Tumor postoperative vaccine as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a tumor postoperative vaccine as well as a preparation method and application thereof. The method comprises the following steps: resuspending tumor cells in a first solution containing metal ions for incubation, collecting the tumor cells, resuspending the tumor cells in a second solution containing mineralized ions for incubation, enabling the metal ions to react with the mineralized ions to obtain metal inorganic salt, forming a mineralized shell layer on the surfaces of the tumor cells, and resuspending the tumor cells to obtain the tumor postoperative vaccine. And due to the mineralized shell layer, the vaccine has the capability of activating a signal channel in an antigen presenting cell through mechanical stimulation, so that an immune system is favorably activated, and the anti-tumor capability of the vaccine is improved. In addition, tumor cells are inactivated in the vaccine preparation process and keep a complete form, and presentation of a complete tumor antigen is facilitated, so that a relatively good anti-tumor effect is obtained.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a tumor post-operative vaccine and a preparation method and application thereof. Background Art

[0002] The current clinical tumor treatment system still uses surgery as the dominant intervention method. However, it is difficult to completely eradicate tumor cells by relying solely on lesion resection and adjuvant therapy. Surgical immunosuppression caused by surgical trauma can lead to the attenuation of immune surveillance function, which is mainly manifested by the expansion of myeloid-derived suppressor cells (MDSCs), the infiltration of regulatory T cells (Tregs), and the imbalance of tumor-associated macrophages (TAMs). The above pathological microenvironment significantly weakens the host's ability to eliminate residual cancer cells, resulting in a local recurrence rate of up to 30%-50% within five years after tumor surgery.

[0003] Dendritic cells (DCs), as powerful professional antigen-presenting cells in the immune system, play a central regulatory role in anti-tumor immune responses. They efficiently present tumor-specific antigens through MHC class I and class II complexes, activating both CD8+ cytotoxic T cells and CD4+ helper T cells, thereby bridging the innate and adaptive immune systems and forming a core hub of anti-tumor immunity. However, in postoperative pathological conditions, the tumor microenvironment can impair DCs' antigen presentation capacity, deplete co-stimulatory signals, and disrupt immune regulatory function through multiple mechanisms, including metabolic reprogramming, aberrant expression of immune checkpoint molecules, and cytokine network imbalance. This functionally inactivated state of DCs not only induces the formation of an immune-tolerant microenvironment but is also closely associated with tumor recurrence and metastatic progression. Current anti-tumor strategies based on enhanced DC function primarily include immune checkpoint inhibition, adoptive cell therapy, and DC-based vaccines, but these approaches face significant challenges in the postoperative setting. For example, immune checkpoint inhibitors, such as PD-1 / PD-L1 inhibitors, rely on sustained exposure to tumor antigens to maintain T cell activation. However, the level of tumor antigen presentation in residual lesions after surgery plummets, hindering the formation of immune synapses and making sustained T cell activation difficult. While adoptive cell transfer can directly replenish immune cells, cell survival rates are currently less than 30% within 72 hours after infusion. Furthermore, metabolic stress in the tumor microenvironment, such as lactate accumulation, and immunosuppressive factors such as TGF-β and IL-10, accelerate DC apoptosis and induce immune editing escape. DC vaccines face the dilemma of impaired antigen presentation in the postoperative environment, with significant downregulation of surface co-stimulatory molecules CD80 / CD86 and MHC class II expression, leading to interrupted T cell activation signaling. These mechanisms collectively result in the inability of existing strategies to effectively maintain DC functional homeostasis, severely hindering the establishment of postoperative anti-tumor immune responses. Summary of the Invention

[0004] The purpose of the present invention is to provide a postoperative tumor vaccine that can effectively activate DCs function and enhance anti-tumor immune response, as well as a preparation method and application thereof.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A post-operative tumor vaccine comprises inactivated tumor cells and a mineralized shell wrapped around the surface of the tumor cells, wherein the main component of the mineralized shell is a metal inorganic salt.

[0007] Optionally, the mineralized shell is doped with a STING agonist.

[0008] Optionally, the metal inorganic salt is generated by reacting the STING agonist with a metal ion, the STING agonist is one or more of 2',3'-cGAMP sodium salt, 3',3'-cGAMP sodium salt and cGAMP disodium salt, and the metal ion is Mg 2+ , Ca 2+ 、Fe 2+ 、Fe 3+ 、Al 3+ and Mn 2+ One or more of .

[0009] Optionally, the metal inorganic salt is calcium phosphate.

[0010] In a second aspect, the present invention further provides a method for preparing the above-mentioned postoperative tumor vaccine, comprising:

[0011] Collect tumor cells;

[0012] resuspending the tumor cells in a first solution containing metal ions, and incubating for a first time to allow the metal ions to adsorb on the surface of the tumor cells, and collecting the adsorbed cells;

[0013] resuspending the adsorbed cells in a second solution containing mineralizing ions and incubating for a second time, so that the metal ions react with the mineralizing ions to obtain the metal inorganic salt and form the mineralized shell;

[0014] The cells are collected and resuspended to obtain the post-tumor vaccine.

[0015] Optionally, the first solution is a calcium chloride solution, the second solution is a 2',3'-cGAMP sodium salt solution, and the mineralizing ions are phosphate ions.

[0016] Optionally, the concentration of the first solution is any value between 80 mmol / L and 120 mmol / L, and the cell density of the tumor cells in the first solution is 1×10 6 / mL~9×10 6 Any value in the range of mmol / mL.

