Dendritic cells loaded with pyroptotic vesicles of tumor cells and applications thereof

Abstract

The present application relates to the field of tumor treatment, in particular to a kind of tumor cell pyroptosis vesicle loaded dendritic cell and its application.The present application utilizes LBP to induce tumor cell pyroptosis, extracts tumor cell pyroptosis vesicle, then fuses the latter with dendritic cell, prepares to obtain tumor cell pyroptosis vesicle loaded dendritic cell, and confirms its effectiveness in treating liver cancer, colorectal cancer and preventing lung cancer, provides a new strategy for the prevention and treatment of cancer, and can be used for tumor biological medicine manufacturing.In addition, the present application further illustrates the mechanism of action of the tumor cell pyroptosis vesicle loaded dendritic cell in treating liver cancer, and lays a theoretical foundation for its practical application in clinical treatment of cancer.

Description

Technical Field

[0001] This invention relates to the field of tumor treatment, and in particular to a dendritic cell loaded with tumor cell pyroptosis vesicles and its application, which can be used in the manufacture of tumor biopharmaceuticals. Background Technology

[0002] Currently, the treatment of solid tumors faces significant challenges due to the tumor's immunosuppressive microenvironment, commonly known as a "cold tumor." How to transform the tumor into an immunopromoting microenvironment (a "hot tumor") is a key issue that the industry needs to address.

[0003] Dendritic cells (DCs) are the most powerful known professional antigen-presenting cells. In individuals without tumors, DCs stimulate the proliferation of cytotoxic lymphocytes (CD8T cells) and enhance their killing function by presenting tumor antigens and secreting cytokines.

[0004] Previous methods for loading tumor antigens onto dendritic cells (DCs) included: (1) loading tumor lysates and apoptotic tumor cell products, and (2) loading DCs with tumor-associated antigen peptide pulses and nucleic acid transfection (mRNA or DNA). The following analysis examines the shortcomings of these two methods: (1) Loading DCs with apoptotic tumor cells does not achieve the effect of activating pro-inflammatory factors because apoptosis is an immune-silent death process. PS outwards (phosphatidylserine): a large number of PS on the surface of apoptotic cells are recognized by DCs, which initiate the cell burial pathway. DCs then secrete anti-inflammatory factors such as IL-10, TGF-β, and IL-27, inducing Treg production. Therefore, when DCs are loaded with apoptotic tumor cells, DCs only take up tumor antigens, but there is no adjuvant signal and they cannot mature (low expression of CD80 / CD86 / MHC). They only perform silent antigen presentation and cannot initiate systemic anti-tumor inflammation.

[0005] (2) Using tumor-associated antigen peptide pulses and nucleic acid transfection (mRNA or DNA) to load DCs has limited coverage and can only play a role in transmitting single antigen information. Therefore, when the prepared DCs are reinfused into tumor patients, they are difficult to overcome the cold tumor immunosuppressive microenvironment and are difficult to effectively activate tumor-killing immune cells such as CD8 T cells and M1 macrophages.

[0006] Chinese patent application CN1686538A discloses dendritic cells loaded with apoptotic heat-shocked tumor cells; Chinese patent application CN103648524A discloses immature dendritic cells loaded with apoptotic tumor cells treated with high hydrostatic pressure. Both of these dendritic cell methods involve direct incubation with apoptotic tumor cells. Since it is impossible to determine whether any surviving tumor cells remain among the apoptotic cells, if surviving tumor cells are mistakenly introduced into the body during the reinfusion of modified dendritic cells, this could actually increase the risk of cancer promotion. Furthermore, apoptotic tumor cells cannot stimulate inflammation, so these methods fail to activate and bind tumor antigens and pro-inflammatory factors. Therefore, when the prepared dendritic cells are reinfused into cancer patients, they struggle to overcome the cold tumor immunosuppressive microenvironment and effectively activate tumor-killing effector immune cells such as CD8 T cells and M1 macrophages.

[0007] Therefore, there is an urgent need to develop safe and effective dendritic cell tumor vaccines. Summary of the Invention

[0008] Based on the above, this invention provides dendritic cells loaded with tumor cell pyroptosis vesicles and their applications. This invention utilizes LBP to induce pyroptosis in tumor cells, extracts pyroptosis vesicles from tumor cells, and then fuses these pyroptosis vesicles with dendritic cells to prepare dendritic cells loaded with tumor cell pyroptosis vesicles. The effectiveness of these cells in treating liver cancer, colorectal cancer, and preventing lung cancer has been demonstrated, providing a new strategy for cancer prevention and treatment, and applicable to the manufacture of tumor biopharmaceuticals. Furthermore, this invention elucidates the mechanism of action of the dendritic cells loaded with tumor cell pyroptosis vesicles in treating liver cancer, laying a theoretical foundation for their practical application in clinical cancer treatment.

[0009] The first objective of this invention is to provide a dendritic cell.

[0010] A second object of the present invention is to provide a pharmaceutical composition.

[0011] A third object of the present invention is to provide the use of the dendritic cells described in the first object or the pharmaceutical composition described in the second object.

[0012] A fourth objective of the present invention is to provide a method for preparing the dendritic cells described in the first objective.

[0013] The above-mentioned objective of this invention is achieved through the following technical solution: The present invention The first aspect The present invention provides a dendritic cell loaded with tumor cell pyroptosis vesicles.

[0014] Pyroptosis is an inflammatory form of programmed cell death mediated by the gasdermin (GSDM) protein. Upon activation by the caspase family of proteases, GSDM proteins form pores approximately 10–15 nm in diameter on the cell membrane, leading to osmotic imbalance, swelling, and eventual rupture. During this lysis process, the cell contents are encapsulated by a lipid bilayer and released as vesicles, known as pyroptosomes. Because pyroptosomes do not contain tumor cells, they do not pose a cancer-promoting risk. Furthermore, in this invention, differential centrifugation is used to remove pyroptosomes that have not been phagocytosed by dendritic cells (DCs).

[0015] Unlike apoptotic bodies formed during apoptosis, pyroptotic vesicles not only encapsulate cellular internal components but also serve as a "natural vaccine" carrier rich in immunostimulatory signals, carrying tumor antigens and immunostimulatory molecules, as detailed below: 1. Tumor-specific antigens Pyroptosis vesicles carry a variety of antigens derived from tumor cells. After dendritic cells (DCs) engulf pyroptosis vesicles, the tumor-associated antigens within the vesicles are processed by lysosomes and the Golgi apparatus to form MHC complexes, which are then presented to DCs and subsequently recognized by T cells, triggering an immune response in the body.

[0016] 2. Damage-related molecular patterns (DAMPs) Pyroptosis vesicles are rich in damage-associated molecular patterns (DAMPs) and other immunostimulatory molecules, such as high-mobility group box 1 (HMGB1). These molecules can induce a strong inflammatory response, directly activating and enhancing the antigen-presenting capacity of dendritic cells (DCs). Reinfusing activated dendritic cells into tumor patients can induce the secretion of various pro-inflammatory factors, thereby inducing pyroptosis proteins in tumor tissue to break the immunosuppressive state of the tumor microenvironment.

[0017] The tumors described in this invention are refractory solid tumors that exhibit a cold tumor state of immunosuppression in clinical practice and do not respond well to immunotherapy.

[0018] In some specific embodiments, the tumor cells are liver cancer cells, more specifically, the tumor cells are Hepa 1-6 cells; in other specific embodiments, the tumor cells are colorectal cancer cells or lung cancer cells.

