected with Atp6v0a4 siRNA and cultured in normal pH (7.2) medium

[0083] Fig.49 Western blotting analysis of indicated proteins in 4T1 cells treated with DMSO or Rabe

[0084] Fig.50 Co-immunoprecipitation and western blotting analysis of the indicated proteins in HEK293T cells co-transfected with the indicated plasmids Fig.51 BCA assay to measure protein content of EVs isolated from the colon of mice injected intraperitoneally with DMSO, Ome or Rabe

[0085] Fig.52 Flow cytometric analysis of the A33 percentage of EVs captured by anti-CD63-coated latex beads from cultured mouse colon supernatants

[0086] Fig.53 Acute colitis was induced in mice with 2.5% DSS for 7 consecutive days. DMSO, Ome or Rabe were injected intraperitoneally for 7 consecutive times, and the changes in body weight were monitored.

[0087] Fig.54 Acute colitis was induced in mice with 2.5% DSS for 7 consecutive days. DMSO, Ome or Rabe were injected intraperitoneally for 7 consecutive times, and the colon length was monitored.

[0088] Fig.55 Acute colitis was induced in mice by 2.5% DSS for 7 consecutive days. DMSO, Ome or Rabe were injected intraperitoneally for 7 consecutive times. Colon tissue sections stained with hematoxylin and eosin (H&E) were monitored.

[0089] Fig.56 CD63 levels in colonic EVs of Rab27af / f and Villin-Cre Rab27af / f mice (OD value 450nm)

[0090] Fig.57 Acute colitis was induced in Rab27afl / fl×Villin-Cre mice with 2.5% DSS for 7 consecutive days. DMSO, Ome or Rabe were injected intraperitoneally for 7 consecutive times, and the changes in body weight were monitored.

[0091] Fig.58 Acute colitis was induced in Rab27afl / fl×Villin-Cre mice with 2.5% DSS for 7 consecutive days. DMSO, Ome or Rabe were injected intraperitoneally for 7 consecutive times, and the colon length was monitored.

[0092] Fig.59 Acute colitis was induced in Rab27afl / fl×Villin-Cre mice by 2.5% DSS for 7 consecutive days. DMSO, Ome or Rabe were injected intraperitoneally for 7 consecutive times. Colon tissue sections stained with hematoxylin and eosin (H&E) were monitored.

[0093] Fig.60 Acute colitis was induced in mice treated with the TGF-βRI inhibitor SB525334 using 2.5% DSS for 7 consecutive days. DMSO, Ome or Rabe were injected intraperitoneally for 7 consecutive times, and the changes in body weight were monitored.

[0094] Fig.61 Acute colitis was induced in mice treated with the TGF-βRI inhibitor SB525334 for 7 consecutive days with 2.5% DSS. DMSO, Ome or Rabe were injected intraperitoneally for 7 consecutive times, and the colon length was monitored.

[0095] Fig.62 Acute colitis was induced in mice treated with the TGF-βRI inhibitor SB525334 for 7 consecutive days with 2.5% DSS. DMSO, Ome or Rabe were injected intraperitoneally for 7 consecutive times. Colon tissue sections stained with hematoxylin and eosin (H&E) were monitored.

[0096] Fig.63 Representative immunofluorescence monitoring CD45 staining and tdTomato expression in the colon of Rosa-LSL-tdTomato mice injected intravenously with iec-ev or iec-ev / Cre

[0097] Fig.64 2.5% DSS induced acute colitis in Rossa-LSL-tdTomato mice for 7 consecutive days, and the corresponding treatment was given every day for 7 consecutive times. The weight changes of mice in each group

[0098] Fig.65 Acute colitis was induced in Rossa-LSL-tdTomato mice with 2.5% DSS for 7 consecutive days. The corresponding treatment was given every day for 7 consecutive days, and the colon length was monitored.

[0099] Fig.66Acute colitis was induced in Rossa-lsl-tdtomato mice with 2.5% DSS for 7 consecutive days. The corresponding treatments were given daily for 7 consecutive days, and the colon tissue sections stained with hematoxylin and eosin (H&E) were monitored.

[0100] Fig.67 4T1 cells with ATP6V1A knockout (Atp6v1a- / -4T1)

[0101] Fig.68 In vivo imaging system detects the distribution of EVs in indicated organs

[0102] Fig.69 In vivo imaging system detects the distribution of EVs in indicated organs

[0103] Fig.70 Dorsal Skinfold Chamber (DSFC) for Multiphoton Intravital Microscopy (MP-IVM) Analysis

[0104] Fig.71 Dorsal Skinfold Chamber (DSFC) for Multiphoton Intravital Microscopy (MP-IVM) Analysis

[0105] Fig.72 Dorsal Skinfold Chamber (DSFC) for Multiphoton Intravital Microscopy (MP-IVM) Analysis

[0106] Fig.73 CCK8 assay to detect the viability of 4T1 and Atp6v1a- / -4T1 cells after 48 h of culture

[0107] Fig.74 The size of the tumor after the mice were injected with 4T1 tumors subcutaneously on each side of the abdomen and DMSO or Rabe was injected.

[0108] Fig.75 The tumor size after injection of DMSO or Rabe in mice with Atp6v1a- / -4T1 tumors subcutaneously on each flank

[0109] Fig.76 Flow cytometry analysis of the percentage of PD-1+, IFN-γ+, and Ki-67+ T cells in the CD45+CD8+ T cell population in dLNs of these mice

[0110] Fig.77 Flow cytometry analysis of the percentage of PD-1+, IFN-γ+, and Ki-67+ T cells in the CD45+CD8+ T cell population in dLNs of these mice

[0111] Fig.78 Flow cytometry quantification of the percentage of the CD45+CD8+ T cell population in dln of mice receiving 4T1 or Atp6v1a- / -4T1 tumors

[0112] Fig.79 Flow cytometry quantification of the percentage of CD45+CD8+ T cell population in dln of mice bearing 4T1 tumors and treated with Rabe

[0113] Fig.80 Measurement of tumor size in 4T1 nude mice

[0114] Fig.81 Measurement of tumor size in Atp6v1a- / -4T1 nude mice

[0115] Fig.82 Representative immunofluorescence staining of TUNEL in tumor tissues of 4T1 tumor-bearing mice treated with Rabe

[0116] Fig.83 Representative immunofluorescence staining of LC3B in tumor tissues of 4T1 tumor-bearing mice treated with Rabe

[0117] Fig.84 Representative immunofluorescence staining of Ki-67 in tumor tissues of 4T1 tumor-bearing mice treated with Rabe

[0118] Fig.85 Representative immunofluorescence staining of TUNEL in tumor tissues of Atp6v1a- / -4T1 tumor-bearing mice treated with Rabe

[0119] Fig.86 Representative immunofluorescence staining of LC3B in tumor tissues of Atp6v1a- / -4T1 tumor-bearing mice treated with Rabe

[0120] Fig.87 Representative immunofluorescence staining of Ki-67 in tumor tissues of Atp6v1a- / -4T1 tumor-bearing mice treated with Rabe

[0121] Fig.88 ELISA analysis of CD9+CD63+ (OD 450nm value) in the serum of 4T1 or Atp6v1a- / -4T1 tumor-bearing mice and 4T1 tumor-bearing mice injected with DMSO or Rabe intratumorally

[0122] Fig.89 ELISA analysis of PD-L1+CD63+EVs (OD 450nm value) in the serum of 4T1 or Atp6v1a- / -4T1 tumor-bearing mice and 4T1 tumor-bearing mice injected intratumorally with DMSO or Rabe

[0123] Fig.90 Determination of tumor size in Pd1- / - mice bearing 4T1 or Atp6v1a- / -4T1 tumors

[0124] Fig.91 Determination of tumor size in Pd1- / - mice bearing 4T1 tumors and injected intratumorally with DMSO or Rabe

[0125] Fig.92 ELISA analysis of CD9+CD63+ and PD-L1+CD63+ EVs (OD 450nm value) in the serum of 4T1 tumor-bearing mice injected intravenously with DMSO or Rabe

[0126] Fig.93 ELISA analysis of tumor size in 4T1 tumor-bearing mice injected intravenously with DMSO or Rabe

[0127] Fig.94 Anti-PD-1 resistance in mice bearing 4T1 tumors

[0128] Fig.95 Anti-PD-1 resistance in mice bearing tumors of size B16F10

[0129] Fig.96 ELISA analysis of CD9+CD63+ and PD-L1+CD63+ EVs (OD value 450nm) in the serum of gastric tumor patients before (day 0) and after (day 2) PPI treatment

[0130] Fig.97 Representative flow cytometry images (left) or statistical analysis (right) of circulating CD8+ T cells in the blood of patients with gastric cancer

[0131] Fig.98 Representative flow cytometry images (left) or statistical analysis (right) of the percentage of PD-1+CD8+T cells and Tim3+CD8+T cells in the blood of patients with gastric cancer

[0132] Fig.99 Representative flow cytometry images (left) or statistical analysis (right) of Ki-67+CD8+T and granzyme B (Gzm B)+CD8+T cells in the blood of gastric cancer patients

[0133] Fig.100 PFS of patients with gastric cancer treated with anti-PD-1 combined with or without PPI

[0134] Fig.101 In vivo imaging system detects the distribution of EVs in indicated organs

[0135] Fig.102 CCK8 assay was used to detect the viability of 4T1 and Atp6v1a- / -4T1 cells treated with RBC-EVs / Dox alone or in combination with Rabe

[0136] Fig.103Determination of the effect of Rabe on RBC-EVs / Dox on the tumor size of 4T1 tumors in nude mice

[0137] Fig.104 Determination of the effect of Rabe on RBC-EVs / Dox on the size of Atp6v1a- / -4T1 tumors in nude mice

[0138] Fig.105 Representative immunofluorescence images of the Rabe assay for RBC-EVs / Dox in nude mice 4T1 tumors and Atp6v1a- / -4T1

[0139] Fig.106 Real-time PCR analysis of miR-155 expression in the liver of mice injected intravenously with rbc-ev / miR-NC-ASO, rbc-ev / miR-NC-ASO and Rabe, rbc-ev / miR-155-aso, or rbc-ev / miR-155-aso and Rabe

[0140] Fig.107 H&E stained liver tissue sections

[0141] Fig.108 ELISA analysis of plasma aspartate aminotransferase and alanine aminotransferase (ALT) levels

[0142] Fig.109 ELISA analysis of serum IL-1β, IL-6 and TNF protein levels

[0143] Fig.110 H&E stained liver tissue sections

[0144] Fig.111 ELISA analysis of plasma aspartate aminotransferase and alanine aminotransferase (ALT) levels

[0145] Fig.112 ELISA analysis of serum IL-1β, IL-6 and TNF protein levels DETAILED DESCRIPTION

[0146] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples, which are only used to explain the present invention and are not intended to limit the scope of the present invention. The materials, reagents, etc. used in the following examples are reagents and materials available from commercial sources unless otherwise specified.