[0017] Optionally, the first time is any value between 20 minutes and 40 minutes, and the second time is any value between 40 minutes and 90 minutes.

[0018] In a third aspect, the present invention also provides an application of the above-mentioned tumor postoperative vaccine, wherein the tumor postoperative vaccine is administered by injection, and the administration frequency is once every three days, and the dosage is 1.0×10 5 / g~1.5×10 5 Any value in g.

[0019] Optionally, the post-tumor vaccine administration is combined with radiation therapy.

[0020] The beneficial effects of the present invention lie in the fact that a mineralized shell coats the vaccine surface. When the vaccine is taken up by antigen-presenting cells, such as dendritic cells and macrophages, the mineralized shell provides mechanical stimulation, activating intracellular signaling pathways and helping to enhance the immune response. Once inside the antigen-presenting cells, the vaccine is transported to lysosomes, where the mineralized shell degrades in the acidic environment of the lysosomes, releasing tumor-associated antigens carried by the tumor cells. This synergistic effect of activated signaling pathways results in anti-tumor effects.

[0021] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is an electron microscopic image of the vaccine after tumor surgery as shown in Example 1 of the present invention;

[0023] Figure 2 This is a flow chart of the method for preparing a postoperative tumor vaccine according to Example 1 of the present invention;

[0024] Figure 3 Graphs showing elemental analysis of the energy dispersive X-ray spectrum of the vaccine for tumor surgery shown in Example 1 of the present invention;

[0025] Figure 4 This is the infrared spectrum absorption diagram of the vaccine and tumor cells after tumor surgery shown in Example 1 of the present invention;

[0026] Figure 5 This is an X-ray diffraction diagram of the vaccine and tumor cells after tumor surgery as shown in Example 1 of the present invention;

[0027] Figure 6 This is a comparison chart of the calcium ion, DNA, RNA and protein contents of the vaccine and tumor cells after tumor surgery shown in Example 1 of the present invention;

[0028] Figure 7 This is a polyacrylamide gel electrophoresis diagram of the vaccine and tumor cells after tumor surgery as shown in Example 1 of the present invention;

[0029] Figure 8 This is a diagram showing the morphological integrity of the vaccine shown in Example 1 of the present invention;

[0030] Figure 9 This is a fluorescent staining image of the vaccine after tumor surgery endocytosis by antigen-presenting cells as shown in Example 1 of the present invention;

[0031] Figure 10This is a comparison chart of calcium ion release of the vaccine after tumor surgery under different environments as shown in Example 1 of the present invention;

[0032] Figure 11 This is a graph analyzing the calcium ion content of antigen-presenting cells after different periods of time of stimulation by the vaccine after tumor surgery as shown in Example 1 of the present invention;

[0033] Figure 12 Flow cytometric histograms of antigen-presenting cells stimulated by the vaccine at different times after tumor surgery as shown in Example 1 of the present invention;

[0034] Figure 13 This is a diagram showing the expression of various signal transduction molecules in antigen-presenting cells after tumor surgery and at different times after stimulation by the vaccine as shown in Example 1 of the present invention;

[0035] Figure 14 This is a diagram showing the expression of various signal transduction molecules after antigen-presenting cells are stimulated with different vaccines or drugs in Example 1 of the present invention;

[0036] Figure 15 This is a cell staining image of samples in the Ctrl group and the CaP@Cell group in the immune activation experiment shown in Example 1 of the present invention;

[0037] Figure 16 Flow cytometry contour plots of samples from each group in the immune activation experiment shown in Example 1 of the present invention;

[0038] Figure 17 This is a statistical diagram of the mature ratio of dendritic cells in each group of samples in the immune activation experiment shown in Example 1 of the present invention;

[0039] Figure 18 This is a statistical graph of TNF-α secretion levels of samples in each group in the immune activation experiment shown in Example 1 of the present invention;

[0040] Figure 19 This is a statistical graph of IFN-γ secretion levels of samples in each group in the immune activation experiment shown in Example 1 of the present invention;

[0041] Figure 20 The in vitro vaccine live-dead staining image and in vivo imaging image in the biosafety experiment shown in Example 1 of the present invention;

[0042] Figure 21 These are multimodal imaging images at different times after the labeled vaccine in Example 1 of the present invention is injected into mice;

[0043] Figure 22 Statistical graph of tumor tissue volume in each group of mice in the recurrence inhibition experiment shown in Example 1 of the present invention;

[0044] Figure 23This is a line graph showing the volume of tumor tissue in each mouse in each group in the recurrence inhibition experiment shown in Example 1 of the present invention;

[0045] Figure 24 This is a statistical graph of the survival rates of mice in each group in the relapse inhibition experiment shown in Example 1 of the present invention;

[0046] Figure 25 This is a statistical graph of weight changes of mice in each group in the relapse inhibition experiment shown in Example 1 of the present invention;

[0047] Figure 26 This is a statistical diagram of the proportions of various immune cells in tumor tissue in the immune cell infiltration experiment shown in Example 1 of the present invention;

[0048] Figure 27 This is a statistical diagram of the proportions of various immune cells in spleen tissue in the immune cell infiltration experiment shown in Example 1 of the present invention;

[0049] Figure 28 These are the tissue staining images and bone reconstruction images in the bone repair experiment shown in Example 1 of the present invention. DETAILED DESCRIPTION

[0050] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0051] In the description of the present invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings and are intended solely to facilitate and simplify the description of the present invention. They are not intended to indicate or imply that the devices or components referred to must have, be constructed, or operate in a specific orientation, and therefore should not be construed as limitations on the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0052] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood in a broad sense. For example, they may refer to fixed, detachable, or integral connections; mechanical or electrical connections; direct or indirect connections through an intermediate medium; and internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on the specific circumstances.