[0019] The present invention The second aspect The present invention provides a pharmaceutical composition comprising the dendritic cells described in the first aspect and an agent capable of inducing pyroptosis in tumor cells.

[0020] In some specific embodiments, the ratio of dendritic cells to the agent that induces pyroptosis of tumor cells described in the first aspect may vary slightly depending on the subject. For example, in mice, the treatment dose is 5 million to 10 million dendritic cells per mouse, and the dose of LBP that induces pyroptosis of tumor cells is 10 mg / kg.

[0021] In some preferred embodiments, the reagent is a chemical reagent, and more preferably, the reagent is lobaplatin (LBP).

[0022] This invention confirms that the dendritic cells described in the first aspect, alone or in combination with LBP, can induce an increase in the expression of GSDME-FL, GSDME-NT, GSDMD-FL, and GSDMD-NT pyroptosis proteins in liver cancer, thereby significantly improving the immunotherapy effect. That is, the dendritic cells and LBP combination described in the first aspect have a synergistic effect.

[0023] It should be understood that, since this invention elucidates the mechanism of action of dendritic cells described in the first aspect at the tissue, cellular, and molecular levels, and demonstrates the synergistic effect of dendritic cells combined with LBP in treating or preventing tumors using DCs loaded with pyroptosis vesicles alone, those skilled in the art can, based on this, select different agents that induce pyroptosis of tumor cells and dendritic cells described in the first aspect to jointly treat different tumors or achieve better therapeutic effects.

[0024] The present invention The third aspect It provides the use of the dendritic cells described in the first aspect or the pharmaceutical composition described in the second aspect in the preparation of an agent for treating tumors and / or cancer.

[0025] In some specific embodiments, the tumor and / or cancer is liver cancer; in other specific embodiments, the tumor and / or cancer is colorectal cancer.

[0026] It should be understood that, since this invention elucidates the mechanism of action of the dendritic cells described in the first aspect at the tissue, cellular, and molecular levels, and also confirms the therapeutic effect of the dendritic cells described in the first aspect and their synergistic effect with LBP in the treatment of liver cancer, those skilled in the art can, based on this, select dendritic cells loaded with pyroptosis vesicles from different tumor cells and / or different agents that induce pyroptosis of tumor cells in combination with the dendritic cells described in the first aspect to jointly treat different tumors and / or cancers in order to achieve better therapeutic effects.

[0027] The present invention The fourth aspectIt provides the use of the dendritic cells described in the first aspect or the pharmaceutical composition described in the second aspect in the preparation of an agent for the prevention of tumors and / or cancer.

[0028] In some specific implementations, the tumor and / or cancer is lung cancer.

[0029] It should be understood that, since this invention elucidates the mechanism of action of dendritic cells described in the first aspect at the tissue, cellular, and molecular levels, those skilled in the art can, based on this, select dendritic cells loaded with pyroptosis vesicles from different tumor cells to prevent different tumors and / or cancers in order to achieve better preventive effects.

[0030] The first of the present invention Five aspects The purpose is to provide the use of the dendritic cells described in the first aspect or the pharmaceutical composition described in the second aspect in any of the following aspects: preparation of an agent that inhibits tumor growth and / or proliferation; preparation of an agent that promotes the aggregation of immune killer cells in tumors; preparation of an agent that inhibits angiogenesis within tumors; preparation of an agent that increases the content of pro-inflammatory cytokines; preparation of an agent that induces pyroptosis of tumor cells; preparation of an agent that promotes the expression of perforin 1; preparation of an agent that increases the number of immune cells; preparation of an agent that promotes the accumulation of macrophages in tumors; preparation of an agent that induces M1 polarization of macrophages.

[0031] In some specific embodiments, the pro-inflammatory cytokines are IFN-γ and TNF-α.

[0032] In some specific embodiments, the dendritic cells of the first aspect or the pharmaceutical composition of the second aspect induce pyroptosis of tumor cells through the following pathway: (1) ROS-NLRP3-Caspase-1-GSDMD pathway: M1 macrophages release large amounts of ROS, which activates the NLRP3 inflammasome, thereby assembling and activating Caspase-1. Activated Caspase-1 directly cleaves GSDMD, releasing its N-terminal pore-forming fragment (GSDMD-NT), which triggers pyroptosis.

[0033] (2) TNF-α-mediated upregulation of GSDME / GSDMD expression and cytotoxic lymphocyte killing pathway: M1 macrophages upregulate TNF-α expression, which promotes the transcription and expression of GSDME and GSDMD. Simultaneously, TNF-α, in conjunction with cytokines such as IFN-γ, promotes the killing of cytotoxic lymphocytes (CD8+). + T cells and NK cells proliferate and activate, causing them to release perforin and granzyme B. After entering tumor cells, granzyme B can directly cleave GSDME, activating GSDME-mediated pyroptosis.

[0034] In some specific embodiments, the immune cells are monocyte-derived macrophages (MoMF) and CD8+ cells. + T cells and NK cells.

[0035] The present invention The sixth aspect The method provides a method for preparing the dendritic cells described in the first aspect, the method comprising the following steps: S1. Induces pyroptosis in tumor cells and extracts pyroptosis vesicles from tumor cells; S2. Pyroptosis vesicles were co-cultured with dendritic cells to obtain dendritic cells loaded with tumor cell pyroptosis vesicles.

[0036] In some specific implementations, step S1 uses differential centrifugation and polyethylene glycol precipitation to extract pyroptosis vesicles, or uses ultra-high speed centrifugation to extract pyroptosis vesicles.

[0037] In some preferred embodiments, after co-culturing the pyroptosis vesicles with dendritic cells in step S2, centrifugation is performed to remove pyroptosis vesicles that have not been phagocytosed by dendritic cells, and the precipitate is collected to obtain dendritic cells loaded with tumor cell pyroptosis vesicles; more preferably, the centrifugation speed is 300 g.

[0038] In some specific embodiments, the dendritic cells described in the first aspect, the pharmaceutical composition described in the second aspect, or the preparation method described in the sixth aspect, wherein the tumor cells are any one or more of liver cancer cells, colorectal cancer cells, and lung cancer cells.

[0039] The beneficial effects of this invention are: This invention provides a novel type of dendritic cell, namely, dendritic cells loaded with tumor cell pyroptosis vesicles. These dendritic cells activate and bind to tumor antigens and pro-inflammatory factors, inhibiting the growth and proliferation of various tumors and exhibiting a certain degree of universality. Furthermore, these dendritic cells can also prevent tumor formation, demonstrating multiple therapeutic effects. Simultaneously, the dendritic cells loaded with tumor cell pyroptosis vesicles exhibit a synergistic effect with agents that induce tumor cell pyroptosis in the treatment of tumors / cancer. This invention also elucidates the mechanism of action of the dendritic cells loaded with tumor cell pyroptosis vesicles, laying a theoretical foundation for their clinical application. This invention further provides a method for preparing the dendritic cells loaded with tumor cell pyroptosis vesicles. This preparation method is simple and facilitates the mass production of the dendritic cells, thereby helping to control production costs. Attached Figure Description

