Dendritic cell vaccine preparation and preparation method thereof

Abstract

The present invention discloses a dendritic cell vaccine preparation and a preparation method thereof, the method comprising the following steps: collecting fresh breast cancer tissue and culturing the breast cancer tissue in a serum-free manner to obtain GREM1-positive triple-negative mammary gland sphere cells; subjecting the GREM1-positive triple-negative mammary gland sphere cells to heat shock and repeated freezing and thawing to obtain a cell lysate; preparing immature dendritic cells; controlling the cell lysate to load the immature dendritic cells to obtain a loaded product; and inducing maturation of the loaded product to obtain a vaccine preparation. The present invention provides a dendritic cell vaccine preparation that can induce an immune response in vivo and in vitro to kill triple-negative breast cancer cells that cause recurrence and metastasis, thereby providing the possibility of overcoming triple-negative breast cancer tolerance and radically curing the recurrence and metastasis of triple-negative breast cancer.

Description

Technical Field

[0001] The present invention relates to the field of biological vaccines, and in particular to a dendritic cell vaccine preparation and a preparation method thereof. Background Art

[0002] Triple-negative breast cancer (TNBC) is defined as breast cancer tissue that is negative for estrogen receptors, progesterone receptors, and proto-oncogenes as determined by immunohistochemical analysis. This condition is associated with a high rate of visceral metastasis, a high risk of early recurrence, and a poor prognosis. While existing conventional treatments, such as surgery, chemotherapy, and radiotherapy, can remove or kill most breast cancer cells, some residual or drug-resistant cells may remain, resulting in a short survival period and a poor prognosis. With the advancement of molecular and cell biology, clinical cancer treatment technologies have emerged and are demonstrating promising results. Cellular immunotherapy for cancer has made significant progress, with the use of a patient's own immune cells achieving excellent results in the clinical treatment of breast cancer. These cells can effectively eliminate residual tumor cells and inhibit tumor recurrence and metastasis without any clinical side effects. Dendritic cells are the largest antigen-presenting cells in the body. They phagocytose and process breast cancer cells, bind to T lymphocytes, and present antigen information to them, which then induce cytotoxicity against tumor cells. Dendritic cell vaccines have been used clinically to treat breast cancer patients. How dendritic cells can achieve better anti-tumor effects depends entirely on the antigens specifically expressed by the tumor. Therefore, screening and finding tumor-specific antigens is a key link in the function of dendritic cells.

[0003] In summary, some breast cancer vaccines in the prior art cannot effectively and accurately eliminate breast cancer cells, and cannot effectively inhibit the recurrence and metastasis of breast cancer. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention proposes a dendritic cell vaccine preparation and a preparation method thereof to solve the problems raised in the above background technology.

[0005] A method for preparing a dendritic cell vaccine preparation, comprising the following steps:

[0006] S1. collecting fresh breast cancer tissue and culturing the breast cancer tissue in a serum-free manner to obtain GREM1-positive triple-negative mammosphere cells;

[0007] S2. heat shocking the GREM1-positive triple-negative mammosphere cells and repeatedly freezing and thawing them to obtain cell lysates;

[0008] S3. Preparation of immature dendritic cells;

[0009] S4, controlling the cell lysate to load the immature dendritic cells to obtain a loaded product;

[0010] S5. performing an induction maturation treatment on the loaded product to obtain a vaccine preparation;

[0011] Wherein, step S1 is specifically as follows:

[0012] Fresh breast cancer tissue was collected, soaked in 75% alcohol for 2-3 minutes, and washed three times with saline containing 2% double antibody. Fat tissue and blood vessels were removed, and the remaining tissue was minced to obtain clean tissue.

[0013] The cleaned tissue was mixed with hyaluronidase and collagenase I in DMEM-F12 digestion medium for digestion, and pipetted with a 1 ml pipette every 15-20 minutes. After digestion, the tissue was filtered and washed with DMEM / F12 containing 10% fetal bovine serum and physiological saline in sequence, and then centrifuged to obtain target cells;

[0014] The target cells were inoculated at a concentration of 1000 cells / ml in serum-free DMEM / F12 and cultured. After 48 hours of culture, half of the medium was replaced until the target cells formed spheres and grew larger. The cells were allowed to stand and the supernatant was aspirated. Accutase was added to the precipitate for digestion and centrifugation to obtain a single cell precipitate.

[0015] The single cell pellet was resuspended in fresh culture medium and inoculated into the same 6-well plate and cultured in a 37°C, 5% CO2 incubator to obtain GREM1-positive triple-negative breast sphere cells;

[0016] The serum-free DMEM / F12 contained 10 μg / mL bFGF, 10 μg / mL EGF, 1× B-27, 1× penicillin / streptomycin, 50 units / mL heparin sodium, 0.2 μM glutamine, and 10 μg / mL Y27632.

