Iterative radiation-resistant tumor cell debris as well as preparation method and application thereof in tumor resistance

Abstract

The invention belongs to the technical field of cancer immunotherapy, and particularly relates to iterative radiation-resistant tumor cell debris, a preparation method thereof and an application thereof in tumor resistance. The anti-radiation tumor cells generated by repeated radiation exposure show higher TMB (Tetramethylbenzidine) and induce stronger immunoreaction, so that a personalized vaccine is developed based on the method, and the frozen fragment vaccine is obtained by freeze thawing and inactivation of the radiation-resistant tumor cells. The RR-FDV exhibits enhanced immunogenicity as compared to a conventional radiosensitive counterpart. In the present disclosure, RR-FDV exhibits superior activation of antigen presenting cells, while in vivo, it recruits more dendritic cells to the vaccination site, promotes DC-mediated antigen presentation, and induces stronger antigen-specific T cell toxicity.

Description

Technical Field

[0001] The present invention belongs to the technical field of cancer immunotherapy, and in particular relates to iterative radiation-resistant tumor cell fragments, a preparation method thereof, and an application thereof in anti-tumor treatment. Background Art

[0002] The advent of cancer immunotherapy has reshaped the treatment paradigm through immune checkpoint blockade (ICB) and T cell-mediated adoptive therapy. In this revolution, tumor vaccines have emerged as precision instruments capable of mobilizing host immunity against malignant tumors by presenting tumor-associated antigens. Effective vaccination depends on two stages: sufficient delivery of immunogenic antigens to activated antigen-presenting cells (APCs), followed by robust priming of tumor-specific T cell clones. At the heart of this process is the immunogenic quality of the vaccine antigen, a key prerequisite for eliciting an immune response, which requires innovative solutions given the suboptimal performance of current platforms.

[0003] In the era of cancer immunotherapy, tumor mutation burden (TMB) has attracted widespread attention due to its correlation with treatment efficacy. Highly mutated malignancies, such as melanoma, show enhanced CD8 + T cell infiltration and superior response to checkpoint inhibitors. TMB is an evolving biomarker for selecting patients for immunotherapy in multiple cancer types, including non-small cell lung cancer, head and neck squamous cell carcinoma, and carcinomas. Mechanistically, somatic mutations generate neoantigens—tumor-specific epitopes that bypass central tolerance, promote immune recognition, and activate T cell responses. These mutation-derived peptides are absent in healthy tissues and are ideal targets for vaccine design. Many attempts have been made to identify neoantigens for vaccine production. However, the heterogeneity of antigens between patients and the high cost of personalized epitope identification have hindered the translation of predefined antigen tumor vaccines from bench to bedside.

[0004] To bridge the gap between personalized immunotherapy and clinical feasibility, a series of efforts have driven the development of whole tumor cell vaccines, which use inactivated tumor cells to exploit the complete tumor antigen spectrum without the need for epitope preselection. Although this approach circumvents the antigen recognition barrier, traditional tumor cell vaccines often fail to fully activate APCs or expand tumor-infiltrating T cell clones, resulting in poor clinical efficacy. Therefore, enhancing the immunogenicity of tumor cells to unleash their full potential is an urgent unmet need.

[0005] Radiation therapy, a cornerstone of traditional cancer treatment, may have unexpected potential to revitalize the design of tumor cell vaccines. Clinical studies have reported enhanced immunotherapy responsiveness following radiotherapy. In an attempt to explain this phenomenon, preclinical models have shown that irradiated tumors exhibit broadened T cell receptor clonality. These observations have drawn attention to the potential for radiation-induced neoantigens. By inducing DNA damage, radiation leaves an imprint on the genome of exposed cells, leading to an increased mutational load. This therapeutic "mutatoxicity" enables the de novo generation of tumor-specific neoantigens. This paradigm shift positions radiation not only as a cytotoxic modality but also as a catalyst for diversifying the antigenic repertoire, motivating our efforts to utilize radiation as a unique tool for engineering antigen-rich tumor cell vaccines.

[0006] The clinical translation of whole-tumor cell vaccines remains hampered by insufficient immunogenicity. Although studies have been conducted on radiation-resistant tumor vaccines, the clinical translation of conventional anti-tumor vaccines remains hampered by immunogenicity barriers. In the era of immunotherapy, the synergistic effects and related mechanisms between radiotherapy and immunotherapy have attracted widespread attention. Evidence suggests that radiotherapy plays a crucial role in anti-tumor immune regulation. Radiation produces an in situ vaccine effect, particularly by triggering DNA damage-induced tumor cell death, releasing tumor antigens, and by inducing immunogenic cell death, generating damage-associated molecular patterns (DAMPs). Furthermore, radiotherapy upregulates major histocompatibility complex class I (MHC-I) receptors and type I interferon, thereby enhancing cross-presentation of tumor antigens. However, few studies have considered that after irradiation, tumor cells can acquire new mutations that may serve as potent neoantigens, and even fewer have examined whether these new mutations in potent neoantigens can play a role in tumor suppression. Summary of the Invention

[0007] The present invention provides iterative radiation-resistant tumor cell fragments and their preparation method and application in anti-tumor, mainly to solve the problem that the clinical transformation of current common anti-tumor vaccines is still limited by insufficient immunogenicity.