[0147] The experimental samples and experimental methods used in the following embodiments are as follows:

[0148] 1. Mice: C57BL / 6J, Balb / c, and nude mice (6-8 weeks old) were purchased from Joint Ventures SipperBK Laboratory Animal Co., Ltd. (Shanghai, China). Rab27afl / fl, Atp6v1a- / -, Cmv-Cre, Villin-Cre, and Rosa-LSL-tdtomato mice were purchased from Cyagen Biosciences (Suzhou, Jiangsu, China). All mice were housed in a specific pathogen-free facility, and the experimental protocols were approved by the Animal Care and Use Committee of Zhejiang University School of Medicine.

[0149] 2. Humans: Human blood samples from patients with gastric cancer treated with PPIs were obtained from the Second Affiliated Hospital of Zhejiang University School of Medicine. The local ethics committee and the review board of the Second Affiliated Hospital of Zhejiang University School of Medicine approved the collection of human samples. Data from 183 patients with advanced lung cancer who received monotherapy with anti-PD-1 or combined with PPI therapy were obtained from Zhejiang Cancer Hospital. All patients were informed of the use of their samples and signed consent forms were obtained.

[0150] 3. Cell culture: 4T1, HeLa, B16F10, A549, MC38, RAW264.7 and THP-1 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). T1, HeLa, B16F10, A549, MC38 and RAW264.7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v v-1) fetal bovine serum (FBS) and 1% penicillin / streptomycin. THP-1 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. To obtain BMDCs, tibiae and femurs were removed using aseptic techniques and the bone marrow was flushed with fresh RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. Bone marrow cells were then plated in RPMI-1640 medium supplemented with GM-CSF (50 ng ml-1) for 6 days and used for subsequent experiments. To obtain mouse primary PEMs, mice (6-8 weeks old) were intraperitoneally injected with 3% liquid thioglycolate medium (Merck, Darmstadt, Germany). After 3 days, the peritoneal lavage fluid was collected and centrifuged. Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. After 2 h, non-adherent cells were removed and adherent monolayer cells were washed with RPMI-1640 medium as plasma membrane (PM).

[0151] 4. Cell therapy: Treatment of cells with 10 μM Rabe or Ome inhibited PPs (Selleck, Shanghai, China). Treatment of cells with 10 μM Cytochalasin D inhibited phagocytosis (MedChemExpress, Shanghai, China). Vesicle-mediated endocytosis and clathrin-mediated endocytosis were inhibited by 10 μM indomethacin (MedChemExpress) and chlorpromazine (Selleck), respectively. Macropinocytosis was inhibited by treatment of cells with 10 μL Y294002 or amiloride (Selleck). Treatment of cells with 10 μM KM91104, Baf-A1, and 10 μM azathioprine (Selleck) inhibited v-ATPase and RAC1 activities, respectively. Cholesterol was extracted from cells by treatment with 10 μM MeβCD (Aladdin). Treatment with 10 μM 2,4-dnp induced an equal pH gradient in cells (Merck). The cells were treated with 10 μM H89 (Selleck) to inhibit PKA activation.

[0152] 5. EV isolation: When isolating EVs, the cell culture supernatant was centrifuged at 500×g for 10 min, 2000×g for 15 min, and 10,000×g for 30 min at 4°C. Then, the supernatant was passed through a 0.22μm syringe filter (Millipore) and collected in a 35ml ultracentrifuge tube (Beckman Coulter, Brea, CA, USA). EVs were concentrated using an SW32Ti rotor (L-90K with SW32Ti rotor, Beckman Coulter) at 100,000×g for 70 min at 4°C. EV protein content was quantified using a BCA protein assay kit (Thermo Fisher Scientific).

[0153] 6. Electron microscopy: For EV negative staining, 200-mesh carbon film was hydrolyzed with a glow discharger at 15 mA for 25 s. The EV solution was pipetted onto a 200-mesh carbon-coated copper grid and kept at room temperature for 1 min. After removing any excess suspension with filter paper, negative staining was performed with 2% uranyl acetate for 1 min at room temperature. Any additional suspension was removed before the grid was air-dried. Images were obtained by electron microscopy (Tecnai G2Spirit 120 kV, Thermo FEI, Hillsboro, OR, USA).

[0154] 7. Nanoparticle tracking analysis: To measure particle size and concentration, nanoparticle tracking analysis was performed on EVs purified from DMSO- and rabe-treated 4T1 cells using a NanoSight NS300 system (Malvern PANalytical) equipped with a 488 nm laser and a high-sensitivity sCMOS camera, and finally analyzed using NTA3.2 or 3.3 software.

[0155] 8. Immunofluorescence staining and confocal microscopy: 4T1 and HeLa cells were plated on glass slides in 24-well plates. Cells were fixed with 4% paraformaldehyde for 30 min at room temperature (RT), permeabilized with 0.1% Triton X-100, blocked with 5% BSA in PBS for 1 h, and stained with specific antibodies. Primary antibodies were detected using iFluor 488 or iFluor 594-conjugated secondary antibodies (Huabio, Hangzhou, Zhejiang, China). Nuclei were stained with DAPI. Cell membranes were labeled with iFluor488phalloidin (Y easen, Shanghai, China). Finally, fluorescence was detected using an Olympus FV3000 laser confocal microscope (Olympus). Images were analyzed using ImageJ software (National Institutes of Health, USA).

[0156] 9. Total internal reflection fluorescence microscopy: 4T1 cells were transfected with mCherry-CD63-HA plasmid for 36 h, cells were incubated in PBS, and then analyzed by TIRF microscopy. For TIRF microscopy using an Olympus IX83 microscope (Olympus), the penetration depth (δ) of the evanescent field used to excite the fluorophore was set to 150 nm. Frames were acquired in 400 images at a frequency of 10 Hz with an exposure time of 100 ms. Fluorescence images were acquired using CellSens software (Olympus). mCherry+CD63 vesicles were quantified using ImageJ software (National Institutes of Health).

[0157] 10. Flow cytometric analysis: To analyze phagocytosis, cells were pretreated with the corresponding inhibitors and then incubated with 10 μg ml-1 PKH26+EVs for 1 h or 50 μg ml-1 TRITC-Dextran (sigma) for 20 min. To analyze the number of EVs in the culture supernatant, EVs were incubated with 4 μm acetaldehyde sulfate beads (Thermo Fisher Scientific) and then collected by centrifugation at 3500 × g for 5 min at 4 °C. Then incubate with the corresponding fluorescent-conjugated primary antibody for 20 min in the dark at 4 °C. Single-cell suspensions prepared from dln or blood were resuspended in PBS supplemented with 1% FBS and stained with the corresponding fluorescent antibodies for 20 min in the dark at 4 °C. The cells or beads were then washed 3 times with PBS and analyzed using a FACS Athena flow cytometer. The antibodies used are shown in Table 1.

[0158] Table 1: Summary of antibodies used

[0159] anti-mouse CD9-PE BioLegend Cat:124805 anti-mouse PD-1-PE BioLegend Cat:135205 anti-mouse CD45-Pacific Blue BioLegend Cat:157211 anti-mouse CD8-APC BioLegend Cat:100711 anti-mouse IFN-γ-PE BioLegend Cat:505807 anti-mouse Ki-67-PE BioLegend Cat:151209 anti-human PD-1-PE BioLegend Cat:379209 anti-human CD45-Pacific Blue BioLegend Cat:982306 anti-human CD8-APC BioLegend Cat:300911 anti-human Gzm B-PE BioLegend Cat:372207 anti-human Ki-67-PE BioLegend Cat:151209

[0160] 11. Plasmids: cDNA sequences were cloned from PEMs cDNA by PCR and inserted into the pcDNA3.1 backbone: ATP6V0A2 and ATP6V0A4. pcDNA3.1-CMV-CFP; UBC-Cre25nt and pL V-CMV-LoxP-DsRed-LoxP-eGFP plasmids were purchased from Addgene (Watertown, USA). All the above structures were verified by sequencing.

[0161] 12. siRNA transfection: TransIT-TKO (Mirus Bio, Madison, Wisconsin, USA) was used for transient siRNA transfection in vitro according to the manufacturer's instructions. The cells were used for experiments 48 h after transfection. The siRNAs used are listed in Table 2.