[0017] According to the method for preparing the dendritic cell vaccine preparation proposed by the present invention, the beneficial effects of the present invention are:

[0018] The dendritic cell vaccine preparation obtained by the method provided by the present invention can induce an immune response in vivo and in vitro to kill triple-negative breast cancer cells that cause recurrence and metastasis, thereby providing a possibility for overcoming triple-negative breast cancer tolerance and radically curing the recurrence and metastasis of triple-negative breast cancer.

[0019] In addition, the method for preparing the dendritic cell vaccine preparation provided by the present invention may also have the following additional technical features:

[0020] Preferably, the ratio of the hyaluronidase to the collagenase I is 1:2-2.5.

[0021] Preferably, step S2 is specifically as follows:

[0022] The GREM1-positive triple-negative mammary gland sphere cells were resuspended in 5 mL of RPMI 1640 medium and dispensed into 1.5 mL EP tubes. The tubes were sealed and placed in a 42°C water bath for heat shock for 2 hours. The tubes were then placed in liquid nitrogen for 10 minutes, taken out and placed at room temperature for 10 minutes. The tubes were frozen and thawed three times to obtain cell lysates.

[0023] Preferably, step S3 is specifically as follows:

[0024] Peripheral blood was collected and centrifuged via Ficoll-Hypaque density gradient to obtain mononuclear cells;

[0025] Mononuclear cells were resuspended in RPMI 1640 medium and added to 6-well plates to adhere;

[0026] After the 6-well plate was placed in a 37°C, 5% CO incubator and incubated for 90 min, the adherent cells were added to complete RPMI 1640 medium for induction culture;

[0027] After the third day of culture, fresh culture medium was replaced and culture was continued for 5 days to obtain immature dendritic cells.

[0028] Preferably, the complete RPMI 1640 culture medium includes 5% autologous serum, 1× glutamine, and 100-2000 IU / mL recombinant human FMS-like tyrosine kinase 3 ligand.

[0029] Preferably, step S5 includes:

[0030] Inducin was added to the loaded product for induction for 24 hours, followed by centrifugation to obtain a mature loaded product. The mature loaded product was washed three times with normal saline and resuspended in normal saline, and then human serum albumin was added at a mass volume ratio of 2% to obtain a vaccine preparation.

[0031] Preferably, the inducing factors are tumor necrosis factor α and lipopolysaccharide or recombinant human interferon γ.

[0032] On the other hand, the present invention further provides a dendritic cell vaccine preparation, which is prepared using the above-mentioned method for preparing the dendritic cell vaccine preparation.

[0033] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1 A graph showing the relative quantitative expression results of GREM1 in triple-negative breast cancer tissues by real-time fluorescence quantitative PCR.

[0036] Figure 2 A diagram showing the mammosphere growth cells cultured from triple-negative breast cancer tissue.

[0037] Figure 3 Flow cytometric analysis of mammosphere cells for markers of breast cancer stem cells.

[0038] Figure 4 The graph shows the results of GREM1 gene PCR detection and protein western blot of the obtained mammary gland sphere cells.

[0039] Figure 5 Figure 2 shows the flow cytometric analysis of the lymphocyte phenotype activated by DC vaccine loaded with triple-negative mammosphere cell lysate.

[0040] Figure 6 Microscopic observation of dendritic cells loaded with GREM1-positive triple-negative mammosphere cell lysate and flow cytometric analysis of the phenotypic flow cytometric results of the DC vaccine prepared.

[0041] Figure 7 A graph showing the cytotoxic activity of CTLs generated by dendritic cells loaded with GREM1-positive triple-negative breast sphere cell lysate against triple-negative breast cancer sphere cells.

[0042] The embodiments of the present invention will be further described below with reference to the accompanying drawings. DETAILED DESCRIPTION

[0043] To make the objects, features, and advantages of the present invention more readily apparent, the following detailed description of the specific embodiments of the present invention is provided in conjunction with the following examples. Several embodiments of the present invention are provided in the examples. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these examples are provided to provide a more thorough and comprehensive understanding of the present invention.

[0044] Example 1

[0045] In Example 1 of the present invention, a method for preparing a dendritic cell vaccine preparation is provided. Before the preparation of the vaccine preparation, a quantitative polymerase chain reaction test of target genes in human breast cancer tissue is required. The specific detection process is as follows:

[0046] Grind 30-50 mg of breast cancer tissue frozen at -80°C in liquid nitrogen. Add 1 mL of tissue lysis buffer and vortex thoroughly to mix thoroughly. Add chloroform in a ratio of 1 mL to 200 μL of chloroform. Cover and shake for 15 seconds. After 2 minutes, centrifuge at 12,000 rpm for 10 minutes. The sample will separate into three layers: a red organic phase, an intermediate layer, and a colorless aqueous phase. Discard the intermediate and colorless aqueous phases and transfer the upper layer to a centrifuge tube for the next step. Add 1 volume of 70% ethanol (prepared in RNase-free water) to the tube and pipette thoroughly. Add the mixture to the adsorption column and centrifuge at 12,000 rpm for 10 minutes. Discard the waste liquid in the collection tube. Then, add 700 μL of buffer RW1 to the adsorption column and centrifuge at 12,000 rpm for 15 seconds. Discard the waste liquid. Return the adsorption column to the 2 mL collection tube. Add buffer RW2 (500μL) to the adsorption column, then centrifuge at 12000rpm / 15 seconds and discard the waste liquid. Put the adsorption column back into the 2mL collection tube. Repeat the above steps. Let the adsorption column stand at room temperature for 1-2 minutes and wait until it is completely dry before proceeding to the next step. Add 1.5 mL of RNase-free water to the adsorption column and place it in a centrifuge tube. Add RNase-free water (30-50μL) to the middle part of the adsorption column, let it stand at room temperature for 1 minute, then centrifuge at 12000rpm / 15 seconds, and finally collect the extracted RNA solution. Place the RNA solution in a -80℃ refrigerator for later use;

[0047] Use a spectrophotometer to measure the concentration and purity of the RNA solution. Adjust the zero setting with RNase-free water containing the RNA. Add 1 μL of RNase-free deionized water or RNA sample to the surface of the measuring base. Once the droplet forms a column on the upper and lower bases, the measurement is complete. The system will generate various parameters related to RNA concentration and quality.

[0048] cDNA reverse transcription was performed using the HiScript III First-Strand cDNA Synthesis Kit (+gDNA wiper). The specific steps are as follows: Prepare a reaction mixture on ice: 2 μL of the 5× gDNA wiper mixture, 1 μg of total RNA, and RNase-free deionized water to 10 μL. Mix thoroughly and incubate at 42°C for 2 minutes. First-Strand cDNA Synthesis: Prepare a reaction mixture: 10 μL of the previous reaction, 2 μL of the 10× RT mixture, 2 μL of the HiScript III enzyme mixture, 2 μL of Oligo(dT) 20VN, 2 μL of random primers, and RNase-free deionized water to 20 μL. Mix thoroughly and perform reverse transcription. Shake thoroughly and incubate at 37°C for 15 minutes. Then, incubate at 85°C for 5 seconds. Store the reverse-transcribed cDNA in a -20°C freezer until needed.

[0049] Real-time quantitative PCR sample testing: All cDNA samples were prepared separately using a real-time quantitative PCR reaction system. The specific system configuration was as follows: 10 μL of 2× AceQ Universal SYBR qPCR Master Mix, 0.4 μL of forward-strand primer, 0.4 μL of reverse-strand primer, and 18 μL of RNase-free deionized water. After mixing, the solution was briefly centrifuged at 5000 rpm. 18 μL of this mixture was added to the corresponding wells of a 96-well PCR plate, followed by 2 μL of the corresponding cDNA. The plate was sealed with sealing film and briefly centrifuged to mix. The prepared PCR plate was then placed on ice and the PCR program was set. PCR reaction: The prepared 96-well PCR plate was placed in a real-time quantitative PCR instrument and the PCR reaction was performed. The PCR reaction program was as follows: 95°C (10 min) followed by 45 cycles of 95°C (15 s) and 60°C (60 s). After the amplification reaction, product specificity was analyzed at (60°C-99°C) to generate a melting curve for the PCR products. The GREM 1 gene was subjected to real-time quantitative PCR reaction, and the internal reference (GAPDH) was used for real-time quantitative PCR reaction. Each sample was tested in 3 wells, and the obtained data were analyzed for relative quantitative expression of genes. Statistical analysis was then performed based on the relative quantitative expression. The specific results are as follows: Figure 1 shown. Figure 1 The relative quantitative expression level of GREM1 gene was analyzed by real-time fluorescence quantitative PCR. Statistical analysis showed that there was a statistically significant difference in GREM1 gene expression (*P<0.05, **P<0.01, ***P<0.001). Figure 1As shown in the data, the quantitative expression of GREM1 gene in triple-negative breast tissue was higher than that in the adjacent cancer group (P<0.001), and the relative quantitative expression of GREM1 gene in non-triple-negative breast tissue was higher than that in the adjacent cancer tissue (P<0.01), indicating that GREM1 gene was specifically expressed in triple-negative breast cancer tissue.

[0050] It should be noted that, in this embodiment, the cell culture plates, cell culture dishes, culture flasks, and cell culture bags used are, for example, 75 cm 2 Cell culture flask, 175cm 2 Cell culture equipment (containers) such as cell culture flasks and 250 mL cell culture bags can be used in the present invention, preferably cell culture plates. The present invention has no particular limitation on the freezing medium, but preferably comprises, for example, 50% calf serum, 40% cell culture medium, and 10% dimethyl sulfoxide. The cell culture medium is more preferably DC cell culture medium.