[0053] In addition, the technical features involved in different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0054] See Figure 1 The postoperative tumor vaccine protected by the present invention includes inactivated tumor cells and a mineralized shell wrapped around the surface of the tumor cells, and the main component of the mineralized shell is a metal inorganic salt.

[0055] The vaccine is coated with a mineralized shell. When the vaccine is taken up by antigen-presenting cells (APCs), such as dendritic cells and macrophages, the mineralized shell provides mechanical stimulation, activating intracellular signaling pathways and helping to enhance the immune response. Once inside the APCs, the vaccine is transported to the lysosomes, where the mineralized shell degrades in the acidic environment of the lysosomes, releasing tumor-associated antigens carried by the tumor cells. This synergistic effect of activated signaling pathways leads to anti-tumor efficacy.

[0056] In some embodiments, the mineralized shell is doped with a STING agonist. After the mineralized shell is released from the lysosomes of antigen-presenting cells, the STING agonist activates the cGAS-STING signaling pathway within the cell, promoting the secretion of type I interferons (such as IFN-β) and enhancing the maturation and antigen presentation capacity of dendritic cells. Activated dendritic cells present tumor antigens to T cells, promoting the proliferation and activation of cytotoxic T cells (CTLs), and exerting anti-tumor effects. At the same time, the vaccine promotes the polarization of macrophages to the pro-inflammatory M1 type, enhancing their phagocytic and anti-tumor functions.

[0057] In some embodiments, the metal inorganic salt is generated by the reaction of a STING agonist with a metal ion, wherein the STING agonist is one or more of 2',3'-cGAMP sodium salt, 3',3'-cGAMP sodium salt, and cGAMP disodium salt, and the metal ion is Mg 2+ , Ca 2+ 、Fe 2+ 、Fe 3+ 、Al 3+ and Mn 2+ One or more of the following. The reaction of a STING agonist with a metal ion to form a metal inorganic salt simplifies the preparation process and facilitates the doping of the STING agonist into the mineralized shell. The sodium salt of a cGAMP derivative provides phosphate ions in solution, facilitating the reaction to form the metal inorganic salt.

[0058] In some embodiments, the inorganic metal salt is calcium phosphate. Phosphate ions are anions with a high content in the body, and calcium ions are metal ions with a high content in the human body and are not prone to toxicity. They are not toxic to the human body after release.

[0059] In a second aspect, the present invention further provides a method for preparing the above-mentioned postoperative tumor vaccine, comprising:

[0060] S1. Collect tumor cells.

[0061] S2. Resuspending the tumor cells in a first solution containing metal ions and incubating for a first time to allow the metal ions to be adsorbed on the surface of the tumor cells, and collecting the adsorbed cells.

[0062] S3. Resuspending the adsorbed cells in a second solution containing mineralizing ions and incubating for a second time to allow the metal ions to react with the mineralizing ions to obtain metal inorganic salts and form a mineralized shell.

[0063] S4. Collect the cells and resuspend them to obtain the post-tumor vaccine.

[0064] By incubating tumor cells in a first solution, the metal ions in the first solution are adsorbed on the surface of the tumor cells, and then the adsorbed cells whose surfaces are connected to the metal ions are resuspended in a second solution, so that the metal ions react with the mineralized ions to generate insoluble or slightly soluble metal inorganic salts. The metal inorganic salts on the cell surface together form a mineralized shell with high mechanical strength, so that the vaccine has the ability to activate the signal pathway in the antigen-presenting cells through mechanical stimulation, thereby improving the anti-tumor ability of the vaccine.

[0065] In some embodiments, the first solution is a calcium chloride solution, the second solution is a 2',3'-cGAMP sodium salt solution, and the mineralizing ions are phosphate ions.

[0066] In some embodiments, the concentration of the first solution is any value between 80 mmol / L and 120 mmol / L, for example, any value between 80 mmol / L, 90 mmol / L, 100 mmol / L, 110 mmol / L, and 120 mmol / L, and the cell density of the tumor cells in the first solution is 1×10 6 / mL~9×10 6 Any value in the range of 1×10 6 / mL, 3×10 6 / mL, 5×10 6 / mL, 7×10 6 / mL and 9×10 6 Any value between 100 and 100 mL / min helps to maintain the integrity of cell morphology and ensure the coverage of tumor cells by the mineralized shell.

[0067] In some embodiments, the first time is any value between 20 minutes and 40 minutes, for example, it can be any value between 20 minutes, 25 minutes, 30 minutes, 35 minutes and 40 minutes. The first time is any value between 40 minutes and 90 minutes, for example, it can be any value between 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes and 90 minutes, which helps to ensure the coverage of the mineralized shell on the tumor cells while maintaining the integrity of the cell morphology.

[0068] In a third aspect, the present invention also provides an application of the above-mentioned tumor postoperative vaccine, wherein the tumor postoperative vaccine is administered by injection, and the administration frequency is once every three days, and the dosage is 1.0×10 5 / g~1.5×10 5 Any value in g, for example, 1.0×10 5 / g, 1.1×10 5 / g, 1.2×10 5 / g, 1.3×10 5 / g, 1.4×10 5 / g and 1.5×10 5 Any value between 100 and 100 g helps to ensure preventive effect while ensuring biosafety.