[0162] Table 2: Summary of siRNAs used

[0163] NC siRNA F:5'-TTCTCCGAACGTGTCACGT-3' Genepharma N / A NC siRNA R:5'-ACGTGACACGTTCGGAGAA-3' Genepharma N / A mAtp6v1a siRNA F:5'-GTCCAACATGATTTCATTCTA-3' Genepharma N / A mAtp6v1a siRNA R:5'-TAGAATGAAATCATGTTGGAC-3' Genepharma N / A mAtp6v0a2 siRNA F:5′-GCCAAATGGTTCTGTAAGATA-3′ Genepharma WELL mAtp6v0a2 siRNA R:5′-TATCTTACAGAACCATTTGGC-3′ Genepharma WELL mAtp6v0a4 siRNA F:5′-GCCTTGTGGATGGTCCTAAAT-3′ Genepharma WELL mAtp6v0a4 siRNA R:5′-ATTTAGGACCATCCACAAGGC-3′ Genepharma WELL mSlc4a7 siRNA F:5′-ACCTTTCCTATCTATCATTAA-3′ Genepharma WELL mSlc4a7 siRNA R:5′-TTAATGATAGATAGGAAAGGT-3′ Genepharma WELL miR-NC-ASOs:5'-CAGUACUUUGUGUAGUACAA-3' Genepharma WELL miR-155-ASOs:5′-ACCCCUAUCACAAUUAGCAUUAA-3′ Genepharma WELL

[0164] 13. Real-time PCR analysis: Total RNA was isolated from cells or tissues using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. For reverse transcription of mature miRNAs, specific primers for miR-155 and U6 (GenePharma, Shanghai, China) were used. For reverse transcription of mRNA, single-stranded cDNA was synthesized using cDNA Synthesis Kit (Vazme, Nanjing, Jiangsu, China). Real-time PCR was performed using SYBR Green Master Mix (Vazyme) with the CFX Touch system Bio-Rad. The primers used are shown in Table 3.

[0165] Table 3: Primer summary

[0166] β-actin-F:5'-GGCTGTATTCCCCTCCATCG-3' Genepharma WELL β-actin-R:5'-CCAGTTGGTAACAATGCCATGT-3' Genepharma WELL Il1b-F:5'-GCAACTGTTCCTGAACTCAACT-3' Genepharma WELL Il1b-F:5'-ATCTTTTGGGGTCCGTCAACT-3' Genepharma WELL Il6-F:5'-TAGTCCTTCCTACCCCAATTTCC-3' Genepharma WELL Il6-F:5'-TTGGTCCTTAGCCACTCCTTC-3' Genepharma WELL Tnf-F:5'-CCCTCACACTCAGATCATCCTTCT-3' Genepharma WELL Tnf-R:5'-GCTACGACGTGGGCTACAG-3' Genepharma WELL miR-155-F:5'-CGGCGGTTAATGCTAATTGTGAT-3' Genepharma WELL miR-155-R:5′-GTGCAGGGTCCGAGGT-3′ Genepharma WELL U6-F:5'-CAGCACATATACTAAAATTGGAACG-3' Genepharma WELL U6-R:5'-ACGAATTTGCGGTCATCC-3' Genepharma N / A

[0167] 14. Western blot analysis: Cell lysates or immunoprecipitates were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Massachusetts, USA). After blocking with 5% BSA, the membranes were incubated with primary antibodies overnight at 4°C and then incubated with the corresponding secondary antibodies at RT for 1 hour. The membranes were washed three times for 10 minutes each, incubated with SuperSignal Chemiluminescent Substrate (Pierce, Dallas, Texas, USA), and scanned with a Tanon 4500 gel imaging system (Tanon, Shanghai, China). The protein band density was semi-quantified using ImageJ software. The antibodies used are shown in Table 1.

[0168] 15. Immunoprecipitation: Cells were lysed in immunoprecipitation buffer containing protease inhibitor cocktail tablets (Roche). The supernatant was collected and centrifuged at 12,000 × g, 4°C for 10 min, and then incubated with protein A / g magnetic beads (Thermo Fisher Scientific) and corresponding antibodies at 4°C overnight. The next day, the cells were washed three times with IP buffer. The immunoprecipitate was boiled and eluted with 1% (w v-1) SDS sample buffer ().

[0169] 16. IBD model experiment: 2.5% DSS was given to induce acute IBD (MP Biomedicals) drinking water for 7 days. The first day that mice started drinking DSS solution was day 0. To analyze the effects of Rabe or Ome on IBD, mice were intraperitoneally injected with DMSO, Ome or Rabe every day for 7 consecutive days. To analyze the effects of iec-ev, Rab27afl / fl×Villin-Cre mice were used to induce acute IBD. In some experiments, mice were intraperitoneally injected with DMSO or Rabe in combination with 30 mg kg-1 TGF-βRI inhibitor SB525334 or IEC-EV.

[0170] 17. Histological analysis: Mouse colon and liver were dissected, fixed with 4% paraformaldehyde, embedded in paraffin, sliced, stained with H&E, and histological changes were observed under a light microscope. Oct Compound (SAKURA, USA), frozen sections, and stained with specific primary antibodies overnight at 4°C. Primary antibodies were detected with iFluor 488 or iFluor 594-conjugated secondary antibodies (Huabio), nuclei were stained with DAPI, and observed with an Olympus FV3000 laser confocal microscope (Olympus). Images were analyzed with ImageJ software.

[0171] 18. Lentivirus production and stable cell line generation: For TP6V1A knockout, 4T1 cells were co-transfected with plv5-Cas9-Blast (Merck) and U6-gRNA: hPGK-puro-2A-tBFP (Merck). After 24 h, single BFP+ cells were sorted into 96-well plates using a Beckman MoFlo Astrios EQ cell sorter (Beckman). Endogenous protein levels were detected by western blot. For RFP overexpression, 4T1 cells were transfected with an RFP expression plasmid and RFP+ cells were selected in the presence of 2.5 μg ml-1 puromycin in the culture medium.

[0172] 19. Tumor model experiments: To observe the effect of Rabe on EVs endocytosis, 1×106 RFP+4T1 cells were injected subcutaneously on day 0 in C57BL / 6J mice. Rabe was injected intratumorally and PKH67+EVs were injected intravenously. DSFCs were mounted on these mice for MP-IVM analysis. To analyze the effect of ATP6V1A on EV endocytosis in vivo, 1×106 4T1 and Atp6v1a- / -4T1 cells were injected subcutaneously on each side of C57BL / 6J mice on day 0. These mice were then injected intravenously with VivoTrack680-labeled 4T1-EVs, and the distribution of EVs was detected using an in vivo imaging system. To analyze the effect of Rabe on EV endocytosis in vivo, 1×106 4T1 or Atp6v1a- / -4T1 cells were injected subcutaneously on both sides of C57BL / 6J mice on day 0. Then, Rabe was injected intratumorally in the flank every 2 days starting from day 9, and the distribution of EVs was detected using an in vivo imaging system. To analyze the effect of A TP6V1A on tumor growth, C57BL / 6J mice were subcutaneously injected with 1×106 4T1 or Atp6v1a- / -4T1 cells on day 0, and tumor size was measured with a V-ernier caliper every other day starting from day 9. To analyze the effect of Rabe on tumor growth, C57BL / 6J mice were subcutaneously injected with 1×106 4T1 or Atp6v1a- / -4T1 cells on day 0, or nude mice were subcutaneously injected with 1×106 4T1 cells on day 0. Then, these mice received intratumoral Rabe treatment every 2 days starting from day 9. Tumor size was measured with a V-ernier caliper every other day starting from day 9. To analyze the role of Rae in the antitumor effect of rbc-ev / Dox, nude mice were injected subcutaneously with 1×106 4T1 or Atp6v1a- / -4T1 cells on day 0. Then, rbc-ev / Dox with or without Rae treatment was intravenously injected every 2 days starting from day 9, and tumor size was measured with a Vernier caliper every other day starting from day 9. According to the standards of the Animal Care and Use Committee of Zhejiang University School of Medicine, the maximum tumor size allowed was 8000 mm3, which was not exceeded in this study.

[0173] 20. ALF model treatment experiment: The ALF model was established by intraperitoneal injection of 50 μg kg-1 lipopolysaccharide (LPS, Sigma-Aldrich) and 400 mg kg-1 d-galactosamine (D-GaIN, Sigma-Aldrich) into mice. Then, rbc-ev / miR-NC-ASO, rbc-ev / miR-155-ASO and Rabe, rbc-ev / miR-155-ASO and Rabe were injected intravenously on days 3, 2, and 1. Mice were killed after 12 hours of treatment.

[0174] 21. BCA method for protein determination: The BCA method uses a kit from Thermo with a catalog number of 23227. The detection method is based on the instructions.

[0175] 22. Cytokine determination: The levels of mouse serum IL-1β, IL-6, TNF (BioLegend, San Diego, California, USA), AST and AL-T (Nanjing Jiancheng, Nanjing, Jiangsu Nanjing, China) were determined by ELISA method according to the manufacturer's instructions.

[0176] 23. Statistical analysis: Unpaired two-tailed Student's t test, one-way analysis of variance (ANOVA) and Tukey's test were used to test the statistical significance of differences between groups. All data are expressed as mean ± SD values ​​and analyzed using GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, California, USA). P < 0.05 was considered significant.