[0051] Serum or plasma can be added to the culture medium during culture. The amount added to the culture medium is not particularly limited, but can range from greater than 0% to 10% by volume. The amount of serum or plasma used can be varied depending on the culture stage, preferably up to 5% (volume ratio). For example, the serum or plasma concentration can be gradually reduced. The source of serum or plasma can be either autologous (meaning the same source as the cultured cells) or non-autologous (meaning a different source than the cultured cells). From a safety perspective, autologous serum or plasma is preferred. Separated and purified serum components, such as human serum albumin, can also be added.

[0052] The present invention can be implemented using various components and culture media. The culture conditions used in the present invention are not particularly limited and can be those commonly used for cell culture. For example, culture can be performed at 37°C and 5% CO2. It is also possible to dilute the cell culture solution by adding fresh culture media at appropriate intervals, replace the culture media, or replace the cell culture equipment.

[0053] The preparation method of a dendritic cell vaccine preparation provided in this Example 1 comprises the following steps:

[0054] S1. collecting fresh breast cancer tissue and culturing the breast cancer tissue in a serum-free manner to obtain GREM1-positive triple-negative mammosphere cells;

[0055] Wherein, the step S1 includes:

[0056] S11. Collect fresh breast cancer tissue, soak it in 75% alcohol for 2-3 minutes, and wash it three times with normal saline containing 2% double-antibody, remove fat tissue and blood vessels, and mince the remaining tissue to obtain clean tissue;

[0057] Specifically, the fresh breast cancer tissue collected was approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University and the patient signed an informed consent. The specific ethics approval number is (2023)CDYFYYLK(08-022). After fineness with physiological saline, 1 gram of the washed tissue was cut into small pieces and completely minced using sterile ophthalmic scissors to obtain the cleaned tissue.

[0058] S12, mixing the cleaned tissue with hyaluronidase and collagenase I in DMEM-F12 digestion medium for digestion, pipetting with a 1 ml pipette every 15-20 minutes, filtering after digestion and washing with DMEM / F12 containing 10% fetal bovine serum and physiological saline in sequence, and then centrifuging to obtain target cells;

[0059] Specifically, the ratio of hyaluronidase to collagenase I is 1:2-2.5. 100 units of hyaluronidase and 200-250 units of collagenase I are added to each ml of DMEM-F12 digestion medium. Digestion is carried out at 37°C for 3-4 hours, and pipetting is performed with a 1 ml pipette every 15-20 minutes. After digestion, the single cell suspension is filtered through a 70 μm nylon mesh, washed with DMEM / F12 / 10% FBS, and then washed with physiological saline. Centrifugation at 1200 pm for 10 minutes can obtain the target cells.

[0060] S13, inoculating the target cells at a concentration of 1000 cells / ml in serum-free DMEM / F12 for culture, changing half of the medium after 48 hours of culture until the target cells form spheres and become larger, letting the medium stand and aspirating the supernatant, adding accutase enzyme to the precipitate for digestion, and centrifuging to obtain a single cell precipitate;

[0061] The serum-free DMEM / F12 medium includes 10 μg / mL bFGF, 10 μg / mL EGF, 1× B-27, 1× penicillin / streptomycin double antibody, 50 units / mL heparin sodium, 0.2 μM glutamine and 10 μg / mL Y27632. After culturing for 48 hours, half of the medium is replaced until the target cells form spheres and become larger. After standing for 5 minutes, the supernatant is aspirated and 1-2 ml of accutase enzyme is added to the lower sediment for digestion. Digestion is carried out in a 37°C incubator for 3-5 minutes to form discrete single cells, which are then centrifuged at 1200 rpm for 5 minutes to obtain a single cell pellet.

[0062] S14, resuspending the single cell pellet in fresh culture medium and inoculating the cells into the same 6-well plate, and continuing to culture in a 37° C., 5% CO 2 incubator to obtain GREM1-positive triple-negative mammary gland sphere cells;

[0063] After obtaining a single cell pellet, resuspend it with fresh culture medium and inoculate it into a 6-well plate. The fresh culture medium here is fresh serum-free DMEM / F12. Pipette the culture medium containing the suspended pellet, centrifuge it at 800 rpm for 2 minutes, remove the supernatant, add fresh culture medium to resuspend it, and inoculate it into the same 6-well plate together. Place it in a 37°C, 5% CO2 incubator for continued culture. Figure 2 As shown in AD, Figure 2 Figures A and D represent the mammospheres derived from triple-negative breast cancer tissue after different culture times. On the third day, obvious cell sphering was observed. As the culture time increased, the mammospheres gradually grew larger. When a small black dot appeared in the center of the mammosphere, the mammospheres were digested and subcultured to obtain GREM1-positive triple-negative mammospheres.