[0040] Figure 1The morphological structure of Hepa1-6 cells described in Example 1 is shown below. Control 1 to Control 3 are three replicates of untreated Hepa1-6 cells (control group), and LBP 1 to LBP 3 are three replicates of Hepa1-6 cells treated with 20 μg / mL LBP (LBP group). Figure 2 The particle size distribution of pyroptotic vesicles in Hepa1-6 cells as described in Example 2; Figure 3 Proteomic analysis of pyroptotic vesicles in Hepa1-6 cells as described in Example 2; Figure 4 Western blot detection of GSDME-FL, GSDME-NT, GSDMD-FL, and GSDMD-NT proteins in Hepa1-6 cell pyroptosis vesicles as described in Example 2; Figure 5 The image shows a fluorescence image of DC cells that have engulfed pyroptosis vesicles as described in Example 3; wherein, green fluorescence represents DiO-labeled pyroptosis vesicles, blue fluorescence represents DC cell nuclei, and the overlapping area of ​​green and blue fluorescence represents DC cells that have engulfed pyroptosis vesicles. Figure 6 The mice described in Example 4 ( n = 10) liver phenotype diagrams, where Sham is the sham surgery group, Model is the model group, DC is the dendritic cell therapy group loaded with tumor cell pyroptosis vesicles, LBP is the lobaplatin therapy group, and DC+LBP is the dendritic cell therapy group loaded with tumor cell pyroptosis vesicles combined with lobaplatin. Figure 7 The mouse liver tissue described in Example 4 ( n =6) HE staining results, where Sham is the sham operation group, Model is the model group, DC is the dendritic cell therapy group loaded with tumor cell pyroptosis vesicles, LBP is the lobaplatin therapy group, and DC+LBP is the dendritic cell therapy group loaded with tumor cell pyroptosis vesicles combined with lobaplatin. Figure 8 The results of immunohistochemical staining of CD34 antigen in mouse liver tissue described in Example 4 are shown. In this group, Sham represents the sham-operated group, Model represents the model group, DC represents the dendritic cell treatment group loaded with tumor cell pyroptosis vesicles, LBP represents the lobaplatin treatment group, and DC+LBP represents the combined treatment group of dendritic cells loaded with tumor cell pyroptosis vesicles and lobaplatin. The blue area represents the cell nucleus. Figure 9The results of Ki67 antigen immunohistochemical staining of mouse liver tissue described in Example 4 are shown below. Sham represents the sham-operated group, Model represents the model group, DC represents the dendritic cell treatment group loaded with tumor cell pyroptosis vesicles, LBP represents the lobaplatin treatment group, and DC+LBP represents the combined treatment group of dendritic cells loaded with tumor cell pyroptosis vesicles and lobaplatin. The blue area represents the cell nucleus, and the brown area represents the immunohistochemically positive area. Figure 10 The mice described in Example 4 ( n =6) Detection results of serum IFN-γ and TNF-α levels, among which, Figure 10 A in Figure 10 A) shows the detection results of IFN-γ content. Figure 10 B in Figure 10 B) Results of TNF-α level detection; Sham group (sham surgery group), Model group (model group), DC group (dendritic cell therapy group loaded with tumor cell pyroptosis vesicles), LBP group (loxaplatin therapy group), DC+LBP group (dendritic cell therapy group loaded with tumor cell pyroptosis vesicles combined with loxaplatin); * P <0.05,** P <0.01, **** P <0.0001, #### P <0.0001; Figure 11 The mouse liver tissue described in Example 4 ( n =3) Detection of pyroptosis marker protein levels, among which, Figure 11 A in Figure 11 A) is a Western blot chromatogram. Figure 11 B in Figure 11 B) ~ Figure 11 G-graph in Figure 11 G) are based on Figure 11 Quantitative results of the expression levels of Cleaved-caspase-3, GSDME-FL, GSDME-NT, Cleaved-caspase-1, GSDMD-FL, and GSDMD-NT proteins in the liver tissue of mice (A); Sham group (sham-operated group), Model group (model group), DC group (dendritic cell therapy group loaded with tumor cell pyroptosis vesicles), LBP group (loxaplatin therapy group), and DC+LBP group (dendritic cell therapy group loaded with tumor cell pyroptosis vesicles combined with lobaplatin);* P <0.05,** P <0.01, *** P <0.001, **** P <0.0001, #### P <0.0001, $P <0.05, $$ P <0.01, $$$$ P <0.0001, ns indicates no significant difference; Figure 12 The mouse liver tissue described in Example 4 ( n =6) The results of the perforin content detection, where Sham is the sham surgery group, Model is the model group, DC is the dendritic cell therapy group loaded with tumor cell pyroptosis vesicles, LBP is the lobaplatin therapy group, and DC+LBP is the dendritic cell therapy group loaded with tumor cell pyroptosis vesicles combined with lobaplatin;** P <0.01, **** P <0.0001, ns indicates no significant difference; Figure 13 The results are single-cell sequencing results of mouse liver tissue described in Example 4, where DC is the mouse sample of the dendritic cell therapy group loaded with tumor cell pyroptosis vesicles, and Tumor is (tumor group). Figure 14 The expression of CD68 and iNOS antigens in mouse liver tissue as described in Example 4 is shown in the figure. Sham is the sham-operated group, Model is the model group, DC is the dendritic cell treatment group loaded with tumor cell pyroptosis vesicles, LBP is the lobaplatin treatment group, and DC+LBP is the dendritic cell treatment group loaded with tumor cell pyroptosis vesicles combined with lobaplatin. Figure 15 The results of colonoscopy in mice described in Example 5 are shown below, where NC represents normal mice, CM represents (tumor model group), and DC represents the dendritic cell therapy group loaded with tumor cell pyroptosis vesicles. Figure 16 This is an image illustrating the effect of dendritic cells loaded with tumor cell pyroptosis vesicles in preventing subcutaneous tumors of LLC lung cancer cells, as described in Example 6. The red box represents the subcutaneous tumor. Detailed Implementation

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0043] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0044] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0045] Unless otherwise stated, the reagents and instruments used in this invention are commercially available.

[0046] Example 1: Induction of Pyroptosis in Tumor Cells 1.1 Drugs and Reagents Hepa1-6 cells (catalog number: CL-0105): purchased from Wuhan Pronosei Life Science Technology Co., Ltd.

[0047] Lobaplatin (LBP): Purchased from Hainan Chang'an International Pharmaceutical Co., Ltd. (specification: 10 mg / vial), used to induce pyroptosis in tumor cells.

[0048] 1.2 Cell Processing Hepa1-6 cells were fed at a rate of 2 × 10⁻⁶ 5 Hepa1-6 cells were seeded at a density of 1 cell / mL in appropriately sized wells of DMEM high-glucose basal medium and cultured for 24 h. Afterward, the cells were divided into a control group and a lobaplatin (LBP) group. The control group received no intervention, while the LBP group was induced with DMEM high-glucose basal medium containing 20 μg / mL LBP. Cells were cultured for another 24 h after each group was treated; subsequently, cells and culture supernatant were collected for further experiments.

[0049] 1.3 Observing cell morphology Observe the cells treated in step "1.2" under a scanning electron microscope and record the morphology and structure of the cells.

[0050] 1.4 Experimental Results like Figure 1 As shown, compared with the Control group, Hepa1-6 cells in the LBP group shrank from their original adherent polygonal shape to a round shape, became significantly swollen, and showed a large number of irregular pores and vesicle-like protrusions on the cell membrane surface. The integrity of the membrane surface structure was completely lost, with extensive cell membrane rupture, accompanied by the complete loss of microvilli and feet, and the release of intracellular substances. This indicates that LBP successfully induced pyroptosis in Hepa1-6 cells. Therefore, subsequent induction of tumor cell pyroptosis based on the above method was carried out.