[0177] Example 1 Determination of the effect of PPIs on EV levels in the supernatant of mouse 4T1 breast cancer cells

[0178] This example sets up several groups of control experiments, with DMSO treatment as the control, and PPIs omeprazole and rabeprazole as the experimental group, and uses flow cytometry, BCA method, transmission electron microscopy, nanoparticle tracking and western blotting analysis to measure data related to EV levels, and then determine the effect of PPIs on EV levels. The specific steps are as follows:

[0179] 1. Experimental content

[0180] ① Mouse 4T1 breast cancer cells were treated with DMSO, PPIs omeprazole (Ome) and rabeprazole (Rabe), respectively, and the supernatants after treatment were extracted. The CD9 percentage of EVs captured by anti-CD63 coated latex beads from the supernatants of 4T1 cells treated with DMSO, Ome or Rabe was analyzed by flow cytometry. Figure 1 As shown in Figure 3, the percentage of CD9 in EVs captured by anti-CD63-coated latex beads in the supernatants of mouse 4T1 breast cancer cells treated with DMSO, Ome, and Rabe were 33.7%, 19.9%, and 16.1%, respectively.

[0181] ② EVs were isolated from equal amounts of 4T1 cells treated with DMSO, Ome or Rabe, and the protein content of these EVs was determined by the BCA method, such as Figure 2As shown in the figure, the ev protein content determined by BCA method was as follows: DMSO treated group, Ome treated group, Rabe treated group.

[0182] ③ Use 200nm transmission electron microscopy to examine the EV morphology of 4T1 cells treated with DMSO or rabe, such as Figure 3 As shown, the EV depletion rate of 4T1 cell supernatants treated with Rabe was higher than that of DMSO-treated cells.

[0183] ④ Analysis of the size distribution of EVs in 4T1 cells treated with DMSO or rabe by nanoparticle tracking, such as Figure 4 As shown, EVs in the supernatant of 4T1 cells treated with Rabe were more narrowly distributed and fewer in number than those in the supernatant of 4T1 cells treated with DMSO.

[0184] ⑤ Western blotting analysis of the indicated proteins was performed on the same number of 4T1 cells treated with DMSO or Rabe and EVs in the supernatants of these cells, as shown in Figure 5 As shown, EVs in the supernatant of 4T1 cells treated with Rabe were reduced compared with those treated with DMSO.

[0185] 2. Experimental results

[0186] PPIs omeprazole (Ome) and rabeprazole (Rabe) can both reduce the EV level in the supernatant of mouse 4T1 breast cancer cells, and Rabe has a stronger inhibitory effect on EV levels than Ome.

[0187] Example 2 Examination of the Effects of PPIs on Multivesicular Bodies (MVBs)

[0188] In this example, two groups of mouse 4T1 breast cancer cells treated with DMSO and Rabe were set up and analyzed by confocal microscopy and TIRF microscopy. Extracellular vesicles (EVs) are mainly produced by outward budding of plasma membrane PM or by endosomal pathways involving fusion of multivesicular bodies (MVBs) with PM. The purpose of this example is to examine whether PPIs reduce supernatant EV levels by inhibiting multivesicular body function. The specific steps are as follows:

[0189] 1. Experimental content

[0190] ① Mouse 4T1 breast cancer cells were treated with DMSO and Rabe respectively, and CD63 in 4T1 cells treated with DMSO or Rabe was analyzed by confocal microscopy. + MVBs, HRS + MVBs, EEA1 + EEs and CD63 +MVBs (multivesicular bodies MVB and endosomes EE, stained with different markers) and LAMP1+lysosomes co-localized, the results are as follows Figure 6 , Figure 7 , Figure 8 , Fig. 9 The images show that there is no significant difference in the co-localization of CD63+MVBs, HRS+MVBs, EEA1+EEs and CD63+MVBs with LAMP1+lysosomes in mouse 4T1 breast cancer cells treated with DMSO and Rabe, respectively, indicating that Rabe does not affect the co-localization of MVBs and LAMP1+lysosomes, indicating that the degradation of MVBs has not changed.

[0191] ② TIRF microscopy was used to analyze the distribution of CD63+MVBs in 4T1 cells treated with DMSO or Rabe. Fig.10 The images showed that the number of CD63+MVBs in the subcytoplasmic membrane region of 4T1 cells treated or not with Rabe was comparable, indicating that PPIs did not affect the fusion of MVBs and PM.

[0192] 2. Experimental results: After Rabe treatment, the degradation of MVBs did not change, nor did it affect the fusion of MVBs and PM, indicating that PPIs are unlikely to regulate EV biogenesis.

[0193] Example 3: Whether the PPI-mediated reduction in supernatant EVs is due to increased cellular EV uptake

[0194] In this example, EVs were first labeled with pkh26, and then three groups of 4T1 cells treated with DMSO, Ome or Rabe were used to uptake the labeled EVs, and the process was analyzed by flow cytometry, confocal microscopy, and real-time fluorescence microscopy. Secondly, the Cre-Loxp system was used to monitor the effect of PPIs on EV uptake in HEK293 cells. Subsequently, a group of control experiments were performed in which Rabe was treated alone or in combination with cytochalasin D. Finally, it was verified that PPIs can promote EV uptake in a variety of cells. The specific steps are as follows:

[0195] 1. Experimental content

[0196] ① First, EVs were labeled with pkh26 by incubating them with EVs at 37°C for 5 min. EVs labeled with pkh26 were analyzed by flow cytometry and confocal microscopy using 4T1 cells treated with DMSO, Ome, or Rabe, respectively. The uptake kinetics of pkh26-labeled EVs by PEMs treated with DMSO or Rabe were analyzed by real-time fluorescence microscopy. The results are shown in Table 1. Fig.11 , Fig.12 , Fig.13As shown. Flow cytometric analysis showed that the percentage of EV uptake labeled with PKH26 in 4T1 cells treated with DMSO, Ome or Rabe was 19.3, 39.4 and 46.6, respectively. Confocal microscopy analysis showed that compared with DMSO, 4T1 cells treated with Rabe significantly increased the uptake of EV labeled with PKH26, and real-time fluorescence microscopy also confirmed that Rabe induced peritoneal macrophages (PEMs) to increase EV uptake.

[0197] ② Next, we developed a Cre-Loxp system to monitor the effects of PPIs on EV uptake in HEK293 cells. Fig.14 4T1 reporter cells were pretreated with DMSO or Rabe and then co-cultured with Cre+4T1 cells. The red fluorescent protein (DsRed) and green fluorescent protein (eGFP) were analyzed by confocal microscopy. The results are shown in Fig.15 As shown. After the uptake of EVs derived from Cre+HEK293 cells, the red-to-green conversion can be detected in HEK293 reporter cells. Through this experiment, we found that more EGFP+ reporter cells pre-treated with Rabe were induced by Cre+HEK293 cells, indicating that the uptake of EVs by Cre+ cells was increased.

[0198] ③ Subsequently, the CD9 percentage of EVs captured from the supernatant of 4T1 cells by anti-CD63-coated latex beads was analyzed by flow cytometry. Rabe alone or in combination with cytochalasin D treatment, the results are shown in Fig.16 Flow cytometry showed that the percentage of EVs in the supernatant of 4T1 cells treated with DMSO or Rabe or DMSO+Cytochalasin D or Rabe+Cytochalasin D were 16.9%, 8.71%, 29.1%, and 28.1%, respectively, indicating that when Cytochalasin D inhibited EV uptake, Rabe no longer reduced the EV level in the supernatant of 4T1 cells.

[0199] ④ As an extension, flow cytometric analysis of PKH26-labeled EVs was performed on a variety of indicator cells treated with DMSO or Rabe, including mouse RAW264.7 macrophages, bone marrow-derived dendritic cells (BMDCs), and MC38 colon cancer cells; human THP-1 monocytic leukemia cells, A549 lung cancer cells, and HeLa cervical cancer cells. The results are shown in Fig.17 As shown. It was confirmed that Rabe can promote EV uptake in the above cells.

[0200] 2. Experimental results: PPIs mediate the reduction of supernatant EVs. Since PPIs promote EV uptake by cells, PPIs can promote EV uptake by a variety of cells.

[0201] Example 4 PPIs promote EV uptake by increasing v-ATPases-dependent macropinocytosis

[0202] This example first verified that Rabe did not affect the membrane fusion of EVs with 4T1 cells and analyzed the role of PPIs in EV endocytosis. Then, the effects of different endocytic pathways on EV uptake were compared and studied using cells with different phagocytic activities. Finally, the plasma membrane (PM) transport of v-ATPases was studied. The specific steps are as follows:

[0203] 1. Experimental methods

[0204] ① Verify that Rabe does not affect EV membrane fusion

[0205] Confocal microscopy analysis of PKH26-labeled EV colocalization in phalloidin-labeled 4T1 cells treated with DMSO or Rabe (phalloidin labeling method is to incubate phalloidin and crawling cells at 4°C overnight). The results are shown in Fig.18 As shown, the results showed that Rabe did not affect the membrane fusion of EV and 4T1 cells.

[0206] ②Analysis of the role of PPIs in EV endocytosis

[0207] Flow cytometry analysis of PKH26-labeled EVs in 4T1 cells treated with Rabe alone or in combination with indomethacin or chlorpromazine. Fig.19 , Fig. 20 Flow cytometric analysis of PKH26-labeled EVs obtained from 4T1 cells treated with Rabe alone or in combination with LY294002 or amiloride showed the following results: Fig.21 Flow cytometry analysis of 4T1 cells treated with DMSO or rabe, without or with ATP6V1A silencing (ATP6V1A silencing method was siRNA interference) for pkh26-labeled EVs, the results are shown in Fig. 22 Western blotting analysis of ATP6V1A protein in 4T1 cells transfected with negative control (NC) or Atp6v1a siRNA was performed. Fig.23 Flow cytometry analysis of 4T1 cells with pkh26-labeled EVs treated with Rabe alone or in combination with KM91104 or Baf-A1 (v-ATases inhibitor) showed the results as shown. Fig.24 As shown, flow cytometry analysis of ATP6V1A on PM of 4T1 cells treated with DMSO or Rabe was performed. Fig.25As shown, confocal microscopy was used to analyze the localization of ATP6V1A in phalloidin-labeled 4T1 cells after treatment with DMSO or Rabe. Fig.26 shown.