[0064] After obtaining GREM1-positive triple-negative mammary sphere cells, a portion of the GREM1-positive triple-negative mammary sphere cells were frozen according to the cell counting results. Cryopreservation medium was added to the cryopreservation tubes, which were placed in a programmed cooling box and placed in a -80°C refrigerator for 24 hours. The cells were then stored in a liquid nitrogen tank for future use.

[0065] S2. heat shocking the GREM1-positive triple-negative mammosphere cells and repeatedly freezing and thawing them to obtain cell lysates;

[0066] Wherein, step S2 is specifically as follows:

[0067] The GREM1-positive triple-negative mammary gland sphere cells were resuspended in 5 mL of RPMI 1640 medium and dispensed into 1.5 mL EP tubes. The tubes were sealed and placed in a 42°C water bath for heat shock for 2 hours. The tubes were then placed in liquid nitrogen for 10 minutes, taken out and placed at room temperature for 10 minutes. The tubes were frozen and thawed three times to obtain cell lysates.

[0068] Furthermore, after preparing GREM1-positive triple-negative mammary gland cells, they need to be subjected to cell testing. The testing process is as follows:

[0069] Take 0.2×10 6 GREM1-positive triple-negative mammosphere cells were stained with CD24-FITC and CD44-PE, or CD133-PE, and isotype control mouse IgG1-FITC and mouse IgG1-PE (Biolegend) respectively, and incubated in a 4°C refrigerator for 30 min. After incubation, the cells were washed once with 1 mL of 1× PBS, centrifuged at 2500 rpm for 3 min, the supernatant was discarded, and the cells were washed again with 1 mL of 1× PBS. The cell pellet was resuspended in 500 μL of flow cytometry buffer and detected on a flow cytometer (FACS AriaII, BD Biosciences, USA). Figure 3Flow cytometry was used to detect markers of breast cancer stem cells. The test results showed that the cells grown into mammospheres were CD44 + / CD24 - 96.7%, CD133 + It was 75.6%, consistent with the specific phenotype of breast cancer stem cells;

[0070] At the same time, if Figure 4 As shown in A, Figure 4 A in the figure represents the statistical analysis results of the relative quantitative expression level of GREM1 gene detected by real-time fluorescence quantitative PCR. There is a statistical difference in the expression of GREM1 gene (*P<0.05, ** represents P<0.01, *** represents P<0.001). Figure 4 As shown in Figure A, the quantitative expression of GREM1 gene in mammosphere cells was higher than that in non-triple-negative breast adherent cells and normal breast cells, indicating that cells growing into mammospheres specifically express the GREM1 gene.

[0071] After that, take 2×10 6GREM1-positive triple-negative mammosphere cells were used for protein extraction by adding pre-prepared protein lysis buffer and lysing the cells on a shaker at 4°C for 10 minutes. The lysed proteins were scraped off with a cell scraper and transferred to a 1.5 mL EP tube. The cells were centrifuged at 13,000 rpm at 4°C for 10 minutes, and the supernatant was gently aspirated into a new EP tube. Protein concentration was determined using the BCA assay. Preparation of SDS-PAGE: Prepare the required protein gel concentration according to the molecular weight of the target protein; Loading: Install the electrophoresis device, add 4°C pre-cooled 1× electrophoresis buffer to the electrophoresis tank, pull out the comb, blow out the remaining gel in the well with a gun, load the sample, and fill the volume difference of each well with 1× loading; Electrophoresis: 80V constant voltage for about 30 minutes, wait for the sample to enter the separation gel, change to 100V, and stop when the sample is 1 cm away from the bottom of the separation gel; Transfer: Take out the gel plate, carefully pry open the plate, cut off the concentrated gel part, soak the separation gel and nitrocellulose membrane in pre-cooled 1× transfer buffer, and balance on a shaker; Assemble the transfer system according to the "sandwich" structure (blackboard, sponge, filter paper, separation gel, nitrocellulose membrane, filter paper, sponge, white board), and ensure that no bubbles are generated during this process; Put it in the transfer tank and pour in pre-cooled 1× transfer Buffer, the entire transfer tank was placed in an ice box, constant voltage 100V, 80min; after the transfer was completed, the nitrocellulose membrane was trimmed, 5% skim milk was added, and the membrane was blocked on a shaker at room temperature for 2 hours; after the blocking was completed, the milk was discarded, the nitrocellulose membrane was washed with deionized water, the deionized water was aspirated, and mouse anti-human GREM1 monoclonal antibody and rabbit anti-human GADPH monoclonal antibody (Wuhan Tri-Initial Co., Ltd.) were added at a ratio of 1:500 and 1:1000, and the membrane was incubated on a shaker at 4°C overnight; the primary antibody was recovered and the membrane was washed 3 times with 1×TBST, each time for 10min; the washing buffer was discarded, and horseradish peroxidase-labeled goat anti-mouse IgG or goat anti-rabbit IgG secondary antibody (Wuhan Tri-Initial Co., Ltd.) was added at a ratio of 1:500 and the membrane was incubated on a shaker at room temperature for 2 hours; the secondary antibody was discarded and the membrane was washed 3 times with 1×TBST, each time for 10min; the ECL color development solution was prepared and added to the nitrocellulose membrane, reacted for 30s, and the image was collected, as shown in the figure. Figure 4 As shown in B. Figure 4 As shown in Figure B, GREM1 protein is hardly expressed in normal breast cells MCF-10A, slightly expressed in non-triple-negative breast cancer adherent cells, and highly expressed in triple-negative breast cancer sphere-forming cells, indicating that GREM1 expression is closely related to the malignancy of breast cancer.