[0069] In some embodiments, post-tumor vaccine administration combined with radiation therapy can help improve the preventive effect.

[0070] Please refer to the following examples for details.

[0071] Example 1:

[0072] See Figure 2 The preparation method of the tumor postoperative vaccine shown in a preferred embodiment of the present application includes:

[0073] S1. Collect tumor cells.

[0074] S2. Resuspending the tumor cells in a first solution containing metal ions and incubating for a first time to allow the metal ions to be adsorbed on the surface of the tumor cells, and collecting the adsorbed cells.

[0075] S3. Resuspending the adsorbed cells in a second solution containing mineralizing ions and incubating for a second time to allow the metal ions to react with the mineralizing ions to obtain metal inorganic salts and form a mineralized shell.

[0076] S4. Collect the cells and resuspend them to obtain the post-tumor vaccine.

[0077] In step S1, mouse osteosarcoma cells (K7M2) in the logarithmic growth phase were used, digested with trypsin, and collected. The cells were washed twice with phosphate buffered saline (PBS), and centrifuged at 1200 rpm for 5 minutes to remove the culture medium and trypsin residues.

[0078] In step S2, the second solution is a CaCl2 solution with a concentration of 100 mmol / L. After resuspending, the cells are counted and the cell density is adjusted to 4×10 6 Cells were incubated at room temperature for 20 minutes. The cell suspension should be gently shaken during the incubation process to ensure a uniform reaction. After the incubation is completed, centrifuge at 1200 rpm for 5 minutes, discard the supernatant, and the pellet is the adsorbed cells.

[0079] In step S3, the second solution is a 50 mmol / L 2',3'-cGAMP sodium salt solution, which is incubated at room temperature for 40 minutes. The cell suspension should be gently shaken during incubation to ensure a uniform reaction. 2',3'-cGAMP sodium salt is a STING agonist and can dissociate into phosphate ions, which serve as mineralizing ions in the second solution.

[0080] In step S4, the cells were centrifuged at 1200 rpm for 5 minutes, the supernatant was discarded to obtain the mineralized cells, washed twice with PBS, and centrifuged again at 1200 rpm for 5 minutes to remove the unreacted solution. The cells in the resulting precipitate were fixed with 4% paraformaldehyde solution for 15 minutes to enhance the stability of the vaccine, washed twice with PBS, and centrifuged again at 1200 rpm for 5 minutes to remove the fixative. Finally, the cells were resuspended in PBS to adjust the cell concentration to 4×10 6 / mL, and dispensed into 1.5mL centrifuge tubes to prepare the tumor postoperative vaccine.

[0081] The tumor postoperative vaccine obtained in this example was named CaP@Cell and stored in a refrigerator at 4°C until use. A small amount of CaP@Cell vaccine was evenly applied to the carbon conductive adhesive on the electron microscope stage. After gold spraying, the overall morphology and surface details of the vaccine were observed under a scanning electron microscope (SEM). Figure 1 , it can be seen that the vaccine retains the complete tumor cell morphology and structure after tumor surgery, and is covered with a mineralized shell on the surface. The elemental composition of the vaccine was then analyzed by Energy Dispersive Spectroscopy (EDS), and the images were merged using the Merge method. Figure 3, it can be seen that the blue-green and red areas in the Merge diagram overlap, proving that calcium (Ca) and phosphorus (P) are highly co-localized on the vaccine surface, thus confirming that the mineralized shell component is calcium phosphate.

[0082] A portion of the CaP@Cell vaccine was taken to form the CaP@Cell group sample, K7M2 live cells were used as the Live Cell group sample, 2',3'-cGAMP sodium salt solution was used as the cGAMP group sample, and a mixed suspension of 2',3'-cGAMP sodium salt and calcium chloride was used as the cGAMP-Ca group sample. Each group of samples was tested on an infrared spectrometer and Fourier transform infrared spectroscopy (FT-IR) absorption graphs were plotted. Figure 4 The FT-IR images revealed characteristic absorption peaks of the CaP@Cell vaccine, which showed significant differences compared with the LiveCell group. The characteristic vibration peaks of inorganic phosphate and organic materials were observed in the analysis, which were located at 500-600 cm-1, indicating PO vibration. -1 The wave number is 1500~1600cm which indicates the vibration of C=O -1 At these wavenumbers, these characteristic peaks indicate that the CaP@Cell vaccine contains additional calcium phosphate and 2',3'-cGAMP compared to living cells. Since the sodium salt of 2',3'-cGAMP reacts with calcium ions to form calcium phosphate, this information, combined with the information in the figure, indicates that the CaP@Cell vaccine contains additional calcium phosphate and 2',3'-cGAMP compared to living cells.

[0083] X-ray diffraction (XRD) patterns of CaP@Cell samples and standard calcium phosphate (PDF#09-0169) were obtained by X-ray diffractometer. Figure 5 By comparing the diffraction peak distribution within the 2θ angle range of 10° to 50°, it can be seen that the characteristic diffraction peaks of the CaP@Cell sample match those of the standard calcium phosphate, indicating that a calcium phosphate mineralization layer has been successfully formed on the vaccine surface.