[0208] Flow cytometric analysis of 4T1 cells treated with DMSO or Rabe or DMSO+indomethacin or Rabe+indomethacin showed that the percentages of EVs labeled with PKH26 in the four groups of cells were 14.6%, 19.3%, 11.8% and 16.0%, respectively. Flow cytometric analysis of 4T1 cells treated with DMSO or Rabe or DMSO+chlorpromazine or Rabe+chlorpromazine showed that the percentages of EVs labeled with PKH26 in the four groups of cells were 13.3%, 18.1%, 10.9% and 15.6%, respectively, indicating that the caveolae-mediated endocytosis inhibitor indomethacin and the clathrin-mediated endocytosis inhibitor chlorpromazine did not affect Rabe-induced EV uptake. However, flow cytometry analysis of 4T1 cells treated with DMSO or Rabe or DMSO+LY294002 or Rabe+LY294002 showed that the percentages of EVs labeled with PKH26 in the four groups of cells were 11.1%, 16.0%, 11.0% and 10.8%, respectively, and flow cytometry analysis of 4T1 cells treated with DMSO or Rabe or DMSO+amiloride or Rabe+amiloride showed that the percentages of EVs labeled with PKH26 in the four groups of cells were 12.4%, 19.2%, 13.4% and 13.2%, respectively, indicating that the macropinocytosis inhibitors LY294002 or amiloride completely abolished the Rabe-induced increase in EV uptake. The enhanced endocytosis of TRITC-Dextran (TRITC is fluorescein, Dextran is a microsphere bead, and TRITC-Dextran is a fluorescent bead) by Rabe-treated PEMs further confirmed the increase in macropinocytosis. V-ATPase is required for macropinocytosis. We found that silencing ATP6V1A (V1A subunit of v-ATPases) prevented Rabe from promoting EV uptake by 4T1 cells. Flow cytometry analysis of 4T1 cells treated with DMSO or Rabe or DMSO+KM91104 or Rabe+KM91104 showed that the percentages of EVs labeled by pkh26 in the four groups of cells were 15.3%, 27.1%, 12.2% and 12.5%, respectively. Flow cytometry analysis of 4T1 cells treated with DMSO or Rabe or DMSO+Baf-A1 or Rabe+Baf-A1 showed that the percentages of EVs labeled by pkh26 in the four groups of cells were 11.7%, 17.5%, 8.52% and 9.57%, respectively, indicating that KM91104 and Baf-A1 prevented Rabe from promoting EV uptake by 4T1 cells. We also observed more ATP6V1A PM localization in rabe-treated 4T1 cells by confocal microscopy analysis.

[0209] ③Study the effects of different endocytic pathways on EV uptake

[0210] We performed this study using cells with different phagocytic activities, including PEMs (high phagocytic activity), 4T1 cells (medium phagocytic activity), and T cells (low phagocytic activity). Flow cytometric analysis of PKH26-labeled EVs was performed using indicator cells treated with DMSO, Baf-A1, indomethacin, or chlorpromazine. Fig. 27 As shown: 56.9%, 45.7%, 50.7% and 46.6% for PEMs, 60.8%, 29.3%, 54.0% and 48.8% for 4T1 cells, and 3.61%, 2.28%, 3.58% and 2.84% for T cells. We found that inhibition of vesicle protein or clathrin-mediated endocytosis moderately or slightly reduced EV uptake in PEMs and 4T1 cells, but had no effect on EV uptake in T cells. Inhibition of macropinocytosis inhibited EV uptake by all cells, with inhibition percentages of 18.57±1.141, 51.16±2.921 and 37.95±6.991 (mean±SD) for PEMs, 4T1 cells and T cells, respectively.

[0211] PM trafficking of v-ATPases would increase PM trafficking of cholesterol and subsequent PM-associated RAC1, a prerequisite for macropinocytosis. We found that Rabe indeed increased PM distribution of cholesterol. Methyl-β-cyclodextrin (MeβCD) can extract cholesterol from the plasma membrane. Flow cytometric analysis of pkh26-labeled EVs from 4T1 cells treated with DMSO or Rabe or DMSO+MeβCD or Rabe+MeβCD showed Fig.28 As shown: the pkh26 uptake rates were 18.4%, 25.2%, 16.9% and 17.9%, respectively. Flow cytometry analysis of pkh26-labeled EVs from 4T1 cells treated with DMSO or Rabe or DMSO + azathioprine or Rabe + azathioprine, the results are shown Fig.29 As shown: the pkh26 absorption rates were 20.1%, 29.6%, 17.7% and 18.0%, respectively. This indicates that MeβCD treatment abolished Rabe-induced EV endocytosis. We also detected an increase in the PM localization of the RAC1 gene, and the RAC1 inhibitor azathioprine abolished Rabe-induced EV endocytosis.

[0212] 2. Experimental results: Rabe treatment did not affect EV membrane fusion, but increased macropinocytosis and PM localization of ATP6V1A, indicating that PPIs increase EV endocytosis by increasing macropinocytosis. Inhibition of vesicle protein or clathrin-mediated endocytosis can moderately or slightly reduce EV uptake in PEMs and 4T1 cells, but has no effect on EV uptake in T cells. Inhibition of macropinocytosis inhibits EV uptake by all cells. The increase in PM v-ATPases is a key step in PPIs-induced macropinocytosis.

[0213] Example 5 PPIs increase the assembly of PM v-ATPases by lowering cytoplasmic pH

[0214] This example first detects whether Rabe activates protein kinase A (PKA), and uses PKA inhibitors and 4T1 cells with slc4a7 knockout to perform EV uptake experiments to verify whether PKA is the mediator of PPIs. Then, control experiments with low pH and high pH culture media were performed to verify whether Rabe induces endocytosis in a pH-dependent manner. Finally, we explored the mechanism of pH-mediated accumulation of v-ATPases on the PM by detecting the mRNA and protein levels of ATP6V1A on the vacuole (VM) and plasma membrane (PM). The specific steps are as follows:

[0215] 1. Experimental methods

[0216] ① Verify the relationship between PKA and the accumulation of v-ATPases on PM

[0217] Western blotting analysis of p-PKA and PKA proteins was performed in 4T1 cells treated with DMSO or Rabe for the indicated time, and the results are shown in Fig.30 As shown, flow cytometry analysis of 4T1 cells treated with PKH26-labeled EVs alone or in combination with H89 was performed. Fig.31 As shown, Western blotting analysis of SLC4A7 expression, the results are as follows Fig.32 Flow cytometric analysis of PKH26-labeled EVs in 4T1 cells transfected with NC or SLC4A7 siRNA followed by DMSO or Rabe treatment was performed as shown. Fig.33 shown.

[0218] In Western blotting analysis of p-PKA and PKA proteins, we did not detect PKA activation by Rabe. 4T1 cells were treated with DMSO or Rabe or DMSO+H89 (H89 is a PKA inhibitor) or Rabe+H89, and flow cytometric analysis of PKH26-labeled EVs showed 21.4%, 31.0%, 16.9% and 25.1%, respectively, indicating that the PKA inhibitor H89 did not eliminate Rabe-induced EV uptake by 4T1 cells. Western blotting analysis of SLC4A7 expression demonstrated that the bicarbonate transporter SLC4A7 is essential for PKA-induced macrophage proliferation. Flow cytometry analysis of PKH26-labeled EVs in 4T1 cells transfected with NC or SLC4A7 siRNA treated with DMSO and Rabe, respectively, showed that 19.6% (DMSO+NCsiRNA), 27.6% (Rabe+NCsiRNA), 11.2% (DMSO+SLC4A7 siRNA) and 21.8% (Rabe+SLC4A7 siRNA), indicating that Rabe still induced EV uptake in SLC4A7-silenced 4T1 cells. Thus, Rabe-mediated accumulation of v-ATPases at the PM is independent of PKA.

[0219] ②Verify the relationship between pH and EV endocytosis

[0220] Confocal microscopy analysis of pH-sensitive fluorescent probe BCECF-AM indicating pH value of 4T1 cells treated with DMSO or Rabe. Fig.34 Confocal microscopy analysis of ATP6V1A localization in phalloidin-labeled 4T1 cells. Fig.35 , Fig.37 As shown, flow cytometry analysis of pkh26-labeled EVs was performed in 4T1 cells cultured in normal pH (7.2) and low pH (6.4) medium. Fig.35 , Fig.36 As shown, or treated with Rabe alone or in combination with 2,4-dinitrophenol (2,4-dnp), the results are as follows Fig.37 , Fig.38 Confocal microscopy analysis of ATP6V1A and LAMP1+ lysosomes in 4T1 cells or ATP6V1A localization in phalloidin-labeled 4T1 cells treated with DMSO or Rabe, respectively. Fig.39 , Fig.40Confocal microscopy analysis of ATP6V1A and LAMP1+ lysosomal colitis in HeLa cells (left) or ATP6V1A localization in phalloidin-labeled HeLa cells (right) transfected without or with ATP6V0A2 plasmid and without (left) or with (right) cholesterol.