[0072] S3. Preparation of immature dendritic cells;

[0073] Wherein, the step S3 includes:

[0074] S31, collect peripheral blood and centrifuge it through Ficoll-Hypaque density gradient to obtain mononuclear cells;

[0075] S32, resuspend the mononuclear cells in RPMI 1640 medium and add them to a 6-well plate to adhere;

[0076] S33. Place the 6-well plate in a 37°C, 5% CO2 incubator and incubate for 90 min. Then, add complete RPMI1640 medium to the adherent cells for induction culture.

[0077] The complete RPMI 1640 culture medium includes 5% autologous serum, 1× glutamine, and 100-2000 IU / mL recombinant human FMS-like tyrosine kinase 3 ligand, and the non-adherent cells are lymphocytes and need to be collected.

[0078] S34. After the third day of culture, the culture medium was replaced with fresh medium and culture was continued for 5 days to obtain immature dendritic cells;

[0079] Specifically, the fresh culture medium here contains 1000 IU / mL recombinant human FMS-like tyrosine kinase 3 ligand.

[0080] S4, controlling the cell lysate to load the immature dendritic cells to obtain a loaded product;

[0081] Specifically, the cell lysate and the immature dendritic cells are loaded in a 37° C., 5% CO 2 incubator for 24 hours to obtain a loaded product.

[0082] S5. Inducing maturation treatment on the loaded product to obtain a vaccine preparation.

[0083] Wherein, step S5 includes:

[0084] Inducers were added to the loaded product for induction for 24 hours, followed by centrifugation to obtain a mature loaded product. The mature loaded product was washed three times with normal saline and resuspended in normal saline, and then human serum albumin was added at a mass volume ratio of 2% to obtain a vaccine preparation. After induction, cell lysate loaded mature dendritic cells were obtained.

[0085] Wherein, the inducing factor is tumor necrosis factor α and lipopolysaccharide or recombinant human interferon γ;

[0086] Specifically, after induction by inducing factors, the cell state is as follows Figure 6 As shown in the figure, DCs cells became larger and had protrusions and dendrites under a 10x microscope ( Figure 6 Middle A), the cell dendrites can be more clearly observed under a 20x objective lens ( Figure 6Middle B) shows that DCs cells have obvious dendritic morphology. Cell lysates loaded with mature dendritic cells were stained with CD86-PE, CD80-PE, CD40-FITC, CD83-PE, CD11c-FITC, and HLA-DR-PerCP (BD Company). Isotype controls were stained with mouse IgG1-FITC, mouse IgG1-PE, and mouse IgG1-PerCP (Biolegend Company). The experimental method was the same as above. Flow cytometry was used to detect the phenotypic changes of mature dendritic cells loaded with cell lysates. Figure 6 C- Figure 6 F in the figure is the phenotypic flow cytometric analysis of the DC vaccine prepared by flow cytometry, and the results show that CD11c + / HLA-DR + 93.4%, CD83 + / CD11c + 73.5%, CD86 + / CD11c + 92.1%, CD40 + / CD80 + The expression of CHS in the cells was 98.5%, which was consistent with the specific phenotype of mature dendritic cells loaded in the cell lysate and high expression of co-stimulatory molecules.

[0087] The IFN-γ secretion level of cytotoxic T lymphocytes was then detected as follows:

[0088] Mixed T lymphocyte activation:

[0089] (1) Cell lysate was loaded with mature dendritic cells and T lymphocytes at a ratio of 1:5, i.e. 2×10 5 The cells in the vaccine preparation were 1×10 6 T lymphocytes were co-cultured in a 6-well plate (3 mL system) in a 37°C, 5% CO2 incubator.

[0090] (2) T lymphocytes were collected after 24, 48, 72, 96 and 120 hours of co-culture, and the placental blue was counted to analyze the proliferation of T lymphocytes.