[0084] The contents of calcium ions, DNA, and RNA in the samples of the CaP@Cell group and the Live Cell group were evaluated by ultraviolet spectrophotometry. The samples were treated with a calcium content detection kit and the absorbance at 575 nm was measured by a microplate reader to obtain a calcium ion standard curve, and the calcium ion content of each sample group was calculated. The DNA and RNA of each sample group were extracted by the phenol / chloroform method, and then their contents were determined by ultraviolet spectrophotometer. The protein content in each group of samples was then detected by the BCA method. Figure 6 As shown in the figure, it can be seen that the calcium ion content of the CaP@Cell group reached (93.3±5.2)μg / 10 6cells, significantly higher than that of the Live Cell group (3.6±0.8)μg / 10 6 The CaP@Cell group showed no statistically significant differences in DNA retention (98.4±3.1%), RNA relative content (97.6±2.8%), and protein content (2.1±0.3 mg) compared to the live cell group (P>0.05). The figures in this example indicate ns, confirming that mineralization treatment can fully retain tumor antigens.

[0085] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect the protein components in the CaP@Cell group and the Live Cell group. Figure 7 Using a prestained protein marker ranging from 10kDa to 250kDa as a reference, the two groups of samples displayed a highly consistent band distribution pattern within the molecular weight range of 45kDa to 220kDa. Image Lab grayscale analysis revealed that the positional deviation of the main bands in the two groups of samples was less than ±2kDa, and the band intensity similarity exceeded 92%, indicating that the mineralization treatment retained greater than 95% of the integrity of the tumor cell antigen protein.

[0086] To verify the in situ mineralization of calcium ions on the tumor cell surface, calcein and DAPI were used to stain the calcium phosphate shell and cell nucleus of CaP@Cell, respectively. After incubating the dye with the vaccine for 15 minutes, the reaction system was rinsed with PBS to obtain the fluorescently labeled CaP@Cell vaccine, which was then observed using a confocal laser scanning microscope (CLSM). Figure 8 The Calcein fluorescence staining image shows that the cell membrane integrity remains well after mineralization treatment, and the green fluorescence is evenly distributed in the cytoplasm area; the DAPI staining image shows that the nuclear morphology of the CaP@Cell vaccine is intact; the bright field image shows that the cells coated with the mineralized layer maintain the original three-dimensional structure. Combined with the 50μm ruler, the diameter of the CaP@Cell vaccine ranges from 50μm to 100μm; the multi-channel fusion image shows that the green fluorescent-labeled cytoplasm and the blue fluorescent-labeled cell nucleus have a high co-localization rate, indicating that the mineralization process has completely retained the original structure and morphology of the tumor cells while forming an inorganic protective layer.

[0087] After incubating the Calcein dye with the CaP@Cell vaccine for 15 minutes, the reaction system was rinsed with PBS, allowing the Calcein to bind to the calcium phosphate shell of the CaP@Cell, labeling the vaccine and obtaining a fluorescently labeled CaP@Cell vaccine. The fluorescently labeled CaP@Cell vaccine was then incubated with mouse bone marrow-derived dendritic cells (BMDCs) at 37°C in a 5% CO2 environment for 24 hours. 30 minutes before the end of the incubation, PE-Cy7 fluorescein conjugated to a CD11c monoclonal antibody and the lysosomal fluorescent probe Lyso-Tracker Red were added to the culture system to label the cell membrane and lysosomes of the BMDCs. After the incubation was completed, the reaction system was rinsed with PBS and observed by CLSM. First, a bright image of the sample was acquired using the bright field imaging mode, and then the target fluorescence signal was collected using a specific fluorescence filter set, and the image was merged using the Merge method. Please refer to Figure 9 Calcein was used as a fluorescent marker for calcium ions, indicating that the tumor cell vaccine had been mineralized. CD11c, a surface marker of DCs, was fluorescently stained as a cell membrane marker for BMDCs. CLSM observations revealed that CaP@Cell localized to the inner side of the CD11c-labeled cell membrane and closely overlapped with the red fluorescent-labeled lysosomes, demonstrating that the CaP@Cell vaccine was effectively taken up by DCs and entered the lysosomes.

[0088] The CaP@Cell vaccine was placed in a simulated lysosome environment at pH 4.5, a simulated tissue microenvironment at pH 7.4, and a simulated tumor microenvironment at pH 6.5. The calcium ion concentration in the supernatant after different incubation times was detected using a calcium assay kit to monitor the amount of calcium ion release in different environments. Figure 10 At pH 4.5, the 24-hour calcium release rate was over 40%, significantly higher than the calcium release rates at pH 6.5 and pH 7.4. This pH-responsive release profile is highly compatible with the acidic environment of dendritic cell lysosomes, demonstrating that the vaccine can achieve controlled antigen release through a lysosomal-specific degradation mechanism. At physiological pH, the cumulative release over 80 hours was less than 10%, effectively mitigating the risk of systemic toxicity.

[0089] BMDCs were plated at 3 × 10 per well. 5Cells were seeded into 6-well culture plates at a density of 100 cells / mL. After 24 hours, CaP@Cell was added at different time points (0 hours, 4 hours, 12 hours, 24 hours, and 48 hours). After incubation, BMDCs were washed with PBS and then stained with Fluo-4, a fluorescent dye for intracellular calcium detection. Figure 11 and Figure 12 Confocal and flow cytometry results showed that the CaP@Cell vaccine increased calcium ion concentration in BMDCs, reaching a peak after 24 hours. This phenomenon confirmed that the CaP@Cell vaccine can activate the downstream PI3K-Akt signaling pathway with calcium ions as the second messenger through the dual mechanisms of mechanical stimulation and chemical release, demonstrating the "mechanical-chemical synergistic effect" of tumor vaccine activation of BMDCs.