[0221] PPIs inhibit the transport of protons from the cytoplasm to the extracellular space, which may reduce the cytoplasmic pH. We detected a decrease in cytoplasmic pH in Rabe-treated 4T1 cells using the pH-sensitive fluorescent BCECF-AM, which prompted us to characterize the role of pH in Rabe-induced EV endocytosis. First, we confirmed that low pH (6.4) medium reduced cytoplasmic pH and analyzed ATP6V1A localization in phalloidin-labeled 4T1 cells by confocal microscopy, demonstrating that low pH induced PM accumulation of ATP6V1A, while the opposite result was obtained in high pH (7.6) medium in the presence of cholesterol. Second, flow cytometry analysis of PKH26-labeled EVs at different pH values ​​showed 10.4% (pH 7.2) and 16.6% (pH 6.4), respectively, indicating increased EV uptake by 4T1 cells. Furthermore, flow cytometric analysis of the uptake of PKH26-labeled EVs by 4T1 cells treated with DMSO or Rabe or DMSO+ or Rabe+ showed that the uptake was 12.2%, 20.7%, 11.7%, and 12.2%, respectively, indicating that the pH gradient induced by the protonophore 2,4-dinitrophenol (2,4-DNP) across the biomembrane was equal, abolishing the Rabe-mediated increase in ATP6V1A PM accumulation and EV uptake. These results suggest that Rabe induces EV endocytosis in a pH-dependent manner.

[0222] ③ Detection of ATP6V1A mRNA and protein levels on VM and PM

[0223] Confocal microscopy analysis of ATP6V1A and LAMP1+ lysosomal colitis in HeLa cells (left) or ATP6V1A localization in phalloidin-labeled HeLa cells (right) transfected without or with ATP6V0A2 plasmid and without (left) or with (right) cholesterol ( Fig.41 Confocal microscopy analysis of the localization of ATP6V1A in phalloidin-labeled HeLa cells (left) or the co-aggregation of ATP6V1A and LAMP1+ lysosomes in HeLa cells transfected or not with ATP6V1A in normal pH (7.2) (left) or high pH (7.6) (right) culture medium. Fig.42 Western blotting verified the overexpression or knockdown of ATP6V0A2 and ATP6V0A4 in HeLa cells. The results are shown in Fig.43 , Fig.44 Confocal microscopy was used to analyze the localization of ATP6V1A in phalloidin-labeled HeLa cells and transfected with Atp6v0a2 or Atp6v0a4 siRNA. Fig.45 , Fig.46 Confocal microscopy analysis of ATP6V1A and LAMP1+ lysosomal colocalization in HeLa cells transfected with Atp6v0a2 siRNA or Atp6v0a4 siRNA and cultured in high pH (7.6) or normal pH (7.2) medium. Fig.47 , Fig.48 Western blotting analysis of the indicated proteins in 4T1 cells treated with DMSO or Rabe showed the following results: Fig.49 The indicated proteins were immunoprecipitated and analyzed by western blotting in HEK293T cells co-transfected with the indicated plasmids. Fig.50 shown.

[0224] By confocal microscopy analysis, we found that Rabe significantly reduced the distribution of LAMP1+ lysosomes (indicating VM) but increased the PM distribution of ATP6V1A. Therefore, we speculated that PM and VM V0 competitively bind ATP6V1A and that reduced VM binding of ATP6V1A might enhance the interaction of ATP6V1A with PM V0. We then tested this hypothesis by overexpression of ATP6V0A2. Since the overexpression effect was low in 4T1 cells, we performed these experiments in HeLa cells, and confocal microscopy analysis of the localization of ATP6V1A in phalloidin-labeled HeLa cells revealed that in cholesterol-treated HeLa cells, the VM localization of ATP6V1A was significantly increased, while the PM localization of ATP6V1A was decreased. In contrast, in HeLa cells cultured in high pH (7.6) medium, overexpression of ATP6V0A4 led to an increase in PM and a decrease in VM localization of ATP6V1A. We obtained opposite results in ATP6V0A2- or Atp6v0a4-silenced 4T1 cells. We also excluded the effect of Rabe on the protein expression of ATP6V0A2 and ATP6V0A4. Furthermore, we confirmed that ATP6V0A2 overexpression reduced ATP6V0A4 which was pulled down by ATP6V1A and vice versa.

[0225] ATP6V1A is the catalytic subunit, ATP6V0A2 is the binding subunit on the endosomal membrane, and ATP6V0A4 is the binding subunit on the cell membrane. ATP6V1A can form a V-ATPases complex when combined with ATP6V0A2 or ATP6V0A4. In summary, the decrease in cytoplasmic pH mediated by PPIs promotes the dissociation of ATP6V1A from VM, thereby enhancing the assembly of PM v-ATPases.

[0226] 2. Experimental results: First, it was confirmed that PKA is not related to the process of PPIs-mediated PM v-ATPases assembly. Further, it was verified that PPIs-mediated EV endocytosis is carried out in a pH-dependent manner, and low pH induces PM accumulation of ATP6V1A. Finally, we found and verified that PPIs-mediated cytoplasmic pH reduction promotes the dissociation of ATP6V1A from VM, thereby enhancing the assembly of PM v-ATPases.

[0227] Example 6 PPIs aggravate IBD by accelerating the clearance of IEC-EV

[0228] In this example, we first introduced Rab27afl / fl×Villin-Cre mice (in which the secretion of A33+IEC-EV (EV derived from intestinal epithelial cells) was specifically inhibited, while CD11c+ev derived from DC (dendritic cells) was not inhibited) to study the role of Rabe and IEC-EV in the development of IBD. To further clarify that Rabe affects the progression of IBD by regulating the uptake of IEC-EV, mice treated with the TGF-βRI (TGF-β receptor) inhibitor SB525334 were introduced, and IEC-EVs from Cmv-cre mice (IEC-EV / Cre) were transferred to rossa-lsl-tdtomato mice (after these mouse cells take up Cre+ev, the cells will express tdTomato23) for control experiments. The specific steps are as follows: 1. Experimental methods

[0229] ① Testing the effects of PPIs on IBD

[0230] Mice were intraperitoneally injected with DMSO, Ome or Rabe, and then EVs were isolated from the colons of these mice. The protein content of these EVs was determined by the BCA method. The results are shown in Fig.51 Flow cytometry analysis of the A33 percentage of EVs captured by anti-CD63-coated latex beads from cultured mouse colon supernatants. Fig.52 Then, mice were induced with acute colitis using 2.5% DSS for 7 consecutive days, and DMSO, Ome or Rabe were injected intraperitoneally for 7 consecutive times. The changes in body weight were monitored, and the results were shown in Fig.53 As shown; monitoring colon length, the results are as follows Fig.54 As shown; and monitoring hematoxylin and eosin (H&E) stained colon tissue sections, the results are as Fig.55 shown.

[0231] The EV protein content measured by BCA method was from high to low in mice injected with DMSO, injected with Ome, and injected with Rabe. The A33 percentage of EV captured by anti-CD63 coated latex beads from the colon supernatant of cultured mice was analyzed by flow cytometry. The results were 23.8% (DMSO), 18.4% (Ome), and 12.2% (Rabe), respectively, indicating that Rabe or Ome treatment reduced the levels of colon EV and A33+IEC-EV. Consistent with these results, monitoring results showed that Rabe and Ome treatment accelerated the development of DSS-induced IBD, manifested by increased weight loss, shortened colon length, and more severe histological damage.

[0232] ②Study the role of IEC-EV in the progression of IBD

[0233] IBD was introduced into Rab27afl / fl×Villin-Cre mice, in which the secretion of A33+IEC-EVs was specifically inhibited, while the secretion of DC-derived CD11c+EVs was not inhibited. The CD63 levels (OD value 450nm) of colonic EVs of Rab27af / f and Villin-Cre Rab27af / f mice captured by anti-A33 or anti-CD11c pre-coated 96-well plates were analyzed by ELISA, as shown in Fig.56 As shown in Figure 2, mice were induced with acute colitis using 2.5% DSS, and DMSO or Rabe were injected intraperitoneally every day for 7 consecutive times. The weight changes of mice in each group were recorded. The results are shown in Figure 2. Fig.57 As shown; the colon length of each group of mice was recorded, and the results were as follows Fig.58 As shown; and record H&E stained colon tissue sections, the results are as follows Fig.59 As shown. In mice treated with the TGF-βRI inhibitor SB525334, 2.5% DSS was used to induce acute colitis in mice for 7 consecutive days, and the corresponding treatment was given every day for 7 consecutive times. The weight changes of mice in each group were monitored, and the results were shown as follows. Fig.60 As shown; the colon length of each group of mice was monitored, and the results were as follows Fig.61 As shown; H&E staining of colon tissue sections of each group of mice was recorded, and the results were as follows Fig.62 Representative immunofluorescence of CD45 staining and tdTomato expression in the colon of Rosa-LSL-tdTomato mice injected intravenously with IEC-EV or IEC-EV / Cre was monitored by transferring IEC-EV from Cmv-cre mice (IEC-EV / Cre) into rossa-lsl-tdtomato mice. Fig.63, 2.5% DSS was used to induce acute colitis in mice for 7 consecutive days, and the corresponding treatment was given every day for 7 consecutive times. The weight changes of mice in each group were recorded. The results are shown in Fig.64 As shown; the colon length of each group of mice was recorded, and the results were as follows Fig.65 As shown; H&E stained colon tissue sections of each group of mice were recorded, and the results were as follows Fig.66 shown.