[0091] T lymphocytes co-cultured for 120 hours were taken for flow cytometry detection, i.e. 0.2×10 6 The cells were stained with CD3-FITC, CD4-PE, CD8-PerCP and IFN-γ-APC (Biolegend), and the isotype controls were mouse IgG1-FITC, mouse IgG1-PE, mouse IgG1-PerCP and mouse IgG1-APC (Biolegend). The experimental methods were the same as above. Figure 5The flow cytometric analysis of lymphocyte phenotypes of mature dendritic cells loaded with cell lysates was performed. The control group was T lymphocytes activated by dendritic cells without loading. + 、CD8 + The percentage of double-positive cells was 4.04%, CD8 + IFN-γ + The percentage of double-positive cells was 0.09%; while the cell lysate loaded mature dendritic cells activated lymphocytes CD3 + 、CD8 + The percentage is 33.6%, CD8 + IFN-γ + The percentage of double-positive cells was 20.3%, and the percentage of IFN-γ produced by mature dendritic cells loaded with cell lysate exceeded that of the control group, which could induce a better cytotoxic response.

[0092] Finally, the vaccine preparation provided by the present invention was subjected to an in vitro tumor killing experiment, as follows:

[0093] In a 6-well plate, RPMI 1640 containing 5% calf serum was still used as the culture medium. The above-mentioned cell lysate-loaded mature dendritic cells and T cells were mixed at a ratio of 1:5 of mature dendritic cells loaded with the above-mentioned antigen epitope peptides: T cells. Recombinant human FMS-like tyrosine kinase 3 ligand (final concentration of 1000U / mL) was added at the same time. The culture was carried out for 3 days. During this period, half of the medium was replaced every other day (RPMI 1640 culture medium containing 5% calf serum and a final concentration of 1000U / mL Flt3L) to obtain CTL cells.

[0094] Corresponding breast cancer cells (MCF-7 and the GREM1-positive triple-negative mammary gland sphere cells cultured in the above examples) were selected as target cells and the cells were cultured with 51 Cr labeled, i.e. target cells (2×10 6 / mL) by mixing with 300 μCi 51 Cr was incubated in RPMI 1640 medium at 37°C for 2 hours. The labeled target cells were washed three times with 1× PBS and finally resuspended in RPMI 1640 (containing 10% calf serum) to a concentration of 2×10 5 The concentration of 2×10 4 Labeled target cells (0.1 mL) were added to the wells of a 96-well plate.

[0095] The CTL cells (effector cells) generated above were added to the corresponding wells at effector-target ratios of 2.5:1, 5:1, 10:1, 20:1, and 40:1, and incubated at 37°C for 4 hours. After incubation, 75 μL of the supernatant was collected and counted using a gamma radiation counter. 51The percentage of Cr release was calculated according to the following formula.

[0096]

[0097] The number of pulses per minute of spontaneous release was obtained by culturing target cells alone (without effector cells), the number of pulses per minute of maximal release was obtained by culturing target cells alone treated with a final concentration of 2% NP-40 (surfactant, Shanghai Bioengineering), and the number of pulses per minute of detection was obtained by culturing target cells with the addition of effector cells.

[0098] Figure 7 Cell lysates loaded with mature dendritic cells are shown. Figure 7 In Figure A, as the effector-target ratio continued to increase, the CTL response to triple-negative breast cancer spheroid cells also specifically increased. Among them, cell lysate-loaded mature dendritic cells triggered a more powerful cytotoxic activity response to triple-negative breast cancer spheroid cells, which was significantly stronger than the CTL cells activated by unloaded dendritic cells.

[0099] Figure 7 Figure B shows the loaded product after induction of maturation, which elicited cytotoxic activity against MCF-7 breast cancer cells. At an effector-target ratio of 10:1 and higher, a specific CTL response was generated against MCF-7 breast cancer cells, with similar cytotoxic activity to DCs loaded with MCF-7 breast cancer cells, a significant difference between the two. However, both exhibited higher cytotoxic activity than T lymphocytes without DC activation. These results indicate that the CTL response generated by cell lysate-loaded mature dendritic cells exhibited more specific cytotoxic activity.

[0100] And according to Figure 7 It can be seen that cell lysates loaded with mature dendritic cells and activated cytotoxic T lymphocytes have a significant killing effect on triple-negative breast sphere cells and breast cancer cells MCF-7. When the effector-target ratio is 40:1, the killing efficiency reaches more than 80%. In addition, cell lysates loaded with mature dendritic cells are co-cultured with T lymphocytes to activate the proliferation of T lymphocytes, and interferon gamma enzyme-linked immunospot analysis can detect the secretion of interferon gamma by specifically activated T lymphocytes, thereby promoting the function of cellular immune response and producing efficient and long-lasting anti-tumor immune effects. Therefore, it is very beneficial to improve the efficacy of patients and prolong their survival.

[0101] Specifically, in another embodiment of the present invention, a dendritic cell vaccine preparation is provided, which is prepared using the preparation method of the dendritic cell vaccine preparation shown in Example 1.

