Method for detecting effectiveness of tumor-specific dc vaccine based on co-culture system

Abstract

The application belongs to the field of tumor immunotherapy and biological detection technology, and provides a tumor-specific DC vaccine effectiveness detection method based on a co-culture system, which comprises the following steps: S1, preparing a patient-derived tumor organoid; S2, preparing a tumor-specific DC vaccine; preparing patient-derived T cells; co-culturing the T cells and the tumor-specific DC vaccine to activate the T cells to have tumor-specific killing effect; S3, co-culturing the tumor organoid obtained in step S1 and the activated T cells obtained in step S2, and observing lymphocyte infiltration and tumor cell killing effect; and S4, DC vaccine effectiveness evaluation. In the method, the patient-derived tumor tissue organoid retains the original genetic background of the patient's tumor, and the correlation between the detection result and the clinical curative effect is significantly better than that of a conventional passaged tumor cell line, thereby providing a solid foundation for evaluating the effectiveness of the DC vaccine in vitro and improving the treatment effect.

Description

Technical Field

[0001] This invention belongs to the field of tumor immunotherapy and biodetection technology. Specifically, this application provides a method for detecting the effectiveness of tumor-specific DC vaccines based on a co-culture system. Background Technology

[0002] Tumor-specific dendritic cell vaccines are an active immunotherapy strategy that loads tumor antigens into mature dendritic cells (DCs) in vitro and then reinfuses them into the patient to elicit a specific anti-tumor immune response. Since Sipuleucel-T (Provenge®) was approved by the FDA for the treatment of prostate cancer in 2010, research on DC vaccines targeting different solid tumors has progressed rapidly, with several DC vaccines targeting tumor-associated antigens such as WT-1, HER2, MAGE-A4, NY-ESO-1, EB virus, and HPV entering clinical trials.

[0003] However, the efficacy evaluation system for DC vaccines is still imperfect, mainly facing the following problems: For example, the commonly used ELISApot assay for IFN-γ only reflects the secretory function of activated T cells and cannot evaluate its direct killing effect on tumor cells; the LDH release assay (lactate dehydrogenase release assay) uses traditional two-dimensional tumor cell lines and lacks the spatial heterogeneity of patient tumor tissue; flow cytometry for CD107a degranulation and other methods cannot simulate the immunosuppressive background of the tumor microenvironment (TME) in vivo; traditional in vitro detection relies on long-passaged tumor cell lines (such as MDA-MB-231 and A549), which lack the clonal heterogeneity, three-dimensional spatial structure (ECM composition, cell polarity), key components of the tumor microenvironment (fibroblast and macrophage infiltration), and patient-specific HLA antigen expression patterns of primary tumor tissue. In contrast, the effects of DC vaccines in vivo are often multifaceted, especially when used in combination therapy. Therefore, it is necessary to establish a multi-dimensional evaluation system and develop standardized quantitative standards.

[0004] Currently, none of the publicly available patents and technologies can solve the core problem of evaluating the in vitro efficacy of DC vaccines. Summary of the Invention

[0005] On the one hand, this application provides a method for detecting the effectiveness of tumor-specific DC vaccines based on a co-culture system, the method comprising: S1. Preparation of tumor organoids derived from patients; S2. Prepare tumor-specific DC vaccines; prepare patient-derived T cells; co-culture and activate T cells with tumor-specific DC vaccines to obtain T cells with tumor-specific killing effects; S3. Co-culturing the tumor organoids obtained in step S1 with the activated T cells obtained in step S2 allows for the observation of lymphocyte infiltration and killing effect on tumor cells. S4, DC vaccine efficacy evaluation.

[0006] Further, step S4 includes: (A) detection of DC cell phenotype and presentation efficiency in DC vaccine; (B) detection of T cell activation using tetramer and enzyme-linked spot analysis system; (C) detection of tumor cell mortality in organoids; (D) detection of cytotoxic T lymphocyte infiltration depth; (E) detection of cytokine profile in the supernatant of the co-culture system in step S2; and (F) detection of cytotoxic T lymphocyte activation rate in the supernatant of the co-culture system in step S2.

[0007] Furthermore, step S3 includes a step of weighting and scoring the detection results of (A)-(F).