[0008] In order to solve the above problems, the present invention adopts the following technical solutions:

[0009] The first aspect provides Radioresistant tumor cell fragments and their application in the preparation of antitumor vaccines.

[0010] Radioresistant tumor cell fragments are primarily prepared by the following method: Primary tumor cells are subjected to several radiation exposure and passage cycles at doses not exceeding 6±0.6 Gy, followed by at least one screening step using high-dose radiation of no more than 10±1 Gy. The term "±0.6 Gy" is used as an example to indicate that actual values ​​may deviate from the measured value due to instrumentation and other factors. This indicates that a certain range of deviation is permitted and the actual value is still used as a reference, but this deviation should be within the scope of the present invention.

[0011] Regarding radiation exposure and subculture cycles, each cycle requires irradiation and subculture. Subculture is typically performed after radiation exposure to a dose not exceeding 6 Gy. Screening can be performed before, during, or after the radiation exposure and subculture cycles, using high-dose irradiation of no more than 10 Gy.

[0012] During the preparation process, the preparation conditions may be further limited according to the circumstances, and any feature of any condition may be selected separately: 1) More specifically, obtaining in vitro primary tumor cells, subjecting the primary tumor cells to X-ray irradiation for primary screening, subjecting them to multiple cycles of X-ray irradiation and subculture, and then subjecting them to X-ray irradiation for secondary screening to obtain radiation-resistant tumor cell fragments; 2) 6 cycles are used when performing multiple cycles of X-ray irradiation and subculture; 3) during screening, the radiation exposure is 6Gy < dose ≤ 10Gy, and the radiation exposure during radiation exposure and subculture is 4Gy < dose ≤ 6Gy, but if The critical point has a certain deviation range, or malicious adjustments that have similar effects to the present invention should all be within the scope of the present invention; the in vitro primary tumor cells are obtained by culturing primary tumor cells from in vitro tumor tissue; further, the following can be adopted: the primary tumor cells are subjected to X-ray irradiation for primary screening and X-ray irradiation for secondary screening with an X-ray energy of 6MV, fresh culture medium is replaced after irradiation, and clonal cell lines are obtained after screening 5 to 7 days; the primary tumor cells are subjected to X-ray irradiation for primary screening and then subjected to multiple cycles of X-ray irradiation and subculture with an X-ray energy of 6MV, and subculture is carried out 48 hours after irradiation.

[0013] X-ray irradiation is a common method. Of course, if other irradiation methods can achieve similar effects to those of the present invention, they should also be within the scope of the present invention.

[0014] Regarding the application of these radioresistant tumor cell fragments, they can be used to treat or prevent tumors. A specific application can be the preparation of anti-tumor vaccines, which can prevent the development of tumors. Furthermore, the anti-tumor vaccine can have the following characteristics: 1) The anti-tumor vaccine is an anti-solid tumor vaccine, and more specifically, the anti-tumor vaccine is an anti-lung adenocarcinoma vaccine, a head and neck squamous cell carcinoma vaccine, or a melanoma vaccine. Vaccines for other types of solid tumors are also within the scope of this invention; 2) The anti-tumor vaccine is an injectable vaccine.

[0015] As for the preparation of the vaccine with radiation-resistant tumor cell fragments, the following specific application example is used, which includes the following steps: 1) obtaining ex vivo tumor tissue for primary tumor cell culture, and obtaining primary tumor cells; 2) subjecting the primary tumor cells and culture fluid obtained in step 1) to in vitro 10Gy X-ray initial irradiation screening; 3) subjecting the surviving cells obtained in step 2) to 6Gy X-ray irradiation induction per cycle and subcultured, recorded as P1; 4) repeating step 3) to obtain tumor cells P10 that have been irradiated and subcultured for 10 cycles with a cumulative 70Gy X-ray irradiation; 5) subjecting the high-dose irradiated tumor cells P10 obtained in step 4) to a secondary screening with 10Gy X-ray irradiation to obtain radiation-resistant tumor cells RR; 6) subjecting the radiation-resistant tumor cells obtained in step 5) to repeated freezing and thawing three times to prepare the radiation-resistant tumor cell fragment vaccine RR FDV.