[0051] Example 2: Preparation of pyroptosis vesicles from tumor cells This embodiment combines differential centrifugation and polyethylene glycol 6,000 precipitation to extract pyroptosis vesicles. The specific steps are as follows: 2.1 Induction of Pyroptosis in Tumor Cells Hepa1-6 cells in the logarithmic growth phase were selected, the culture medium in the culture flask was aspirated, and PBS was added for rinsing. After discarding the PBS, DMEM high glucose basal medium containing 10 μg / mL and 20 μg / mL lobaplatin was added respectively, and the cells were cultured for another 24 h. Then, intervention was performed, and Hepa1-6 cells that were not induced by LBP were used as the control group (Control group).

[0052] In addition, in order to obtain pyroptosis vesicles with higher pyroptosis protein content, this embodiment also selected time points of 0 h, 12 h, and 24 h to screen for suitable intervention times.

[0053] 2.2 Extraction of pyroptosis vesicles After 24 hours, the liquid in the bottle was collected and centrifuged at 250×g for 5 min. At this time, the tumor cells were in the precipitate, while the pyroptotic vesicles were in the supernatant due to their small size. The supernatant was aspirated and passed through a sterile 8 μm filter membrane. The liquid after passing through the filter membrane was mixed with the prepared PEG 6,000 solution at a volume ratio of 1:2 and placed in a 50 mL centrifuge tube. The mixture was then incubated at 4°C for 24 hours. After 24 hours of incubation, the mixture was centrifuged at 3,000×g for 20 min, and the liquid was discarded. Each tube was washed with 4 mL of PBS. The precipitate obtained by centrifugation was mixed by pipetting and centrifuged again at 3,000×g for 20 min. The resulting precipitate was the pyroptotic vesicles of Hepa1-6 cells. The precipitate was resuspended in an appropriate amount of 1640 basal culture medium and stored at 4°C.

[0054] 2.3 Verification of pyroptosis vesicles (1) The diameter range of pyroptosis vesicle particles prepared by treating tumor cells with 20 μg / mL LBP for 24 h was analyzed using a particle size analyzer.

[0055] (2) Proteomic analysis of Hepa1-6 pyroptosis vesicles: To explore the molecular characteristics of pyroptosis vesicles isolated from the supernatant of Hepa1-6 cells after 24 h of LBP intervention, AstralDIA quantitative proteomics technology was used to detect them and analyze the tumor antigen information and related signaling molecules obtained by DC cells after taking up Hepa1-6 pyroptosis vesicles. Specifically, WikiPathway enrichment analysis was performed on the differentially expressed proteins of pyroptosis vesicles from Hepa1-6 cells after LBP intervention (LBP group) and extracellular vesicles of Hepa1-6 cells without LBP intervention (Control group).

[0056] (3) GSDME-FL, GSDME-NT, GSDMD-FL, and GSDMD-NT are key proteins for pyroptosis execution. Therefore, in this embodiment, Western blot (WB) technology is used to detect the above four proteins in the pyroptosis vesicles of Hepa1-6 cells prepared in step “2.2” to verify whether the prepared pyroptosis vesicles carry key signals for pyroptosis execution, and thus verify the successful construction of pyroptosis vesicles.

[0057] 2.4 Experimental Results Particle size results as follows Figure 2 As shown, two peaks appeared, at 200 nm and 1,000 nm, indicating that the pyroptosis vesicle particles have two main size peaks, one at 200 nm and the other at 1,000 nm. The proteomics sequencing results are as follows... Figure 3 As shown, differentially expressed proteins were significantly enriched in the oxidative stress response pathway. P <0.01), inflammatory response pathway, histone modifications, P <0.01), all three pathways are closely related to the induction of pyroptosis and immune regulation. Western blot results are as follows: Figure 4 As shown, compared with extracellular vesicles extracted from Hepa1-6 cells without LBP intervention (Control group), the expression levels of GSDME-FL, GSDME-NT, GSDMD-FL, and GSDMD-NT proteins were significantly increased in the 10 μg / mL and 20 μg / mL LBP groups, indicating successful preparation of pyroptotic vesicles from Hepa1-6 cells. Since the expression levels of the above four proteins were higher when the LBP concentration was 20 μg / mL, 20 μg / mL LBP was preferentially used to prepare pyroptotic vesicles from tumor cells.

[0058] Furthermore, with the extension of LBP treatment time, the protein content of GSDME-FL, GSDME-NT, GSDMD-FL, and GSDMD-NT in pyroptotic vesicles of Hepa1-6 cells increased accordingly, that is, the expression levels of the above four proteins were highest when LBP intervention lasted for 24 h. Figure 4 Therefore, pyroptosis vesicles of tumor cells were prepared by treating tumor cells with 20 μg / mL LBP for 24 h.

[0059] In summary, this embodiment successfully prepared pyroptosis vesicles of Hepa1-6 cells and optimized their preparation method.

[0060] Example 3: Preparation of dendritic cells loaded with tumor cell pyroptosis vesicles 3.1 Preparation of Hepa1-6 cell-loaded pyroptotic vesicle dendritic cells This embodiment provides a method for preparing dendritic cells loaded with Hepa1-6 cell pyroptosis vesicles, the specific steps of which are as follows: Pyroptosis vesicles extracted from Hepa1-6 cells cultured in one T75 flask (see Examples 1-2 for specific preparation steps) were divided into three equal portions. The pyroptosis vesicles were resuspended in DC complete culture medium and then added to three T75 flasks containing DCs (each containing approximately 7 million DCs) and DC complete culture medium, respectively, so that the pyroptosis vesicles and DC cells were co-cultured.

[0061] After one day of co-culturing, the culture medium in the flask was completely replaced with DC complete medium containing LPS at a final concentration of 1 μg / mL. The cells at the bottom of the flask were gently pipetted, and then the culture medium was added to a 15 mL centrifuge tube. The tube was centrifuged at 300 g for 5 min, and the precipitated DCs were collected for subsequent experiments. Previous experiments showed that during centrifugation at 300 g, pyroptotic vesicles were in the supernatant and separated from the centrifuged DCs.

[0062] 3.2 Validation of Hepa1-6 cell pyroptosis vesicle dendritic cells This embodiment utilizes the lipophilic fluorescent dye DiO to prepare green fluorescently labeled pyroptosis vesicles, visually demonstrating the phagocytosis of vesicles by dendritic cells (DCs) to verify the successful construction of dendritic cells loaded with Hepa1-6 cell pyroptosis vesicles. The specific steps are as follows: DiO is dissolved in DMSO, added to the pyroptotic vesicle solution (preparation method is the same as that described in Example 2) at a concentration of 0.1%, and labeled at 37°C for 1 h. The labeled pyroptotic vesicles are added to a DC culture flask for 3 h of culture, followed by centrifugation at 1,000 r / min, the supernatant is discarded to remove the DiO dye not phagocytosed by DC cells. The mixture is then washed once with PBS buffer, added with 4% paraformaldehyde by mass fraction for fixation for 20 min, centrifuged at 1,000 r / min, rinsed twice with PBS buffer, added with DAPI for staining for 10 min at room temperature, centrifuged at 1,000 r / min, washed twice with PBS, and subjected to fluorescence imaging using a fluorescence microscope. At this point, only the pyroptotic vesicles internalized by DC cells are retained.