[0008] Furthermore, the total score in the weighted scoring steps is 10 points, and the full scores for steps (A)-(F) are 1.5, 2, 3, 1.5, 1, and 1 points respectively.

[0009] Furthermore, the tumor cell mortality rate in organoids was detected using CellTox™ Green staining or the Annexin V-EGFP-PI method.

[0010] Furthermore, confocal microscopy Z-axis scanning was used to detect the depth of cytotoxic T lymphocyte infiltration (D).

[0011] Furthermore, the cytokine profile in the supernatant of the co-culture system in step (E) S2 includes the detection of TNF-α and γ-IFN.

[0012] Furthermore, the culture time in step S3 is 60-84 hours.

[0013] Furthermore, the tumor-specific DC vaccine includes: preparing patient-derived DC cells; sensitizing DC cells with tumor-specific antigen peptides or tumor tissue lysates to obtain the tumor-specific DC vaccine.

[0014] Furthermore, tetramer assays were used to detect tumor-specific antigenic peptides on activated T cells, and the positive counts of TNF-α and γ-IFN secreted by activated T cells were detected using an enzyme-linked spot assay system (ELISPOT).

[0015] On the other hand, this application provides the application of the above method in evaluating the effectiveness of active immunization-designed peptide vaccines, mRNA vaccines, and DNA vaccines.

[0016] Some of the abbreviations used in this application are as follows: Patient-Derived Organoid (PDO); Dendritic Cell (DC); DC Vaccine Efficacy Score (DVS); Cytotoxic T Lymphocyte (CTL).

[0017] The beneficial effects of this application include: Greater clinical applicability: Patient-derived tumor tissue organoids retain the patient's original tumor genetic background, and the correlation between test results and clinical efficacy is significantly better than that of conventionally passaged tumor cell lines.

[0018] Multi-dimensional comprehensive evaluation: It integrates 6 independent indicators to avoid the one-sidedness of a single indicator.

[0019] A comprehensive evaluation system (DVS) and a DC vaccine efficacy grading system were established: the comprehensive evaluation system and grading standards are also applicable to similar testing methods. The grading standards are also applicable, or partially applicable, to other peptide vaccines, mRNA vaccines, and DNA vaccines based on active immunization design.

[0020] An in vivo validation method for a dual humanized animal model (patient-specific HLA subtype immune reconstituted mice + patient-derived tumor cell PDX) was established. Attached Figure Description

[0021] Figure 1 Image of passaged cells.

[0022] Figure 2 This is a diagram illustrating the process of culturing tumor organoids.

[0023] Figure 3 Image showing the results of Beyo3D™ DiO staining.

[0024] Figure 4 The image shows the results of cell viability assays using CellTiter-Glo® reagent.

[0025] Figure 5 This is the result of tumor cell mortality detection in PDO.

[0026] Figure 6 This is a photograph of tumor growth in Example 4.

[0027] Figure 7 Photograph of tumor tissue after receiving the DC vaccine. Detailed Implementation

[0028] Example 1: Establishment of Patient-Derived Tumor Organoids (PDOs) The patient (male, 67 years old, advanced squamous cell carcinoma of the lung. Tumor tissue sample collected on March 23, 2025) and the processing of the tumor tissue.

[0029] Under sterile conditions (in a biosafety cabinet), first wash the tumor tissue sample with phosphate-buffered saline (PBS) containing penicillin-streptomycin (PS) + amphotericin B to remove blood contamination, repeating at least 3 times; then wash in 75% alcohol for 1-2 minutes, repeating twice; finally wash with PBS or saline containing penicillin-streptomycin (PS) + amphotericin B 1-2 times.

[0030] Using sterilized tweezers and scissors, first remove non-tumor tissue and necrotic tissue, then trim the removed tissue into 1mm pieces. 3 Small pieces.

[0031] Add the mixed enzyme digestion solution (containing 1 mg / ml type I collagenase, 1 mg / ml type IV collagenase, 0.1 mg / ml hyaluronidase, and 1 mg / ml neutral protease), and digest in a water bath at 37°C for 2-3 hours, shaking the centrifuge tube continuously during digestion.