[0016] As for the anti-tumor vaccine, it can be a tumor prevention vaccine or a tumor treatment vaccine, and can play a corresponding role when any of the aforementioned radioresistant tumor cell fragments is used as an effective component.

[0017] The second aspect provides Use of the radioresistant tumor cell fragments in any of the aforementioned scenarios in combination with Freund's adjuvant in the preparation of an anti-solid tumor vaccine. The radioresistant tumor cell fragments are prepared from primary tumor cells that have undergone several radiation exposure and passage cycles with doses not exceeding 6±0.6Gy and have been screened at least once with high-dose radiation not exceeding 10±1Gy. In this application, the preparation conditions may be further limited as appropriate, and any feature within any condition may be selected independently:

[0018] Among them, the anti-solid tumor vaccine is at least one of the anti-lung adenocarcinoma vaccine, the head and neck squamous cell carcinoma vaccine, or the melanoma vaccine. Of course, other similar types of vaccines should also be included.

[0019] Second, the combination of radioresistant tumor cell fragments and Freund's adjuvant significantly enhanced the vaccine's effectiveness. This combination significantly enhanced the tumor-suppressing effect compared to radioresistant tumor cell fragments alone. While the immune adjuvant itself has no tumor-suppressing effect, combining it with radioresistant tumor cell fragments significantly improved the tumor-suppressing effect.

[0020] Third, Freund's adjuvants are generally FDA-approved adjuvants, which can significantly improve the effectiveness of vaccines. Of course, other types of Freund's adjuvants are not excluded.

[0021] As for the anti-solid tumor vaccine, it can be a tumor prevention vaccine or a tumor treatment vaccine. When any of the aforementioned radioresistant tumor cell fragments is used as an effective component, it can play a corresponding role and can inhibit the growth of tumor cells.

[0022] The third aspect provides a vaccine against solid tumors containing radioresistant tumor cell fragments and a method for preparing the same. The radioresistant tumor cell fragments are prepared from primary tumor cells that have undergone several radiation exposure and passage cycles with a dose not exceeding 6±0.6 Gy and have been screened at least once with a high-dose radiation dose not exceeding 10±1 Gy.

[0023] The anti-solid tumor vaccine is the aforementioned radioresistant tumor cell fragments in any of the aforementioned situations that have been subjected to multiple freeze-thaw cycles. Furthermore, to enhance the vaccine's efficacy, the vaccine also includes a Freund's adjuvant. The anti-tumor vaccine is an anti-solid tumor vaccine, preferably a lung adenocarcinoma vaccine, a head and neck squamous cell carcinoma vaccine, or a melanoma vaccine.

[0024] As for the preparation method of anti-solid tumor vaccine, the radioresistant tumor cell fragments in any of the above situations are inactivated by several freeze-thaw cycles to prepare the vaccine. The tumorigenicity is eliminated by freeze-thaw inactivation, while the tumor antigen spectrum is completely retained. Compared with conventional radiosensitive vaccines, this RR-FDV vaccine exhibits a stronger ability to activate antigen-presenting cells in vitro. In vivo, it can effectively recruit dendritic cells to the vaccination site, and the presented new antigens deliver co-stimulatory signals to T cells, thereby subsequently activating antigen-specific cytotoxic T lymphocyte responses. Animal experiments further confirmed that RR-FDV can not only induce long-lasting immune memory, but also reshape the immunosuppressive tumor microenvironment, significantly inhibit tumor growth, and prolong the survival time of tumor-bearing mice. In this preparation method, the preparation conditions can be further limited according to the circumstances, and any feature in any condition can be selected separately: 1) the anti-tumor vaccine is an anti-solid tumor vaccine, preferably an anti-lung adenocarcinoma vaccine or a head and neck squamous cell carcinoma vaccine or a melanoma vaccine, and the anti-tumor vaccine can be used to prevent the occurrence of lung adenocarcinoma or head and neck squamous cell carcinoma or melanoma; 2) Freund's adjuvant is added during vaccine preparation; 3) the vaccine is an intravenous vaccine.

[0025] Repeated radiation exposure can cultivate radioresistant tumor cells with higher TMB. Based on this discovery, a personalized vaccine was developed by cyro-pulsing radioresistant tumor cells in liquid nitrogen and then thawing them at room temperature to obtain a frozen fragment vaccine (RR-FDV). RR-FDV can effectively activate APCs and promote the activity of cytotoxic T lymphocytes both in vitro and in vivo. Therapeutic administration of RR-FDV can induce long-term antitumor immunity, reshape the immunosuppressive microenvironment, and prolong survival.