[0102] In summary, the dendritic cell vaccine preparation obtained by the method provided by the present invention can induce an immune response in vivo and in vitro to kill triple-negative breast cancer cells that cause recurrence and metastasis, thereby providing a possibility for overcoming triple-negative breast cancer tolerance and radically curing the recurrence and metastasis of triple-negative breast cancer.

[0103] Throughout this specification, reference to terms such as "one embodiment," "some embodiments," "examples," "specific examples," or "some examples" means that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[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 present invention. 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 present invention shall be determined by the appended claims.

Claims

1. A method for preparing a dendritic cell vaccine preparation, characterized in that: The method comprises the following steps: S1. collecting fresh breast cancer tissue and culturing the breast cancer tissue in a serum-free manner to obtain GREM1-positive triple-negative mammosphere cells; S2. heat shocking the GREM1-positive triple-negative mammosphere cells and repeatedly freezing and thawing them to obtain cell lysates; S3. Preparation of immature dendritic cells; S4, controlling the cell lysate to load the immature dendritic cells to obtain a loaded product; S5. performing an induction maturation treatment on the loaded product to obtain a vaccine preparation; Wherein, step S1 is specifically as follows: Fresh breast cancer tissue was collected, soaked in 75% alcohol for 2-3 minutes, and washed three times with saline containing 2% double antibody. Fat tissue and blood vessels were removed, and the remaining tissue was minced to obtain clean tissue. The cleaned tissue was mixed with hyaluronidase and collagenase I in DMEM-F12 digestion medium for digestion, and pipetted with a 1 ml pipette every 15-20 minutes. After digestion, the tissue was filtered and washed with DMEM / F12 containing 10% fetal bovine serum and physiological saline in sequence, and then centrifuged to obtain target cells; The target cells were inoculated at a concentration of 1000 cells / ml in serum-free DMEM / F12 and cultured. After 48 hours of culture, half of the medium was replaced until the target cells formed spheres and grew larger. The cells were allowed to stand and the supernatant was aspirated. Accutase was added to the precipitate for digestion and centrifugation to obtain a single cell precipitate. The single cell pellet was resuspended in fresh culture medium and inoculated into the same 6-well plate and cultured in a 37°C, 5% CO2 incubator to obtain GREM1-positive triple-negative breast sphere cells; The serum-free DMEM / F12 contained 10 μg / mL bFGF, 10 μg / mL EGF, 1× B-27, 1× penicillin / streptomycin, 50 units / mL heparin sodium, 0.2 μM glutamine, and 10 μg / mL Y27632.

2. The method for preparing the dendritic cell vaccine preparation according to claim 1, wherein: The ratio of the hyaluronidase to the collagenase I is 1:2-2.

5.

3. The method for preparing the dendritic cell vaccine preparation according to claim 1, wherein: Step S2 is specifically as follows: The GREM1-positive triple-negative mammary gland sphere cells were resuspended in 5 mL of RPMI 1640 medium and dispensed into 1.5 mL EP tubes. The tubes were sealed and placed in a 42°C water bath for heat shock for 2 hours. The tubes were then placed in liquid nitrogen for 10 minutes, taken out and placed at room temperature for 10 minutes. The tubes were frozen and thawed three times to obtain cell lysates.

4. The method for preparing the dendritic cell vaccine preparation according to claim 1, wherein: Step S3 is specifically as follows: Peripheral blood was collected and centrifuged via Ficoll-Hypaque density gradient to obtain mononuclear cells; Mononuclear cells were resuspended in RPMI 1640 medium and added to 6-well plates to adhere; After the 6-well plate was placed in a 37°C, 5% CO incubator and incubated for 90 min, the adherent cells were added to complete RPMI 1640 medium for induction culture; After the third day of culture, fresh culture medium was replaced and culture was continued for 5 days to obtain immature dendritic cells.

5. The method for preparing the dendritic cell vaccine preparation according to claim 4, characterized in that: The complete RPMI1640 culture medium includes 5% autologous serum, 1× glutamine, and 100-2000 IU / mL recombinant human FMS-like tyrosine kinase 3 ligand.

6. The method for preparing the dendritic cell vaccine preparation according to claim 1, characterized in that: Step S5 includes: Inducin was added to the loaded product for induction for 24 hours, followed by centrifugation to obtain a mature loaded product. The mature loaded product was washed three times with normal saline and resuspended in normal saline, and then human serum albumin was added at a mass volume ratio of 2% to obtain a vaccine preparation.

7. The method for preparing the dendritic cell vaccine preparation according to claim 6, characterized in that: The inducing factors are tumor necrosis factor α and lipopolysaccharide or recombinant human interferon γ.

8. A dendritic cell vaccine preparation, characterized in that The dendritic cell vaccine preparation is prepared by the preparation method of the dendritic cell vaccine preparation according to any one of claims 1 to 7.

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