[0032] After digestion, 1640 medium containing 10% FBS + 1% PS was added to terminate digestion. The cell suspension was filtered through 40 μm or 100 μm to collect single cell suspensions. The cells were centrifuged at 1500 rpm for 10 min, resuspended in 1640 medium containing 10% FBS + 1% PS, and counted.

[0033] Primary cell culture: Take well-counted primary tumor cells at a ratio of 1×10 5 Inoculate into 100mm culture dishes / T75 flasks; incubate at 37℃ in a 5% CO2 incubator for routine culture.

[0034] Change the medium every 3 days and observe and record the growth using an inverted microscope; after more than 90% of the culture has merged and a large number of clones have formed, rinse the remaining culture medium with PBS / physiological saline and incubate with 1 ml of 0.25% trypsin / ethylenediaminetetraacetic acid (EDTA) solution at room temperature for 1-3 min.

[0035] When the cells become rounded and begin to separate from the substrate, gently tap the culture dish or flask and remove the separated cells by aspiration. Add complete 1640 medium to stop digestion and transfer the mixture to a 15 mL centrifuge tube; centrifuge at 1500 rpm for 10 min; after centrifugation, resuspend the cell particles in complete medium and seed proportionally for further passage or cryopreservation. Passaged cells are as follows... Figure 1 As shown (A1, A2, and A3 represent different batches).

[0036] Treatment of feeder cells (3T3-J2): Resuscitation of feeder cells (3T3-J2): The feeder cells were removed from liquid nitrogen and immediately placed in a 37°C water bath. They were shaken rapidly until completely thawed, then quickly removed, dried, and sterilized with alcohol before being transferred to a biosafety cabinet. The feeder cells were placed in complete culture medium and cultured at 37°C in a 5% CO2 incubator according to standard procedures, with the culture medium changed every other day. Mitomycin C treatment of feeder cells (3T3-J2): When the feeder cells reached a confluence of 70%-90%, the culture medium was removed, and the cells were washed 2-3 times with PBS solution; 6 μg / ml of mitomycin C was added, and the cells were treated for 3 hours; the mitomycin C solution was removed, and the cells were washed 2-3 times with PBS solution; conditioned medium was added, and the cells were cultured in a 37℃, 5% CO2 incubator for routine culture; on the second day of culture, the feeder cells were irradiated with ultraviolet light for 10 minutes, and then cultured in a 37℃, 5% CO2 incubator for routine culture.

[0037] Cell co-culture: Take the counted tumor cells as follows: 1×10 5 The cells were seeded onto the feeder layer and cultured in ADMEM / F12 medium (containing 50% Wnt3a, 1 mM HEPES, glutamine (1X, Invitrogen), 10 mM nicotinamide, 1 mM N-acetylcysteine, penicillin-streptomycin, 10 nM gastrin, and 50 ng / mLEGF). The cells were then incubated at 37°C in a 5% CO2 incubator for routine culture. The remaining cells were seeded at the same concentration onto culture dishes and incubated at 37°C in a 5% CO2 incubator for routine culture.

[0038] The medium was changed every 3 days, and the growth was observed and recorded using an inverted microscope. After more than 90% of the co-cultured cells had merged and a large number of clones had formed, the microtumor cells were passaged using 200 UI of type IV collagenase for further research.

[0039] After centrifugation, the cell particles are resuspended in complete culture medium and inoculated in proportion for continued co-culture or cryopreservation.

[0040] The process of tumor organoid culture, such as Figure 2 As shown: A. Initial cellular state, B. Intermediate process, C. Tumor organoids.

[0041] After culturing in U-shaped 96-well plates for 72 hours, the culture medium was aspirated, and Beyo3D™ DiO staining working solution was added directly for staining for 15 minutes. Results showed that the Beyo3D™ Cell Membrane Green Fluorescent Staining Kit (DiO) provided clear and bright staining for 3D cells (e.g., ...). Figure 3 (As shown).

[0042] Example 2: Establishment of a tumor organoid-T cell co-culture system Isolation and culture of DC cells and loading of tumor-specific antigen peptides: 100 mL of peripheral blood was collected from the patient (alimited in 5 mL anticoagulant tubes), and mononuclear cells were isolated using countercurrent centrifugation. Cells were counted using a hemocytometer, and the cell density was adjusted to 1 × 10⁻⁶ cells / mL with 1640 cell culture medium. 6 Incubate at 37°C in a 5% CO2 incubator with cells / mL.