[0063] The results are shown in Figure 5 , after co-culture of pyroptotic vesicles and DC cells for 3 h, some DC cells contain vesicles labeled with fluorescent dye, indicating that dendritic cells loaded with Hepa1-6 cell pyroptotic vesicles are successfully prepared.

[0064] Example 4 Therapeutic effect of dendritic cells loaded with tumor cell pyroptotic vesicles on orthotopic liver tumors 4.1 Construction and treatment of mouse orthotopic liver tumor-bearing model In this example, 50 SPF-grade 6-week-old male C57BL / 6J mice were purchased from Guangdong Provincial Medical Laboratory Animal Center, with the animal qualification certificate number: SYXK (Yue) 2022-00022026569214.

[0065] Aseptic ophthalmic scissors are used to make an approximately 1 cm incision along the abdominal midline on the upper abdomen of a C57BL / 6J mouse. A sterile cotton swab is used to squeeze out the left liver lobe of the mouse, and then the cotton swab is used to gently pull out the left liver lobe. A micro-syringe is used to draw 20 μL of mouse hepatoma cell Hepa1-6 suspension and 5 μL of Matrigel for injection into the left liver lobe (the number of inoculated cells is 3×10 6 cells / mouse), and a slight white bulge is visible at the injection site after injection. After confirming no liquid leakage after injection, the left liver lobe of the mouse is placed back in situ, and sutured layer by layer. The inner layer is sutured with 7-0 absorbable surgical suture, and the outer layer is sutured with 5-0 absorbable surgical suture. After suturing, the wound is disinfected with iodophor, and the mouse is placed back into the cage box to await its awakening.

[0066] In this example, the above tumor-bearing mice are randomly divided into 5 groups (Sham group, Model group, DC group, LBP group and (DC+LBP) group) for subsequent experiments. The treatment methods for each group are as follows: Sham operation group (Sham group): 10 days after simulated modeling, 0.9% normal saline is intraperitoneally injected; 12 days later, PBS buffer is intraperitoneally injected (once every 10 days, 2 times in total); 10 days after completing 2 injections of PBS buffer, 0.9% normal saline is intraperitoneally injected.

[0067] Model group: 10 days after tumor bearing, mice were injected intraperitoneally with 0.9% saline. 12 days later, mice were injected intraperitoneally with PBS (10 times per day, 2 times in total). 10 days after the completion of the 2 PBS buffer injections, mice were injected intraperitoneally with 0.9% saline.

[0068] Dendritic cell therapy group loaded with tumor cell pyroptosis vesicles (DC group): 10 days after tumor bearing, mice were intraperitoneally injected with DC vaccine (10 days / time, 2 times in total). The preparation method of the DC vaccine was the same as described in Examples 1 to 3.

[0069] Lobaplatin treatment group (LBP group): Mice were intraperitoneally injected with LBP (10 mg / kg, once) 12 days after tumor bearing.

[0070] Dendritic cells loaded with tumor cell pyroptosis vesicles and treated with lobaplatin (DC+LBP group): 10 days after tumor bearing, mice were intraperitoneally injected with DC vaccine (once every week, for a total of 2 times). The preparation method of the DC vaccine was the same as described in Examples 1 to 3. 12 days after the completion of the 2 DC vaccine injections, LBP (10 mg / kg, once in total) was injected intraperitoneally.

[0071] 4.2 Observation of in situ hepatic tumors After the experiment, samples were collected. Mice were anesthetized with isoflurane and then euthanized by dislocation of the neck. Blood was collected, centrifuged, and serum was stored at -80°C. The weight of the experimental mice, as well as the weight of the spleen, thymus, and liver, were weighed and recorded. The liver was photographed.

[0072] 4.3 HE staining of mouse liver Fixation: Mouse liver tissue was fixed in 4% formaldehyde solution for 24 hours to maintain the integrity of the tissue structure.

[0073] Gradient dehydration: The fixed liver tissue was placed in ethanol at different concentrations (70% ethanol for 15 min, 80% ethanol for 20 min, 90% ethanol for 20 min, 95% ethanol for 20 min, and 100% ethanol twice for 20 min each time) to remove water from the liver tissue. Transparency: The dehydrated liver tissue was immersed in xylene twice (10 minutes each time) to make it transparent, which facilitated the next step of paraffin impregnation.

[0074] Paraffin infiltration: The transparent tissue was removed and embedded in paraffin at 60°C, allowing it to solidify on a low-temperature instrument platform.

[0075] Sectioning: The embedded liver tissue was cut into 4μm thick sections.

[0076] Spreading the slide: Using ophthalmic forceps, quickly transfer the selected slide to 42°C warm water for spreading. Attaching the slide: Using a glass slide, approach the slide along the water surface and slowly move it to attach the tissue slide to the glass slide.

[0077] Baking: Baking the slides in a 70 ℃ oven for 1 hour to ensure that the slides adhere firmly to the glass slide.

[0078] Dewaxing: The slides with attached sections were placed in xylene (5 min, twice), anhydrous ethanol (5 min, twice), 90% ethanol (5 min), and 75% ethanol (5 min) in sequence to remove paraffin from the tissue sections.

[0079] Staining: ① Hematoxylin staining: Stain with hematoxylin solution for 3 minutes, rinse with tap water to stain the cell nuclei blue. ② Differentiation: Place the tissue sections in hematoxylin differentiation solution for 3 seconds, then rinse with running water until the blue color reappears to remove excess hematoxylin dye. ③ Eosin staining: Stain the tissue sections in eosin solution for 5 minutes to stain the cytoplasm red, then dehydrate with anhydrous ethanol (5 minutes, twice) to remove water from the sections.

[0080] Mounting: Place the slide in xylene twice to clear it.

[0081] 4.4 Immunohistochemical staining of mouse liver 1. Dewaxing of paraffin sections to water and antigen retrieval.

[0082] 2. Permeability: Permeability is required for intracellular antigens. Incubate with 0.5% Triton X-100 in PBS at room temperature for 15 min, then rinse with PBS 3 times for 5 min each time.

[0083] 3. Blocking: Add 10% goat serum and 0.1% Tween-20 PBS. Place the sample in a humidified chamber, adding a small amount of water, and incubate overnight at 4°C.

[0084] 4. Primary antibody incubation: Add 50 μL of diluted primary antibody to the sample slide and incubate overnight at 4°C in a humidified chamber.

[0085] 5. Secondary antibody incubation: Wash the sample slides with PBS three times, 5 min each time. Add 50 μL of diluted secondary antibody labeled with fluorescein and incubate in a humidified chamber at room temperature in the dark for 1 h.

[0086] 6. Nuclear staining: Wash the sample slides with PBS three times, 5 min each time. Add 50 μL of DAPI (diluted with PBS to 1 μg / mL) and incubate in a humidified chamber at room temperature in the dark for 8 min.

[0087] 7. Mounting: Wash the slides twice with PBS, 5 min each time. Add water-soluble mounting medium and cover with a coverslip.

[0088] 8. Detection: Observation under a fluorescence microscope. Different dyes are selected for excitation light at different wavelengths.

[0089] 4.5 Detection of IFN-γ and TNF-α in mouse serum Serum IFN-γ and TNF-α in mice of each group were detected by ELISA.