[0234] In Rab27afl / fl×Villin-Cre mice, monitoring results showed that Rabe was hardly involved in the development of IBD. IEC-EVs inhibited the development of IBD through TGF-β13. We also found that Rabe no longer affected the severity of IBD in mice treated with the TGF-βRI inhibitor SB525334, further supporting the central role of IEC-EVs in this process. To clarify that Rabe affects the progression of IBD by regulating the uptake of IEC-EVs, we transferred IEC-EVs from Cmv-cre mice (IEC-EV / Cre) to rossa-lsl-tdtomato mice. We found by representative immunofluorescence that IEC-EV / Cre could be taken up by intestinal CD45+ and CD45- cells, and Rabe significantly enhanced this effect. In addition, monitoring results showed that the inhibitory effect of IBD progression by transferring IEC-EVs / Cre significantly suppressed the development of IBD in mice treated with Rabe or OME. Therefore, these results indicate that PPIs promote the clearance of IEC-EVs mediated by uptake, thereby aggravating IBD symptoms.

[0235] 2. Experimental results: IEC-EV has a protective effect on IBD, while PPIs aggravate IBD by accelerating the clearance of IEC-EV.

[0236] Example 7 PPIs enhance anti-tumor immunity by promoting TEV clearance

[0237] This example first images the endocytosis of TEV by tumor cells and evaluates the effect of Rabe on this process. Secondly, the effect of macropinocytosis-mediated tumor-derived EV (TEV) clearance on anti-tumor immunity is evaluated, and the direct effect of micropinocytosis on tumor cells is excluded by imaging. The specific steps are as follows:

[0238] 1. Experimental methods

[0239] ①Verify that Rabe accelerates TEV endocytosis in tumor cells

[0240] RFP+4T1 cells were subcutaneously inoculated into the back of mice, and Rabe was injected intratumorally. Then, we established 4T1 cells with ATP6V1A knockout (Atp6v1a- / -4T1) ( Fig.67), and 4T1 or Atp6v1a- / -4T1 tumors were subcutaneously inoculated on each side of the mice. Then, these mice were intravenously injected with 4t1-ev labeled with VivoTrack 680 (labeling method: VivoTrack 680 and EV were incubated at room temperature for 20 minutes), and the distribution of EVs in the indicated organs was detected using an in vivo imaging system (method: EVs labeled with VivoTrack 680 were intravenously injected into mice. Mice were euthanized 24 hours later, and tumors, brain, heart, lung, liver, spleen, kidney and gastrointestinal organs were collected and images were taken under the IVIS system (PerkinElmer)). The results are shown in Fig.68 Then, the mice were injected with 4T1 or Atp6v1a- / -4T1 subcutaneously in bilateral flank tumors, and Rabe was injected intratumorally in one flank tumor. The distribution of EVs in the indicated organs was detected using an in vivo imaging system. The results are shown in Fig.69 As shown. RFP+4T1 (red fluorescent protein marker) was injected subcutaneously into the bilateral flank tumors of mice, and Rabe was injected intratumorally into one flank tumor. Then, these mice were intravenously injected with PKH67-labeled 4T1-EV, and dorsal skinfold chambers (DSFC) were installed on these mice for multiphoton intravital microscopy (MP-IVM) analysis (specific reference for the method: Imaging windows for long-term intravital imaging. IntraVital 3, e29917 (2014).), and the experimental results are shown in Fig.70 , Fig.71 , Fig.72 shown.

[0241] Through the in vivo imaging system, we found that the uptake of 4T1-ev by Atp6v1a- / -4T1 was significantly inhibited. After intratumoral injection of Rabe and intravenous injection of 4T1-evs labeled with VivoTrack 680, we found that 4T1 tumors with Rabe injection internalized more 4T1-evs than 4T1 tumors without Rabe injection, which was not detected in Atp6v1a- / -4T1 tumor mice. These results indicate that Rabe promotes TEV uptake by tumors through macropinocytosis. According to MP-IVM analysis, Rabe led to accelerated EV uptake, accelerated the arrival of the absorption platform, and enhanced the absorption intensity from 0 to 14 hours after receiving the injection.

[0242] ②Evaluate the impact of macropinocytosis-mediated TEV clearance on anti-tumor immunity

[0243] The CCK8 method (kit: Yeasen 40203ES60) was used to detect the viability of 4T1 and Atp6v1a- / -4T1 cells after 48 h of culture. Fig.73Mice were injected subcutaneously with 4T1 or Atp6v1a- / -4T1 tumors on each flank, or 4T1 tumor-bearing mice were injected intratumorally with DMSO or Rabe as indicated. Fig.74 , Fig.75 As shown, flow cytometry analysis of the percentage of PD-1+, IFN-γ+, and Ki-67+ T cells in the CD45+CD8+ T cell population in the dLNs of these mice showed Fig.76 , Fig.77 Flow cytometry quantification of the percentage of CD45+CD8+ T cell populations in dln of mice receiving 4T1 or Atp6v1a- / -4T1 tumors, or mice receiving 4T1 tumors and treated with Rabe, respectively. Fig.78 , Fig.79 DMSO or Rabe was injected intratumorally, and then the tumor size of 4T1 tumor-bearing nude mice or Atp6v1a- / -4T1 tumor-bearing mice was measured. The results are shown in Fig.80 , Fig.81 shown.

[0244] Although Atp6v1a- / -4T1 and 4T1 cells showed comparable viability in vitro, CCK8 assay showed that Atp6v1a- / -4T1 tumors exhibited accelerated tumor growth relative to 4T1 tumors. We also detected a lower proportion of CD8+T cells in the draining lymph nodes (dLNs) of 4T1 Atp6v1a- / - tumor-bearing mice. In addition, increased exhaustion markers and decreased activation and proliferation markers were detected in CD8+T cells in these mice, indicating that the overall function of CD8+T cells was weakened, which was not conducive to anti-tumor activity. In contrast, we observed delayed tumor growth in 4T1 tumor-bearing mice treated with intratumoral Rabe. Accordingly, we found an increased proportion of CD8+T cells in the draining lymph nodes of these mice, decreased exhaustion markers in CD8+T cells, and enhanced activation and proliferation markers. This corresponds to the result that intratumoral injection of Rabe does not restrict 4T1 tumor growth in thymic-deficient mice. Furthermore, Rabe did not affect the progression of Atp6v1a- / -4T1 tumors, suggesting that Rabe-mediated suppression of antitumor T-cell responses is dependent on macropinocytosis.

[0245] ③Study the effect of micropinocytosis on tumor cells

[0246] 4T1 tumor-bearing mice treated with Rabe ( Fig.82 , Fig.83 , Fig.84 ) or 4T1 or Atp6v1a- / -4T1 tumor-bearing mice ( Fig.85 , Fig.86 , Fig.87 )TUNEL in tumor tissues ( Fig.82 , Fig.85)、LC3B( Fig.83 , Fig.86 ) and Ki-67( Fig.84 , Fig.87 ) to detect cell apoptosis, autophagy, and proliferation, respectively. No decrease in TUNEL staining and autophagosomes, and no increase in Ki-67 staining were found in 4T1 tumor tissues of Rabe-treated mice, indicating that Rabe does not affect tumor cell apoptosis and proliferation and nutrient-sensitive autophagy of tumors. In addition, we found that Atp6v1a- / -4T1 tumors had similar apoptosis, proliferation, and autophagy as 4T1 tumors. These results suggest that micropinocytosis may not directly affect the biological properties of tumor cells.

[0247] 2. Experimental results: Rabe can lead to accelerated EV absorption by tumor cells, accelerate the arrival of the absorption platform and enhance the absorption intensity. Rabe-mediated inhibition of anti-tumor T cell responses depends on macropinocytosis. Micropinocytosis may not directly affect the biological characteristics of tumor cells. In summary, PPIs enhance anti-tumor immunity by promoting TEV clearance.

[0248] Example 8 PPIs can reduce the resistance of tumor patients to anti-PD-1 treatment

[0249] This example establishes a control experiment of Atp6v1a- / -4T1 tumor-bearing mice and 4T1 tumor-bearing mice, and whether Rabe is administered or not, which proves that Rabe can effectively prevent PD-L1+TEV-induced immunosuppression. In addition, the effect of PPIs on PD-1 treatment resistance is further studied by collecting and analyzing the blood of tumor patients treated with PPIs. The specific steps are as follows:

[0250] 1. Experimental methods

[0251] ① Verify that Rabe prevents PD-L1+TEV-induced immunosuppression

[0252] ELISA analysis was performed on CD9+CD63+ and PD-L1+CD63+ EVs (OD 450nm values) in the serum of 4T1 or Atp6v1a- / -4T1 tumor-bearing mice and 4T1 tumor-bearing mice injected intratumorally with DMSO or Rabe. The results were as follows: Fig.88 , Fig.89 The tumor size of Pd1- / - mice bearing 4T1 or Atp6v1a- / -4T1 tumors was measured, and the results are shown in Fig.90 As shown; the tumor size of Pd1- / - mice bearing 4T1 tumors and injected with DMSO or Rabe intratumorally was measured, and the results are shown Fig.91ELISA analysis of CD9+CD63+ and PD-L1+CD63+ EVs (OD 450nm value) and tumor size in the serum of 4T1 tumor-bearing mice injected intravenously with DMSO or Rabe was performed to detect the effect of Rabe on CD9+CD63+ and PD-L1+CD63+ EVs, as well as tumor size. The results are shown in Fig.92 , Fig.93 shown.

[0253] Before significant differences in tumor size were observed, the number of serum CD9+ and PD-L1+ EVs in Atp6v1a- / -4T1 tumor-bearing mice was significantly higher than that in 4T1 tumor-bearing mice. The results were opposite in 4T1 tumor-bearing mice treated with Rabe compared with those not treated with Rabe. In Pd1- / - mice, Atp6v1a- / -4T1 and 4T1 tumors or 4T1 tumors showed similar growth trends with or without Rabe treatment. Systemic administration of Rabe also reduced serum CD9+ and PD-L1+ EVs and slowed the growth of 4T1 tumors.