[0043] Induction experiment: Add cell growth factors 100 ng / mL rhGM-CF and 50 ng / mL rhIL-4. Change half the medium every other day, and observe cell growth and morphological changes daily.

[0044] Transfection of DC cells with tumor-specific antigen peptides or tumor tissue lysate: On day 6 of culture, add 5 μg / ml of tumor-specific antigen peptides or tumor tissue lysate to sensitize the growing DC cells.

[0045] Add cell growth factors rhTNF-α, IL-1β, and PG-E2 to allow sensitized dendritic cells (DCs) to grow into further mature DCs. Change half the medium every other day.

[0046] Sampling is used for DC cell counting, maturity detection, and tetramer detection.

[0047] T cell isolation and purification: Peripheral blood was collected from the patient, and Ficoll was used to isolate PBMCs. Cell counting: Cells were counted using a hemocytometer, and the cell density was adjusted to 1×10⁻⁶ cells / mL with 1640 cell culture medium. 6 Cells / mL, incubated in a 37℃ 5% CO2 incubator.

[0048] PBMCs were induced and cultured with 500 U / mL rhIL-2, 0.5 μg / mL anti-CD3, and 0.5 μg / mL anti-CD28.

[0049] Two days later, the culture medium was changed to contain only 500 U / mL rhIL-2 and cultured for 7 days to differentiate into T cells.

[0050] Activation and expansion of tumor-specific T cells: T cells were co-cultured with mature DC cells in a 24-well culture plate and 500 U / mL rhIL-2 was added.

[0051] After one day of culture, supernatants from each group were collected. T cell proliferation was detected using the CCK8 assay kit; cytokines such as TNF, IL-12, and γ-IFN were detected using ELISA. Cultured T cells were collected for flow cytometry analysis of T cell phenotype, tetramer analysis of tumor antigen-specific T cells, and ELISPOT analysis of activated T cell function.

[0052] Construction of the PDO-T cell three-dimensional co-culture system: Tumor organoids from Example 1 were used, and the tumor organoids and the aforementioned T cells were co-cultured at an effector-to-target cell ratio (E:T ratio) gradient of 10:1. Culture conditions were 37℃ and 5% CO2. Samples were taken after 24h, 48h, and 72h of co-culture for analysis.

[0053] Example 3: DC vaccine efficacy testing indicators and evaluation system DC cell phenotype and presentation efficiency assay: The DC cells cultured in Example 2 were used.

[0054] DC phenotypic detection: Take 1×10 4 The cells were placed in a flow cytometry tube and centrifuged at 1500 rpm for 5 min, and the supernatant was discarded.

[0055] Resuspend the cells in 100 μL of PBS, add 1 μL of fluorescently labeled antibodies: HLA-DR, CCR7, CD86, CD80 fluorescent antibodies, and stain at room temperature in the dark for 30 min.

[0056] After washing and centrifuging again, each tube was resuspended in PBS. Data were then analyzed using flow cytometry.

[0057] Tetramer assay for DC presentation efficiency: MHC tetramer technology can directly assess the uptake and presentation efficiency of DC cells by detecting the expression level of MHC-antigen peptide complex on the surface of DC cells.

[0058] Take 1×10 6 Centrifuge each cell in a flow cytometry tube at 1500 rpm for 5 min, discarding the supernatant. Resuspend the cells in flow cytometry buffer and adjust the cell concentration to 1 × 10⁶ cells / mL. 6 cells / 100 μL.

[0059] Add PE fluorescently labeled tumor antigen-specific MHC tetramer, and simultaneously add surface-labeled antibodies: anti-CD11c-APC, anti-CD80-FITC, anti-CD86-PE, anti-CD83-PerCP-Cy5.5, etc., incubate at 4℃ in the dark for 30-60 minutes, and then perform flow cytometry detection.

[0060] Data collection: 10,000-50,000 CD11c samples were collected for each sample. + The event was analyzed by counting the percentage of MHC tetramer-positive dendritic cells (DCs). The higher the percentage, the higher the efficiency of DCs in uptake and presenting antigens. Generally, >10% is considered to be a good antigen loading efficiency (Table 1).

[0061] Table 1 Results of DC cell phenotypic detection

[0062] .