[0090] 4.6 Western blot detection of pyroptosis markers in mouse liver tissue The detection steps for GSDME-FL, GSDME-NT, GSDMD-FL, and GSDMD-NT proteins are the same as described in Example 2. The antibodies used for detecting Cleaved-caspase-1 and Cleaved-caspase-3 proteins are (RabbitAnti-GSDMDAntibody, Abcam, ab219800; RabbitAnti-GasderminEAAntibody, CST, 40618S; CleavedCaspase-3Antibody, CST, 9661; CleavedCaspase-1Antibody, CST, 89332T; Caspase1 / P20 Polyclonal antibody, Proteintech, 22915-1-AP). The remaining steps are the same as described in Example 2. Furthermore, this example also includes a quantitative analysis of the detection results.

[0091] 4.7 Detection of Perforin 1 in Mouse Liver Tissue The left lobe of the liver was collected from the Sham group, and tumor tissue from the left lobe of the liver was collected from the other groups. The expression level of Perforin1 in the liver tissue homogenate of each group of mice was detected by ELISA.

[0092] 4.8 Single-cell sequencing of mouse liver tissue After dissociating mouse liver tissue samples, single-cell suspensions were obtained. These suspensions were then subjected to quality control and counting. Cells that passed the tests were washed, resuspended, and their concentration was adjusted to 700-1,200 cells / μL.

[0093] Using BGI C4’s droplet generation system (Disc-seq), single-cell suspensions are mixed with mRNA-capturing magnetic beads with unique barcodes (used to label the mRNA of individual cells) and indexcarry magnetic beads. Oil droplets are generated by a negative pressure device, with each droplet encapsulating one cell and multiple magnetic beads.

[0094] The raw data was filtered, compared with the genome, quantified transcripts, and identified by the BGI official analysis software DNBelab_C_Series_HT_scRNA-analysis-software (https: / / pypi.org / project / DNBC4tools / ), and finally the gene expression matrix of each cell was obtained.

[0095] 4.9 Detection of CD68 and iNOS antigens in mouse liver tissue Immunofluorescence staining was used to detect CD68 and iNOS antigens.

[0096] 4.10 Experimental Results Observation results of liver in situ tumors as follows Figure 6 As shown, no tumor formation was observed in the livers of mice in the Sham group; multiple tumor foci were observed in the livers of mice in the Model group, indicating successful modeling. Compared with the Model group, liver tumor growth was significantly inhibited in mice treated with DC, LBP, and a combination of DC and LBP. From the perspective of liver appearance, the tumor-inhibiting effects of the DC and LBP groups were similar, while the DC+LBP group showed the best effect. It should be understood that because in situ tumors grow invasively within the liver parenchyma, gross observation alone is insufficient to fully assess the anti-tumor effect; further comprehensive analysis using HE staining and other indicators is necessary.

[0097] HE staining results are as follows Figure 7 As shown, compared with the Model group, the tumor area in the liver tissue of mice in the DC group, LBP group, and DC+LBP group was reduced. Furthermore, in the Model group, tumor cells were densely packed, with deeply stained nuclei, an increased nucleocytoplasmic ratio, significant cellular atypia, and abundant neovascularization; conversely, in the tumor tissues of the DC group, LBP group, and DC+LBP group, tumor cell atypia was reduced, cell packing was loose, nuclear pyknosis was observed, and the number of neovascularizations was decreased. Further, significant lymphocyte infiltration was observed in the tumor tissues of the DC group and DC+LBP group, indicating an enhanced tumor immune response. These results suggest that, compared with the Model group and LBP group, DC loaded with tumor cell pyroptosis vesicles, and the combined use of DC and LBP, are more effective in inhibiting the growth of mouse liver tumors, especially when DC and LBP are used in combination.

[0098] Tumor growth is usually accompanied by angiogenesis and abnormally active cell proliferation. CD34 protein is a commonly used marker of microvascular density, and its positive staining area can reflect the degree of tumor angiogenesis; Ki67 protein is a cell proliferation-related nuclear antigen, and its positive rate can assess the proliferative activity of tumor cells.

[0099] Immunohistochemical staining results as follows Figures 8-9As shown, compared with the Sham group, the Model group had an increased number of CD34-positive staining areas in its liver tissue. Figure 8 The number of Ki67-positive cell nuclei also increased. Figure 9 The results indicate that tumor angiogenesis and cell proliferation are active in the tumor-bearing state, consistent with theoretical findings. Compared with the Model group and LBP group, the positive staining of CD34 and Ki67 in liver tumor tissues of the DC group and DC+LBP group was reduced, meaning that angiogenesis and cell proliferation in the livers of mice in the DC group and DC+LBP group were inhibited, with the DC+LBP group being more preferred. These results suggest that DCs loaded with tumor cell pyroptosis vesicles can effectively inhibit the progression of hepatocellular carcinoma by suppressing tumor angiogenesis and liver cancer cell proliferation.

[0100] IFN-γ (interferon-γ) and TNF-α (tumor necrosis factor-α) are two key pro-inflammatory cytokines, mainly secreted by immune cells (such as T cells, natural killer cells, and macrophages). They play a central role in regulating immune responses, resisting pathogens, and tumor surveillance. Therefore, this embodiment detected IFN-γ and TNF-α cytokines in the serum of tumor-bearing mice.

[0101] Detection of IFN-γ and TNF-α in mouse serum as follows Figure 10 As shown, from Figure 10 As shown in Figure A, compared with the Sham group, the serum IFN-γ level in the Model group mice did not change significantly. Compared with the Model group and the LBP group, the serum IFN-γ content in the DC group mice was significantly increased, suggesting that DC treatment can enhance the Th1 immune response and the killing function of cytotoxic lymphocytes. Figure 10 B indicates that, compared with the Sham group, the serum TNF-α level in the Model group mice was significantly increased. Compared with the Model and LBP groups, the serum TNF-α levels in the DC group and the DC+LBP group mice were significantly increased, with the increase in the DC+LBP group being significantly smaller than that in the DC group. This result suggests that when DC loaded with tumor cell pyroptosis vesicles is used in combination with LBP, LBP may partially weaken the DC-induced TNF-α upregulation effect. This reflects that LBP, as a chemotherapeutic drug, inhibits immune cells in addition to tumor cells, lacks target specificity, and therefore has certain side effects. The above results suggest that DC loaded with tumor cell pyroptosis vesicles can effectively inhibit the progression of hepatocellular carcinoma by upregulating the levels of pro-inflammatory cytokines (IFN-γ and TNF-α) and activating the body's immune response.

[0102] Western blot results of pyroptosis markers in mouse liver tissue are as follows: Figure 11 As shown, from Figure 11 A~ Figure 11As shown in Figure D, compared with the Sham group, the expression levels of Cleaved-caspase-3 and GSDME-FL proteins in the liver tissue of the Model group did not change significantly, while the expression level of GSDME-NT increased, suggesting that there may be a certain degree of GSDME-related pyroptosis activation under tumor-bearing conditions. Compared with the Model group, the expression levels of Cleaved-caspase-3, GSDME-FL, and GSDME-NT proteins were all increased in the DC group, LBP group, and DC+LBP group, especially in the DC+LBP group. Among them, the expression level of GSDME-NT, a key protein in pyroptosis execution, was highest in the DC+LBP group, followed by the LBP group, and relatively lowest in the DC group. In summary, both DCs and LBPs loaded with tumor cell pyroptosis vesicles can activate the GSDME-mediated pyroptosis pathway. On this basis, the activation level was further enhanced after the combination of DC and LBP.