[0090] To verify the temporal activation characteristics of the PI3K-Akt signaling pathway activated by the CaP@Cell vaccine, the expression of key signaling molecules was detected by Western Blot (WB) after BMDCs were incubated with the CaP@Cell vaccine for different time periods. Figure 13 , WB results showed that CaP@Cell significantly upregulated the expression of PI3K-Akt key signal transduction molecules P-PI3K, P-Akt, P-mTOR and P-IRF3, and reached a peak after 24 hours of co-incubation, which proved that the PI3K-Akt pathway of BMDCs was activated. Phosphorylated IRF3 (p-IRF3, 50kDa) is an important transcription factor that is mainly involved in the production of interferon-I (IFN-I) and plays a core role in the innate immune response. The activation of this pathway synergizes with the cGAS-STING pathway to enhance the synthesis and secretion of IFN-I. After CaP@Cell vaccine intervention, the expression of phosphorylated FAK was significantly increased, and its downstream protein RhoA also showed an increase, both of which peaked at 24 hours. Phosphorylated MYPT1 and MLC2 are mainly involved in cytoskeleton regulation and also increased compared with 0 hours. This demonstrates that the activation of the RhoA-MYPT1-MLC2 pathway contributes to the enhanced expression of actin and myosin, promoting cytoskeleton remodeling of BMDCs.

[0091] To further evaluate CaP@Cell's activation of the cGAS-STING pathway in BMDCs, inactivated K7M2 cells were co-incubated with BMDCs to form the Ctrl group; irradiated K7M2 cells were co-incubated with BMDCs to form the IR@Cell group; the vaccine was prepared with calcium ions alone and co-incubated with BMDCs to form the Ca@Cell group; K7M2 cells were incubated with a free STING agonist and inactivated, then co-incubated with BMDCs to form the STING group; and the CaP@Cell vaccine was co-incubated with BMDCs to form the CaP@Cell group. Western blotting was performed to examine the expression of STING-TBK1-IRF3 pathway-related signaling molecules in BMDCs in each of these groups, and quantitative analysis was performed. Figure 14 It can be seen that the expression levels of phosphorylated STING, TBK1 and IRF3 in the CaP@Cell group were significantly higher than those in the IR@Cell, Ca@Cell and STING groups. The level of phosphorylated IRF3 in the CaP@Cell group was about 5.8 times higher than that in the control group. This proves that when DCs come into contact with CaP@Cell, they first experience extracellular Ca under mechanical stimulation. 2 + influx. Then DCs phagocytize CaP@Cell and release Ca 2 + and cGAMP, jointly activate the PI3K-Akt and cGAS-STING signaling pathways, and promote the synthesis of IFN-I.

[0092] To observe the morphological changes of BMDCs after being activated by CaP@Cell vaccine, BMDCs with DAPI-labeled nuclei were cultured at 2×10 5 Cells were seeded at a density of 100 cells / mL into confocal culture dishes and incubated with CaP@Cell for 24 hours. After incubation, BMDCs were washed with PBS. Next, BMDCs were fixed with 4% paraformaldehyde for 15 minutes and permeabilized with 0.5% TritonX-100 for 20 minutes. Actin-Tracker Red and non-muscle Myosin IIA were diluted in PBS containing 4% bovine serum albumin and incubated with the cells in the dark for 50 minutes at room temperature. After three PBS washes, BMDCs were imaged and analyzed by CLSM. See Figure 15 , it can be seen that BMDCs treated with the CaP@Cell vaccine exhibited significant morphological changes compared to the control group. Vaccine-treated BMDCs showed actin remodeling and formed more branched protrusions, which may help increase the contact area between BMDCs and other immune cells, thereby more effectively delivering tumor antigens and activating T cells. Figure 15The Merge image in Figure 3 shows the spatial relationship between the red actin fibers and the blue cell nuclei, indicating that vaccine-treated cells undergo cytoskeletal changes, which is important for mature BMDCs to play a role in immune responses.

[0093] To further explore the immune activation effect of CaP@Cell on BMDCs, BMDCs were cultured at a rate of 3×10 5 Cells were seeded at a density of 100 cells / well in a 6-well plate. After 24 hours, the culture medium was replaced with 2 mL of fresh culture medium containing different treatment materials (PBS, IR-Cell, Ca@Cell, 2',3'-cGAMP, CaP@Cell) and cultured for another 24 hours. BMDC cell surface markers CD86, MHCII, and CD11c were labeled with various fluorescently labeled antibodies. The expression levels of BMDC maturation markers in each sample group were analyzed by flow cytometry and quantitatively analyzed. Figure 16 The percentage of MHCII-positive cells or CD86-positive cells among CD11c-positive cells is marked with numbers in the figure. It can be seen that the content of mature BMDCs in the CaP@Cell group samples is higher, which proves the upregulation of cell surface molecules involved in costimulatory signaling, indicating the enhancement of BMDCs' antigen presentation ability. Figure 17 By counting CD86, MHCII, and CD11c-positive cells and analyzing the percentage of mature BMDCs in each group, it can be seen that the proportion of mature BMDCs after vaccine treatment reached the highest level in the CaP@Cell group, significantly higher than in the other treatment groups. Furthermore, the ability of untreated, irradiated, calcium-treated, treated with a free STING agonist, and treated with the vaccine of the present invention to catalyze BMDC maturation increased. This confirms that the mineralization vaccine effectively increases the proportion of mature BMDCs by upregulating the costimulatory molecule CD86 and the antigen-presenting molecule MHC II.