[0063] Tetramer and ELISPOT assays for T cell activation: T cell phenotype and tetramer assays were performed using T cells sensitized with tumor-specific DC cells cultured in Example 2: cell concentration was adjusted to 1×10⁻⁶. 6 -5×10 6 Cells / mL: 100 μL of cell suspension was added to a flow cytometry tube, along with fluorescently labeled MHC tetramer. The cells were incubated at 4°C in the dark for 30-60 minutes. Simultaneously, surface-labeled antibodies and T-cell activation antibodies were added: anti-CD8-FITC (human), anti-CD3-PerCP-Cy5.5, CD4, CD16, CD28, CD56, CD25, CD45RO, CD19, CD127, CD44, CD62L, etc. The cells were incubated at 4°C in the dark for 30 minutes. After washing with flow cytometry buffer, the cells were resuspended in 200 μL of fixative (PBS + 1% paraformaldehyde (PFA)) and analyzed using a flow cytometer.

[0064] ELISPOT assay: Performed according to the kit instructions, adjusting the cell concentration to 2.5 × 10⁻⁶. 5 Cell suspension was added to microplate wells pre-coated with anti-IFN-γ antibody or TNF-α at a concentration of / mL. T cells were then added and stimulated with the antigen. After incubation at 37°C and 5% CO2 for 16–20 hours, the cells were removed. Biotin-labeled detection antibody was added sequentially; then enzyme-labeled streptavidin was added; and finally, a chromogenic substrate was added to initiate an enzymatic reaction, forming insoluble colored spots at the original locations of the secreting cells. The spots were analyzed using the ELISPOT enzyme-linked spot analysis system to obtain the results. Each colored spot corresponds to an active cell that secreted the target protein during culture, and the spot count directly reflects the frequency of functional secreting cells in the sample.

[0065] The results are shown in Table 2.

[0066] Table 2 Results of T cell activation detection .

[0067] Tumor cell mortality rate in PDO: CellTox Green dye assay: In Example 2, after co-culturing for 72 hours, CellTox™ Green dye was added to detect cytotoxicity, followed by CellTiter-Glo® reagent in the same well to determine cell viability. Figure 4 The number of dead / live PDO cells was quantitatively counted using a high-content imaging system (such as the PerkinElmer Opera Phenix).

[0068] Tumor killing assay – Annexin V-EGFP-PI method: Prepare tumor cell suspensions from co-cultured tumor organoids and count the cells; seed tumor cell suspensions into 96-well plates, and resuspend the cells in 100 μL of 1×Binding Buffer. Add 5 μL Annexin V-FITC and 10 μL PI Staining Solution, and mix gently. Incubate in the dark at room temperature for 10-15 min.

[0069] Add 400 μL of 1× Binding Buffer, mix well, and place on ice. Analyze using flow cytometry or fluorescence microscopy within 1 hour. Analyze using software such as CellQuest, and plot a two-color dot plot with FITC on the x-axis and PI on the y-axis. In a typical experiment, cells can be divided into three subpopulations: viable cells show only very low background fluorescence, early apoptotic cells show only strong green fluorescence, and late apoptotic cells show dual staining with both green and red fluorescence. Figure 5 (and Table 3).

[0070] Table 3. Results of tumor killing detection .

[0071] CTL wetting depth: Method 1: Tumor cell suspensions were prepared from tumor organoids co-cultured for 72 hours, and T cells were detected by flow cytometry to determine the number and types of T cells in the tumor organoids after co-culture (Table 4).

[0072] Table 4. Number and types of T cells in tumor organoids after co-culture. .

[0073] Method 2: Z-axis confocal microscopy scanning. The PDO-T cell three-dimensional co-culture system from Example 2 was used, and the entire PDO-containing three-dimensional tissue was fixed in 4% paraformaldehyde (PFA) at room temperature for 30 minutes. The following fluorescent antibodies were used for staining: anti-CD8-PE, DAPI, and anti-EpCAM-Alexa Fluor 647. Z-axis confocal microscopy was performed with a step size of 5 μm and a total depth of 100-400 μm using Imaris or FIJI software to measure the maximum depth (μm) of CTL infiltration into the PDO and / or the number of infiltrating CTLs / mm³.