[0103] The pyroptosis pathway is divided into the GSDMD-mediated pyroptosis pathway and the GSDME-mediated pyroptosis pathway. The former is activated by inflammation-related Cleaved-caspase-1 / 4 / 5 / 11, mediating infectious / sterile inflammatory pyroptosis, while the latter is activated by apoptosis-effect Cleaved-caspase-3, mediating pyroptosis under chemotherapy or apoptotic stimulation. To further explore the mechanism of action of dendritic cells loaded with tumor cell pyroptosis vesicles, this example detected the expression levels of Cleaved-caspase-1, GSDME-FL, GSDME-NT, GSDMD-FL, GSDMD-NT, and Cleaved-caspase-3 proteins in mouse liver tissue after different treatments.

[0104] from Figure 11 A, Figure 11 E~ Figure 11As shown in G, compared with the Sham group, the expression levels of Cleaved-caspase-1, GSDMD-FL, and GSDMD-NT proteins in the liver tissue of Model group mice did not change significantly. Compared with the Model group, the expression levels of Cleaved-caspase-1, GSDMD-FL, and GSDMD-NT proteins were significantly increased in the DC group and DC+LBP group, while the expression levels of these proteins in the LBP group did not change significantly. More specifically, the expression level of the key pyroptosis protein GSDMD-NT was highest in the DC+LBP group, followed by the DC group, while the LBP group was similar to the Model group. This means that compared with LBP treatment alone, DCs loaded with tumor cell pyroptosis vesicles alone, and DCs loaded with tumor cell pyroptosis vesicles combined with LBP treatment, are more effective in stimulating the immune system's killing effect on tumors. LBP monotherapy did not significantly induce macrophage infiltration or M1 polarization in tumor tissue. LBP treatment inhibited tumor cell growth and also inhibited immune cells and their secreted anti-tumor factors.

[0105] comprehensive Figure 11 The test results showed that DCs loaded with tumor cell pyroptosis vesicles could induce GSDME and GSDMD-mediated pyroptosis in liver tumor tissues of hepatocellular carcinoma mice, and the pyroptosis induction effect could be further enhanced when the DCs were used in combination with LBP.

[0106] Perforin 1 is a key effector molecule released by cytotoxic lymphocytes. Its main function is to form pores on the target cell membrane, mediating the entry of granzymes such as GzmB into the target cell cytoplasm. GzmB then cleaves and activates GSDME, inducing pyroptosis. Furthermore, IFN-γ and TNF-α can both upregulate GSDME expression, promoting GzmB-induced pyroptosis. The above serological results show that treatment with DCs loaded with tumor cell pyroptosis vesicles significantly increased the levels of IFN-γ and TNF-α in hepatocellular carcinoma mice. Figure 10 Western blot (WB) experiments further confirmed that significant pyroptosis occurred in the liver tumor tissue of mice after this treatment. Figure 11 To further elucidate the molecular pathways and mechanisms by which DCs loaded with pyroptosis vesicles activate pyroptosis in liver cancer cells, this embodiment used ELISA to detect the expression level of Perforin1 in liver tissue homogenates from each group of mice.

[0107] ELISA test results as follows Figure 12As shown, compared with the Sham group, the expression level of Perforin1 in the liver tissue of the Model group did not change significantly, suggesting that the baseline level of Perforin1 in tumor tissue under tumor-bearing conditions is low. Compared with the Model group, the expression levels of Perforin1 in the liver tissue of the DC group, LBP group, and DC+LBP group were significantly increased. Among them, the expression levels of the DC group and DC+LBP group were similar and higher than those of the LBP group, indicating that DC loaded with tumor cell pyroptosis vesicles, LBP, and the combination of both can upregulate the expression of Perforin1 in the tumor tissue of hepatocellular carcinoma mice, and may promote GSDME-mediated pyroptosis through the Perforin1-GzmB pathway. It is worth noting that the combined application of DC loaded with tumor cell pyroptosis vesicles and LBP has a similar effect on increasing Perforin1 as DC alone, while the upregulation effect of LBP alone is relatively weak, indicating that LBP failed to effectively activate immune cells that release Perforin1.

[0108] To further elucidate the mechanism of action of dendritic cells (DCs) loaded with tumor cell pyroptosis vesicles at the cellular level, this embodiment also performed single-cell sequencing on liver tissue from mice bearing hepatic orthotopic tumors after treatment with the aforementioned DC cells, using liver tissue from untreated tumor-bearing mice as a control. The single-cell sequencing results are as follows: Figure 13 As shown, monocyte-derived macrophages (MoMF) and CD8 cells... + The abundance of T cells and NK cells increased significantly after DC treatment, indicating that DCs loaded with tumor cell pyroptosis vesicles can enhance the body's anti-tumor immune response, which is beneficial for the immune system to clear and suppress tumor cells.

[0109] Given that macrophages are the most abundant and functionally complex group of immune cells in the liver, their polarization state (M1 / M2 type) directly affects tumor progression and the direction of the immune response. Specifically, M1 macrophages are induced by cytokines such as IFN-γ and LPS, and their main functions are to activate and maintain inflammatory responses, kill pathogens (especially intracellular bacteria), and inhibit tumors; they can secrete a large number of pro-inflammatory factors (such as TNF-α, IL-1β, IL-6, and IL-12) and reactive oxygen species / nitrogen mediators, enhancing antigen presentation capacity. M2 macrophages, on the other hand, are induced by cytokines such as IL-4 and IL-13, and their main functions are to inhibit inflammation, promote tissue repair and angiogenesis, and regulate immune tolerance; they can secrete anti-inflammatory factors (such as IL-10 and TGF-β) and arginase-1, participating in wound healing and fibrosis, but can also promote tumor growth and allow chronic infection. Based on the fact that M1 macrophages are helpful in fighting tumors, and that CD68 and iNOS antigens are commonly used markers for M1 macrophages, this embodiment uses immunofluorescence staining to detect the expression of CD68 and iNOS antigens in liver tissues of each group.

[0110] Immunofluorescence staining results as follows Figure 14 As shown, compared with the Sham group, the Model group had a reduced number of CD68-positive cells (macrophages) in liver tissue, while the positive signal of the M1 macrophage marker iNOS did not change significantly, and no significant expression of iNOS was observed in the liver tissue of either group. After treatment with DCs loaded with tumor cell pyroptosis vesicles or with the aforementioned DCs and LBP, CD68... + iNOS + The increased number of double-positive cells, along with their co-localized distribution, indicates that the positive cells observed under the microscope were predominantly M1-type macrophages. Conversely, after LBP intervention, no significant expression of iNOS positive signals was observed, suggesting that the observed macrophages were not in an M1-type polarized state. These results indicate that DCs loaded with pyroptosis vesicles can induce macrophage accumulation and M1-type polarization in liver tumor tissue, thereby promoting anti-tumor immune responses. Moreover, the combined treatment of DCs and LBP further promotes macrophage accumulation and M1-type polarization; conversely, LBP treatment has no significant inducing effect on macrophage accumulation and M1-type polarization.