[0094] The supernatant of each group of samples was taken to quantitatively analyze the secretion levels of cytokines TNF-α and IFN-γ. Figure 18 and Figure 19 , it can be seen that the secretion of cytokines TNF-α and IFN-γ in the CaP@Cell group was significantly higher than that in the Ctrl, IR@Cell, Ca@Cell, and STING groups. These results indicate that the CaP@Cell vaccine enhances the anti-tumor immune response by promoting the secretion of cytokines TNF-α and IFN-γ.

[0095] Combined with the results of the above immune activation experiments, it can be seen that the CaP@Cell vaccine significantly promoted the maturation and immune activation of BMDCs.

[0096] To evaluate the safety of the vaccine, live / dead cell staining and in vivo tumorigenesis experiments were performed. Tumor cells and CaP@Cell were co-incubated with Calcein-AM / propidium iodide for 30 minutes, washed with PBS, and then formed into live cell and CaP@Cell groups, respectively. CLSM was used to observe the live / dead status of the vaccine. Live K7M2 cells were labeled with wild-type luciferase, and the CaP@Cell vaccine was prepared from these labeled cells. 2×10 6 Live cells and vaccines were injected into the left hind limbs of mice to form Live Cell group and CaP@Cell group mouse samples, and fluorescence signals were collected using IVIS imaging system on the 1st and 7th day after injection. Figure 20 Live / dead staining confirmed the complete inactivation of the mineralized vaccine. The CaP@Cell group showed no fluorescence signal for 7 days, while the fluorescence intensity in the live cell group increased over time, confirming its lack of tumorigenicity in vivo. These in vitro and in vivo experiments confirm the CaP@Cell vaccine's lack of tumorigenicity and excellent biosafety.

[0097] In order to track the distribution and retention of CaP@Cell vaccine in mice, the CaP@Cell vaccine was labeled with fluorescent dye Cypate to form Cy-CaP@Cell, and radioactive isotopes were used to 177 Lu-labeled CaP@Cell vaccine formation 177 Lu-CaP@Cell, the labeled vaccine was injected into the tumor of the mouse, and the vaccine tracking imaging and analysis were performed by multimodal imaging (IVIS / SPECT / CT) at different time points. Figure 21 The results of in vivo imaging system (IVIS) showed that the Cypate-labeled Cy-CaP@Cell vaccine could be visualized in the tumor site for 7 days. 177 Lu-labeled 177 Lu-CaP@Cell vaccine was traced, and its dynamic distribution characteristics were consistent with the IVIS imaging results. 177 The decay law of the radioactive signal of Lu-CaP@Cell and 177 The Lu nuclide theoretical decay curve is highly consistent, confirming that the radioactive labeling system has good tracing stability.

[0098] To evaluate the inhibitory effects of different treatment regimens on the postoperative recurrence of osteosarcoma in a mouse model, 2×10 6 Mouse osteosarcoma models were established using K7M2 cells and randomly divided into five groups.3 Afterwards, a recurrence inhibition experiment was conducted. The Ctrl group did not remove the tumor tissue. Three days after the operation, the experimental group injected normal saline into the tumor site every two days, for a total of three injections, with each injection of 25μL. The remaining groups underwent surgery to remove the tumor tissue. Three days after the operation, the same volume of treatment fluid as the Ctrl group was injected into the surgical site every two days, for a total of three injections. Some groups also received radiation therapy. The radiation therapy started three days after the operation, and 6Gy of X-ray irradiation was received every two days, for a total of three times. The group that received radiation therapy with normal saline as the treatment fluid was marked as the Surgery+RT group. Each time, 25μL of 2×10 6 The group that received 5 mg / kg of 2',3'-cGAMP in saline as the treatment solution and did not receive additional irradiation was labeled as the Surgery+IR@Cell group. The group that received 5 mg / kg of 2',3'-cGAMP in saline as the treatment solution and did not receive additional irradiation was labeled as the Surgery+STING group. 6 The group that received CaP@Cell vaccine and normal saline and radiation therapy was labeled as Surgery+CaP@Cell+RT group. The tumor volume, survival rate and body weight changes of each mouse in each group were calculated and plotted as a line graph. Figure 22 and Figure 23 The tumor volume of the Surgery+CaP@Cell+RT group was effectively controlled and significantly reduced compared with the Surgery+STING group and the Surgery+IR@Cell group. Figure 24 Surgery + CaP@Cell + RT treatment significantly improved the survival rate of treated mice, indicating that CaP@Cell combined with radiotherapy has a good effect in inhibiting tumor recurrence. In the untreated control group, the survival rate dropped to 0% on day 29, while the radiotherapy alone, Cell-IR, and cGAMP groups reached 0% on days 31, 43, and 37, respectively. In contrast, the survival rate of the CaP@Cell + RT group remained at 60% on day 51. See Figure 25 No significant changes in body weight were observed among the groups, indicating that CaP@Cell combined with radiotherapy has good biosafety.

[0099] Combined with the results of the above recurrence inhibition experiments, it can be seen that the CaP@Cell vaccine can effectively reduce the recurrence rate of tumors after surgery, improve the survival rate and prolong the survival period.