[0074] ELISPOT Cytokine Profiling (Multiple Detection): Method 1: ELISPOT method. The supernatant from PDO-T cells co-cultured in Example 2 for 72 hours was used to detect cytokines IFN-γ and TNF-α at 24h, 48h, and 72h (Table 5). Table 5. Results of ELISPOT assay for cytokine profiles .

[0075] Method 2: ELISA method to detect cytokines such as TNF-α and γ-IFN using a kit.

[0076] T cell phenotype analysis after co-culture: In Example 2, after PDO-T cells were co-cultured for 72 hours, T cells were recovered from the liquid layer above the co-culture wells and analyzed by flow cytometry (Table 6).

[0077] Table 6 Results of T cell phenotypic analysis .

[0078] DVS scoring framework: The DVS is a weighted sum of 6 indicators, with a total score of 10 (Table 7): Table 7 DVS Scoring Framework .

[0079] DVS determination criteria and DC vaccine classification (Table 8): Table 8 DVS Judgment Criteria and DC Vaccine Grading Criteria .

[0080] The above analysis, using six indicators—DC cell maturation phenotype and tumor-specific antigen presentation efficiency detection, tetramer and ELISPOT method detection of DC cell activation into tumor-killing T cells, activated T cell PDO killing rate of tumor organoids, depth of immune cell infiltration in tumor organoids after co-culture of tumor organoids and T cells, detection of functional cytokine secretion after T cell activation, and T cell phenotype analysis and activation rate detection—to comprehensively evaluate the efficacy of tumor-specific DC vaccines from multiple levels, including the mechanism of action of DC cells, immune activation, and direct tumor-killing efficacy. This establishes a method for evaluating the effectiveness of tumor-specific DC vaccines based on organoid-T cell co-culture systems during the development of tumor-specific DC vaccines.

[0081] Different scores and weights were determined based on the role of each indicator in evaluating the effectiveness of tumor-specific DC vaccines. The DVS score can predict the clinical response rate. If the predicted clinical response rate is low, the strategy can be adjusted and the design or optimization can be carried out during the research and development stage.

[0082] This evaluation system has been validated in ongoing studies of tumor-specific DC vaccines for triple-negative breast cancer. Designs with higher overall scores showed significantly better clinical efficacy in triple-negative breast cancer patients compared to designs with relatively lower overall scores. It has also been partially validated in studies of tumor-specific mRNA vaccines for squamous cell carcinoma of the lung. This indicates that comprehensive evaluation using this system in the early stages of tumor-specific DC vaccine development is crucial for early development decisions and later clinical trial predictions.

[0083] Example 4: In vivo validation in a dual-humanized animal model An immune reconstitution mouse model was established using patient-derived PBMCs or PBMCs from healthy volunteers with the same HLA subtype as the patient. Severely immunodeficient mice were used as hosts, and PBMCs were implanted into the mice via tail vein injection or intraperitoneal injection. Following PBMC transplantation, the reconstituted immune cells were predominantly human T cells. These cells were detectable in the peripheral blood of the mice approximately four weeks post-transplantation. Patient-derived tumor cells from Example 1 were then subcutaneously inoculated.

[0084] Inoculation with patient-derived, antigen-loaded dendritic cells (DCs) as described in Example 2, via subcutaneous or intralymphatic injection (1-5 × 10⁻⁶ DCs). 5 (cells / animal). Vaccination frequency: 3 times on days 0, 7, and 14.

[0085] Sample collection: Blood samples were collected before DC cell inoculation and on days 7, 14, and 21 after inoculation to separate PBMCs for testing.

[0086] On day 21, tumor tissue was removed, and after measurement and photography, tissue dissection was performed immediately. Tumor cells and immune cells were detected using the method described in Example 3.

[0087] result: Tumor tissue growth after inoculation Figure 6 As shown.

[0088] Photos of tumor tissue after DC vaccine administration, as shown Figure 7 As shown.

[0089] Table 9 shows the apoptosis of tumor cells in tumor tissue after DC vaccination.

[0090] Table 9. Statistics on tumor cell apoptosis in tumor tissues after DC vaccine administration. .

[0091] The proportions of immune cells in tumor tissue are shown in Table 10.

[0092] Table 10. Statistics on the proportion of immune cells in tumor tissue .