[0254] ②Verify that Rabe eliminates resistance to anti-PD-1 therapy

[0255] Mice bearing 4T1 breast cancer cells or B16F10 melanoma cells were intraperitoneally injected with isotype control antibody Rat IgG2a or anti-PD-1, and simultaneously intravenously injected with DMSO or Rabe. Fig.94 , Fig.95 shown.

[0256] Anti-PD-1, together with systemic injection of Rabe, significantly inhibited the growth of 4T1 tumors resistant to anti-PD-1 therapy. Similar results were obtained in B16F10 tumor-bearing mice.

[0257] ③Verify the functional relevance of PPIs on TEV in cancer patients

[0258] Serum CD9 in patients with gastric cancer before (day 0) and after (day 2) PPI treatment + CD63 + and PD-L1 + CD63 + ELISA analysis was performed on EVs (OD value 450nm) to detect the difference between CD9+CD63+ and PD-L1+CD63+ EVs before and after treatment. The results are as follows Fig.96As shown; representative flow cytometry graphs (left) or statistical analysis (right) of the percentages of circulating CD8+T cells, PD-1+CD8+T and Tim3+CD8+T cells and Ki-67+CD8+T and granzyme B (Gzm B)+CD8+T cells in the blood of gastric tumor patients were performed to detect changes in different subpopulations of CD8+T cells to see if they are beneficial to anti-tumor. The results are shown as follows: Fig.97 , Fig.98 , Fig.99 The progression-free survival (PFS) of patients with gastric cancer treated with anti-PD-1 combined with or without PPI is shown in Fig.100 shown.

[0259] PPI treatment increased the proportion of circulating CD8+ T cells, reduced exhaustion markers, and enhanced activation and proliferation markers of CD8+ T cells. Therefore, PPIs may alleviate anti-PD-1 treatment resistance in tumor patients by reducing systemic TEV. The survival of 183 patients with advanced lung cancer who received single-agent anti-PD-1 treatment as second-line or subsequent treatment was retrospectively analyzed. During treatment, 28 patients also took Ome (20 mg, bid) orally due to gastric ulcers. Compared with patients treated with anti-PD-1 alone, these 28 patients had a better progression-free survival (PFS) than those without PPI treatment. In conclusion, Rabe attenuates resistance to anti-PD-1 treatment by removing immunosuppressive TEV.

[0260] 2. Experimental results: Rabe can prevent PD-L1+TEV-induced immunosuppression and eliminate resistance to anti-PD-1 therapy. Its mechanism is to weaken resistance to anti-PD-1 therapy by eliminating immunosuppressive TEV.

[0261] Example 9 PPIs can enhance the delivery of therapeutic drugs by EV

[0262] This example first studies whether Rabe improves the anti-tumor results of chemotherapy-loaded red blood cells (rbc-EV) by systemic administration of doxorubicin (DOX) loaded into RBC-EV (RBC-EV / DOX). Secondly, it is tested whether Rabe can improve the therapeutic effect of rbc-ev / miR-155-ASOs (antisense oligonucleotides of miR-155) to study whether Rabe enhances EV-mediated delivery of therapeutic drugs by promoting EV endocytosis. The specific steps are as follows:

[0263] 1. Experimental methods

[0264] ① Study whether PPIs can improve the delivery efficiency of EVs

[0265] Mice were first injected subcutaneously with 4T1 tumors and then intravenously injected with DMSO or Rabe. Then, these mice were intravenously injected with VivoTrack 680-labeled rbc-evs, and the distribution of EVs in the indicated organs was detected using an in vivo imaging system. The results are shown in Fig.101 Then, we loaded doxorubicin into rbc-ev (rbc-ev / Dox) and used CCK8 method to detect the viability of 4T1 and Atp6v1a- / -4T1 cells treated with RBC-EVs / Dox alone or in combination with Rabe. The results are shown in Fig.102 The effect of Rabe on RBC-EVs / Dox on the size of 4T1 tumors in nude mice and Atp6v1a- / -4T1 tumors was determined. The results are shown in Fig.103 , Fig.104 As shown, representative immunofluorescence images were measured, and the results are shown in Fig.105 shown.

[0266] Using an in vivo imaging system, we confirmed that systemic administration of Rabe promoted the uptake of RBC-EVs by 4T1 tumors. Then, doxorubicin was loaded into RBC-EVs (rbc-EV / Dox), and we found that Rabe treatment produced more pronounced cytotoxicity in 4T1 cells than in Atp6v1a- / -4T1 cells caused by rbc-EV / Dox. Correspondingly, Rabe significantly enhanced the antitumor effect of RBC-EVs / Dox on 4T1 tumors in nude mice, but had no effect on Atp6v1a- / -4T1 tumors. In addition, we detected increased Dox uptake and apoptosis in Rabe-treated 4T1 tumors, which were significantly abolished in Atp6v1a- / -4T1 tumors.

[0267] ② Test whether Rabe can improve the therapeutic effect of rbc-ev / miR-155-ASOs

[0268] Real-time PCR analysis of miR-155 expression in the liver of mice intravenously injected with rbc-ev / miR-NC-ASO, rbc-ev / miR-NC-ASO and Rabe, rbc-ev / miR-155-aso, or rbc-ev / miR-155-aso and Rabe. The results are shown in Fig.106As shown. ALF mice induced by D-GalN and LPS (D-GalN / LPS) were intravenously injected with rbc-ev / miR-NC-ASO, rbc-ev / miR-155-ASO and Rabe, rbc-ev / miR-155-ASO, or rbc-ev / miR-155-ASO and Rabe. ELISA analysis of H&E-stained liver tissue sections, plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, and serum IL-1β, IL-6, and TNF protein levels was performed, and the results were shown as follows, respectively. Fig.107 , Fig.108 , Fig.109 As shown. ALF was introduced into Atp6v1a- / - mice by treatment with D-GalN and LPS (D-GalN / LPS), chemical agents used to induce liver injury, followed by intravenous injection of rbc-ev / miR-NC-ASO, rbc-ev / miR-155-ASO and Rabe, rbc-ev / miR-155-ASO, or rbc-ev / miR-155-ASO and Rabe. H&E-stained liver tissue sections, plasma AST and ALT levels, and serum IL-1β, IL-6, and TNF protein levels were analyzed again by ELISA, and the results are shown in Fig.110 , Fig.111 , Fig.112 shown.

[0269] Rabe had a synergistic effect on the downregulation of miR-155 levels in rbc-ev / miR-155-aso-induced ALF liver. Consistent with this result, Rabe significantly improved the therapeutic effects of RBC-EVs / miR-155-ASOs on ALF, as evidenced by liver histopathology, function, and proinflammatory cytokine levels. However, we were unable to observe these effects in Atp6v1a- / - mice. In summary, Rabe enhances EV-mediated delivery of therapeutic drugs by promoting EV endocytosis.

[0270] 2. Experimental results: PPIs can enhance the delivery effect of EV on therapeutic drugs, Rabe can improve the therapeutic effect of rbc-ev / miR-155-ASOs, and Rabe enhances EV-mediated delivery of therapeutic drugs by promoting EV endocytosis.

[0271] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto, and any equivalent modifications or substitutions that can be easily thought of by any technician familiar with the technical field within the technical scope disclosed by the present invention should be included in the protection scope of the present invention.

Claims

1. A use of a proton pump inhibitor for preparing an anti-tumor preparation, characterized in that: The proton pump inhibitor is selected from one or more of omeprazole, lansoprazole, esomeprazole, pantoprazole, rabeprazole, dacilprazole or a pharmaceutically acceptable salt thereof.

2. The use according to claim 1, characterized in that: The proton pump inhibitor improves anti-tumor immunity by promoting the clearance of extracellular vesicles.

3. A use of a proton pump inhibitor for preparing a preparation for reducing drug resistance to anti-PD-1 therapy, characterized in that: The proton pump inhibitor is selected from one or more of omeprazole, lansoprazole, esomeprazole, pantoprazole, rabeprazole, dacilprazole or a pharmaceutically acceptable salt thereof.

4. The use according to claim 3, characterized in that: The proton pump inhibitors attenuate resistance to anti-PD-1 therapy by clearing extracellular vesicles.

5. Use of a proton pump inhibitor for preparing a preparation for promoting drug delivery, characterized in that: The proton pump inhibitor is selected from one or more of omeprazole, lansoprazole, esomeprazole, pantoprazole, rabeprazole, dacilprazole or a pharmaceutically acceptable salt thereof.

6. The use according to claim 5, characterized in that The drug is delivered via extracellular vesicles as carriers; the proton pump inhibitor promotes the endocytosis of extracellular vesicles, thereby promoting the delivery of drugs by extracellular vesicles.

7. Use of a proton pump inhibitor for preparing a preparation for reducing the content of extracellular vesicles in a living body, characterized in that: The proton pump inhibitor is selected from one or more of omeprazole, lansoprazole, esomeprazole, pantoprazole, rabeprazole, dacilprazole or a pharmaceutically acceptable salt thereof.

8. The use according to claim 7, characterized in that The proton pump inhibitors increase the endocytosis of extracellular vesicles by increasing macropinocytosis, thereby reducing the extracellular vesicle content.

9. The use according to claim 8, characterized in that The proton pump inhibitors promote the assembly of PM v-ATPases by lowering the pH, thereby increasing macropinocytosis.

10. A product for anti-tumor, or drug delivery, or reducing drug resistance to anti-PD-1 therapy, characterized in that These include proton pump inhibitors.

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