[0111] Based on the above experimental results, this embodiment demonstrates that: treatment with dendritic cells loaded with tumor cell pyroptosis vesicles or treatment with dendritic cells combined with LBP effectively inhibits the proliferation of hepatocellular carcinoma. Figure 6Among them, the treatment effects of both the DC+LBP group and the DC group were superior to those of the LBP group, and the DC+LBP group showed the best inhibitory effect on hepatocellular carcinoma, indicating that the combination of dendritic cells loaded with tumor cell pyroptosis vesicles and LBP has a synergistic effect. Furthermore, this embodiment also explored the mechanism of action of dendritic cells loaded with tumor cell pyroptosis vesicles, specifically as follows: Dendritic cells loaded with tumor cell pyroptosis vesicles were drained to the mesenteric lymph nodes via intraperitoneal injection, thereby presenting tumor-associated antigens carried by the pyroptosis vesicles to T cells, promoting CD8... + T cell proliferation and activation enhance the release of effector molecules such as perforin, granzyme B, IFN-γ, and TNF-α. Among these, granzyme B cleaves GSDME after entering tumor cells, inducing pyroptosis; IFN-γ and TNF-α synergistically upregulate the expression of GSDME and GSDMD, inducing macrophage aggregation and M1 polarization. M1 macrophages release large amounts of ROS, which in turn activate the NLRP3 inflammasome, assembling and activating Caspase-1 to directly cleave GSDMD, triggering pyroptosis.

[0112] Example 5: The therapeutic effect of dendritic cells loaded with tumor cell pyroptosis vesicles on colorectal cancer AOM (azomethane), as a DNA methylating agent, induces gene mutations in colonic epithelial cells (especially at the base of crypts), such as beta-catenin and p53 gene mutations, thereby initiating the carcinogenesis process. DSS (sodium dextran sulfate), a sulfated polysaccharide, induces acute colitis by disrupting the intestinal epithelial barrier; it can also continuously stimulate AOM-induced mutant cells, accelerating their proliferation, deterioration, and ultimately tumor formation. In summary, the repeated inflammation-repair cycle mediated by AOM-DSS promotes the proliferation of mutant cells and tumor formation.

[0113] Based on the above, this embodiment uses AOM and DSS to construct a mouse colorectal cancer (CRC) model. One mouse from each group was used to obtain tumor cells from orthotopic colorectal cancer tissue via enzymatic digestion. These cells were then further induced to form dendritic cells (DCs) loaded with pyroptosis vesicles for treatment of the model. The preparation process of the DCs was the same as described in Examples 1-3, and the specific experimental procedures are as follows: Phase 1 – Tumor Initiation (Week 1): SPF-grade 6-week-old male C57BL / 6J mice were injected intraperitoneally with AOM at a dose of 10 mg / kg.

[0114] Phase 2 – Inflammation Promotion (Weeks 2-4): Starting on day 7 after AOM injection, 2%–3% DSS was dissolved in sterile drinking water and allowed mice to drink freely for 7 days until the mice showed symptoms of acute colitis such as weight loss, bloody stools, and diarrhea.

[0115] Phase 3 – Maintenance and Tumor Formation (Weeks 5-11): Mice were given 2% DSS solution for 7 days in the drinking water; then switched back to regular water. At weeks 8 and 10, mice were intraperitoneally injected with 8 million DC cells loaded with pyroptosis vesicles of in situ colorectal cancer cells. The mice were fed until week 11, and then colorectal endoscopy was performed to observe the occurrence of colorectal tumors.

[0116] In this embodiment, a healthy mouse group (NC group) was used as a negative control, and a mouse group with colorectal cancer that did not receive any treatment (CM group) was used as a positive control. Six biological replicates were set up for each experimental group.

[0117] The results are as follows Figure 15 As shown, compared to the NC group, the CM group mice had multiple tumor foci in the colorectal region. After treatment with DC cells loaded with LLC tumor cell pyroptosis vesicles, the tumor growth of mice with colorectal cancer was significantly inhibited, indicating that the DC cells can be used to treat colorectal cancer.

[0118] Example 6: The preventive effect of dendritic cells loaded with tumor cell pyroptosis vesicles on lung cancer. This embodiment investigates the antitumor effect of dendritic cells loaded with LLC cell pyroptosis vesicles. The specific steps are as follows: Dendritic cells loaded with LLC cell pyroptosis vesicles were prepared according to the steps described in Examples 1-3. The difference from Examples 1-3 is that LLC cells were used instead of Hepa1-6 cells in this embodiment. SPF-grade 6-week-old male C57BL / 6J mice were subcutaneously injected with 6 million DC cells per mouse at the axillary site. Seven days later, SPF-grade 6-week-old male C57BL / 6J mice were subcutaneously injected with 1 million LLC cells per mouse at the axillary site. Two weeks later, tumor growth in the mice was observed.

[0119] The results are as follows Figure 16 As shown, mice injected with dendritic cells loaded with LLC cell pyroptosis vesicles for prophylaxis did not develop subcutaneous tumors, while all control mice developed subcutaneous tumors. This indicates that the DC injection can present LLC-related tumor antigens to mouse CD8 cells. + The cells can act as an early warning system for subsequently injected LLC cells, thereby achieving the effect of preventing lung cancer.

[0120] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A dendritic cell, characterized in that, The dendritic cells are loaded with tumor cell pyroptosis vesicles.

2. A pharmaceutical composition, characterized in that, The composition comprises the dendritic cells of claim 1 and an agent capable of inducing pyroptosis in tumor cells.

3. The pharmaceutical composition according to claim 2, characterized in that, The reagent is lobaplatin.

4. Use of the dendritic cells of claim 1 or the pharmaceutical composition of any one of claims 2 to 3 in the preparation of agents for treating tumors and / or cancer.

5. Use of the dendritic cells of claim 1 or the pharmaceutical composition of any one of claims 2 to 3 in the preparation of an antitumor and / or cancer agent.

6. Use of the dendritic cells of claim 1 or the pharmaceutical composition of any one of claims 2-3 in any of the following aspects: preparation of an agent that inhibits tumor growth and / or proliferation; preparation of an agent that promotes the aggregation of CD8+ T and NK cells in tumors; preparation of an agent that inhibits angiogenesis within tumors; preparation of an agent that increases the content of pro-inflammatory cytokines; preparation of an agent that induces pyroptosis in tumor cells; preparation of an agent that promotes perforin expression; preparation of an agent that increases the number of immune cells; preparation of an agent that promotes the accumulation of macrophages in tumors; preparation of an agent that induces M1 polarization of macrophages.

7. The method for preparing dendritic cells according to claim 1, characterized in that, The method includes the following steps: S1. Induces pyroptosis in tumor cells and extracts pyroptosis vesicles from tumor cells; S2. Co-culture the pyroptosis vesicles extracted in step S1 with dendritic cells to obtain dendritic cells loaded with tumor cell pyroptosis vesicles.

8. The preparation method according to claim 7, characterized in that, Step S1 involves extracting pyroptosis vesicles using differential centrifugation and polyethylene glycol precipitation, or using ultra-high speed centrifugation.

9. The preparation method according to claim 7, characterized in that, After co-culturing the pyroptosis vesicles with dendritic cells as described in step S2, differential centrifugation was performed to remove the pyroptosis vesicles that were not phagocytosed by the dendritic cells. The precipitate was then collected to obtain dendritic cells loaded with tumor cell pyroptosis vesicles.

10. The dendritic cells of claim 1, or the pharmaceutical composition of any one of claims 2-3, or the preparation method of any one of claims 7-9, characterized in that, The tumor cells are any one or more of liver cancer cells, colorectal cancer cells, and lung cancer cells.

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