[0093] The proportions of immune cells in peripheral blood are shown in Table 11.

[0094] Table 11. Statistics on the proportion of immune cells in peripheral blood .

[0095] The results of peripheral blood cytokine detection are shown in Table 12.

[0096] Table 12 Results of Cytokine Detection in Peripheral Blood .

[0097] The results above indicate that: by establishing an immune-reconstituted mouse model using patient-derived PBMCs or PBMCs from healthy volunteers with the same HLA subtype, subcutaneous inoculation with the same patient-derived tumor cells can successfully construct an animal model for in vivo evaluation of DC vaccine efficacy. Subcutaneous or intralymphatic injection of patient-derived DC cells loaded with tumor-specific antigens showed a specific killing effect on tumor tissue, with significant tumor volume reduction and varying degrees of apoptosis in the tumor cells. In the model control group (not inoculated with antigen-loaded DC cells, results not listed), no tumor volume reduction was observed, and tumor cell apoptosis was minimal.

[0098] Following inoculation with patient-derived dendritic cells (DCs) loaded with tumor-specific antigens, mice developed a specific immune response. Different immune cells, including activated cytotoxic T cells, were detected in the tumor tissue. Cytokines secreted by tumor-specific antigen-loaded immune cells and activated T cells were also detected in the peripheral blood. In contrast, mice in the model control group (not inoculated with antigen-loaded DCs, results not shown) showed very few activated cytotoxic T cells in their tumor tissue, no tumor-specific antigen-loaded immune cells were detected in their peripheral blood, and very few cytokines were secreted by activated T cells. These results indicate that tumor-specific antigen-loaded DCs specifically activate the immune system in vivo, producing immune cells with specific cytotoxic effects capable of specifically killing tumor tissue.

Claims

1. A method for detecting the effectiveness of tumor-specific DC vaccines based on a co-culture system, characterized in that, The method includes: S1. Preparation of tumor organoids derived from patients; S2. Prepare tumor-specific DC vaccines; prepare patient-derived T cells; co-culture and activate T cells with tumor-specific DC vaccines to obtain T cells with tumor-specific killing effects; S3. Co-culturing the tumor organoids obtained in step S1 with the activated T cells obtained in step S2 allows for the observation of lymphocyte infiltration and killing effect on tumor cells. S4, DC vaccine efficacy evaluation.

2. The method according to claim 1, characterized in that, Step S4 includes: (A) detection of DC cell phenotype and presentation efficiency in DC vaccine; (B) detection of T cell activation using tetramer and enzyme-linked spot analysis system; (C) detection of tumor cell mortality in organoids; (D) detection of cytotoxic T lymphocyte infiltration depth; (E) detection of cytokine profile in the supernatant of the co-culture system in step S2; and (F) detection of cytotoxic T lymphocyte activation rate in the supernatant of the co-culture system in step S2.

3. The method according to claim 2, characterized in that, Step S4 includes a weighted scoring of the detection results of (A)-(F).

4. The method according to claim 3, characterized in that, The total score for the weighted scoring steps is 10 points, with the full scores for steps (A)-(F) being 1.5, 2, 3, 1.5, 1, and 1 points respectively.

5. The method according to claim 2, characterized in that, Tumor cell mortality in organoids was detected using CellTox™ Green staining or the Annexin V-EGFP-PI method.

6. The method according to claim 2, characterized in that, The depth of cytotoxic T lymphocyte infiltration was detected using confocal microscopy Z-axis scanning.

7. The method according to claim 2, characterized in that, The detection step S2 involves detecting the cytokine profile in the supernatant of the co-culture system, including the detection of TNF-α and γ-IFN.

8. The method according to claim 1, characterized in that, The incubation time in step S3 is 60-84 hours.

9. The method according to claim 1, characterized in that, The preparation process of tumor-specific DC vaccines includes: preparing patient-derived DC cells; sensitizing DC cells with tumor-specific antigen peptides or tumor tissue lysates to obtain tumor-specific DC vaccines.

10. The method according to claim 2, characterized in that, Tetramer assays were used to detect tumor-specific antigenic peptides on activated T cells, and an enzyme-linked dot assay system was used to detect the positive counts of TNF-α and γ-IFN secreted by activated T cells.

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