[0100] From the in vitro cell experiments, it can be seen that CaP@Cell can enhance the immune response and improve the immune effect of the matrix on tumor cells when acting alone on DCs. Radiotherapy can regulate tumor phenotype, enhance antigen presentation and tumor immunogenicity, increase the production of cytokines, change the tumor microenvironment, and thus kill tumors by enhancing the immune system. In recent years, researchers have begun to notice that compared with the use of radiotherapy or immunotherapy alone, the combination of the two can lead to a more effective anti-tumor response. Combining the results of in vitro cell experiments and recurrence inhibition experiments, it can be seen that the post-operative tumor vaccine of the present invention has the function of improving the patient's immunity to tumor cells alone, and can play a synergistic role with radiotherapy.

[0101] Mice in each group were sacrificed, and tumor tissue and spleen tissue were collected for immune cell infiltration experiments. CTLs were detected using a CD8 and IFN-γ dual-labeling strategy, and Tregs were identified by combined CD25 and Foxp3 labeling. Flow cytometry was used to quantify the content of immune cell subsets in both tissues. Figure 26 and Figure 27 It can be seen that the CaP@Cell vaccine significantly increased the infiltration ratio of cytotoxic T cells in tumor tissue and spleen tissue, while reducing the proportion of regulatory T cells, indicating that it achieves synergistic immunotherapy enhancement through the dual mechanism of reshaping the local immunosuppressive microenvironment of the tumor and activating the systemic anti-tumor immune response.

[0102] When the mouse died or the tumor volume reached 1500mm 3 At 14:00, the animals were euthanized, and tumor tissue was obtained by dissection and placed in 4% paraformaldehyde for H&E staining. The invasion of tumor cells into the tibia was observed under an optical microscope. The left leg was obtained by dissection and placed in 4% paraformaldehyde for micro-CT reconstruction of bone microstructures to analyze whether the CaP@Cell vaccine can ameliorate bone damage caused by osteosarcoma tumor cells. Results are shown in the figure. Figure 28 , minimal hematoxylin staining (light staining) and regular cell nuclei were observed at the surgical site in the CaP@Cell+RT group. In contrast, the other groups showed high staining (dark staining) and irregular cell nuclei, which are characteristics of tumor tissue. These results indicate that CaP@Cell+RT treatment effectively eliminated residual tumor cells. Micro-CT and 3D reconstruction analysis showed that the tibial boundary integrity of the CaP@Cell group was significantly better than that of the other groups, and the bone volume fraction (BV / TV) of the CaP@Cell group reached 51.6%, which was about 3.8 times higher than that of the Ctrl group (13.5%), and the trabecular number (Tb.N) of the CaP@Cell group was 2.0mm -1 , which was significantly higher than the 1.1 mm of the free STING group. -1This indicates that the CaP@Cell group had the least bone destruction in the joints compared to the other groups, followed by the Surgery+STING group, which received a free STING agonist after surgery. This suggests that the calcium phosphate mineral layer of the vaccine inhibits osteoclast activity through biomimetic bone integration, while the sustained-release cGAMP promotes bone repair by activating the STING pathway and upregulating osteoblast-related factors.

[0103] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0104] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A post-operative tumor vaccine, characterized in that: The invention comprises inactivated tumor cells and a mineralized shell wrapped on the surface of the tumor cells, wherein the main component of the mineralized shell is a metal inorganic salt.

2. The postoperative tumor vaccine according to claim 1, characterized in that The mineralized shell is doped with a STING agonist.

3. The postoperative tumor vaccine according to claim 2, characterized in that The metal inorganic salt is generated by the reaction of the STING agonist with a metal ion, wherein the STING agonist is one or more of 2',3'-cGAMP sodium salt, 3',3'-cGAMP sodium salt and cGAMP disodium salt, and the metal ion is Mg 2+ , Ca 2+ 、Fe 2+ 、Fe 3+ 、Al 3+ and Mn 2+ One or more of .

4. The postoperative tumor vaccine according to claim 1, wherein The metal inorganic salt is calcium phosphate.

5. A method for preparing a postoperative tumor vaccine according to any one of claims 1 to 4, characterized in that: include: Collect tumor cells; resuspending the tumor cells in a first solution containing metal ions, and incubating for a first time to allow the metal ions to adsorb on the surface of the tumor cells, and collecting the adsorbed cells; resuspending the adsorbed cells in a second solution containing mineralizing ions and incubating for a second time, so that the metal ions react with the mineralizing ions to obtain the metal inorganic salt and form the mineralized shell; The cells are collected and resuspended to obtain the post-tumor vaccine.

6. The preparation method according to claim 5, wherein The first solution is a calcium chloride solution, the second solution is a 2',3'-cGAMP sodium salt solution, and the mineralized ions are phosphate ions.

7. The preparation method according to claim 6, wherein The concentration of the first solution is any value between 80 mmol / L and 120 mmol / L, and the cell density of the tumor cells in the first solution is 1×10 6 / mL~9×10 6 Any value in the range of mmol / mL.

8. The preparation method according to claim 5, wherein The first time is any value between 20 minutes and 40 minutes, and the second time is any value between 40 minutes and 90 minutes.

9. A use of the tumor postoperative vaccine according to any one of claims 1 to 4, characterized in that: The vaccine is administered by injection after tumor surgery. The administration frequency is once every three days. The dosage is 1.0×10 5 / g~1.5×10 5 Any value in g.

10. The use according to claim 9, characterized in that The vaccine administration after tumor surgery is combined with radiotherapy.

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