[0026] In this study, cumulative X-ray irradiation can induce tumor cells to produce hypermutated complexes and neoantigens, while tumor cell fragments retain a rich tumor antigen repertoire and immunogenicity to a large extent. Using radiation-resistant tumor cell fragments as a tumor vaccine can more effectively activate immunity, thereby achieving the prevention and treatment of tumors. This study uses radiation as a scalable strategy to overcome the immunogenicity limitations of autologous cancer vaccines, providing a clinically feasible model for personalized immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The construction process and corresponding characteristics of RR FDV. (A) Schematic diagram of the establishment of a radiation-resistant cell line; (B) Cell viability test of LLC-OVA-RS and LLC-OVA-RR at different radiation doses (n = 5); (C-D) WES bioinformatics analysis of TMB and SNPs, normalized to the RS cell line (n = 3); (E) Schematic diagram of the FDV preparation workflow.

[0028] Figure 2 (A) Schematic diagram of the SCC7 tumor vaccine treatment protocol. C3H mice were intradermally immunized with SCC7-RS FDV (n=8), SCC7-RR FDV (n=8), or PBS (n=7) on days -15, -12, and -9, and then immunized with 10×10 5 SCC7 tumor cells were inoculated subcutaneously in the flank; (B) Kaplan-Meier analysis of tumor-free mice from the corresponding treatment groups shown in (A); (C) Kaplan-Meier survival plot of the corresponding treatment groups described in (A); (D) C57BL / 6 mice were intradermally immunized with B16-OVA-RRFDV (n=8) or PBS (n=6) on days -15, -12, and -9, and then inoculated subcutaneously in the flank with 10×10 5 B16 tumor cells or LLC tumor cells, B16 tumors were dissected from the corresponding treatment groups; (E-F) are the tumor growth curves of B16 (E) and LLC (F) in the corresponding treatment groups shown in (D); (G) is a schematic diagram of the treatment plan and analysis of the re-challenge SCC7 subcutaneous tumor model (n=3); (H) is the percentage of tumor-free mice in the corresponding treatment groups shown in (G); (I-J) are flow cytometric analysis and quantitative statistics of memory T cells in the lymph nodes of mice treated in (G), CD4 + T cells (I) and CD8 + The proportion of memory cells in T cells (J) and representative scatter plots.

[0029] Figure 3 RR-FDV activates APC in vitro and in vivo. (A) GSEA pathway enrichment analysis of RR and PBS; (B-E) GSEA pathway features enriched in RR and RS; (F) RT-qPCR analysis of activation markers in DCs treated with PBS, RS FDV, or RR FDV, normalized to PBS (n=3); (G-H) Flow cytometric analysis of CD86 and CD80 expression in BMDCs (G) and BMDMs (H) treated with PBS, LLC-OVA-RS FDV, or LLC-OVA-RR FDV (n=3), MFI, mean fluorescence intensity; (I) Schematic diagram and analysis of the LLC vaccine immunization experimental treatment plan (n=4); (J) Flow cytometric gating strategy for detecting dendritic cells at the inoculation site of mice treated in (I); (K-O) Flow cytometric analysis of the percentage of DCs in the inoculation site of mice treated in (I), CD86 (L), CD80 (M), and MHC on DCs (K). II(N) and SIINFEKL(O) expression.

[0030] Figure 4 Figure 2 shows immune analysis of lymph nodes and spleen after vaccination. (A-D) Flow cytometric analysis of CD80 (A), CD86 (B), MHC II (C), and SIINFEKL (D) expression on DCs in lymph nodes of mice 48 hours after treatment with B16-OVA-RS FDV or B16-OVA-RR FDV (n=4); (E-G) Flow cytometric analysis of MHC II (E), SIINFEKL (F), and CCR7 (G) expression on DCs in lymph nodes 120 hours after treatment with B16-OVA-RS FDV or B16-OVA-RR FDV (n=4); (H-J) Flow cytometric analysis of CD86 expression on DCs in spleen of mice 48 hours after treatment with B16-OVA-RS FDV or B16-OVA-RR FDV + Flow cytometric analysis of T cell subsets, CD8 + Expression of CD69 on T cell subsets (H), CD8 + IFNγ + (I) and CD8 + B + (J) Representative dot plots and ratios of CTLs (n=4).

[0031] Figure 5 RR-FDV inhibits tumor growth and regulates TME. (A) Schematic diagram and analysis of the treatment plan for the B16-LUC subcutaneous tumor model; (B-C) B16-LUC tumor growth curve (B) and in vivo bioluminescence images (C) from the corresponding treatment groups shown in (A) (n=8); (D) Schematic diagram of the treatment plan and analysis of the LLC vaccine and adjuvant immunization experiment; (E-F) LLC tumor growth curve (E) and tumor weight (F) from the corresponding treatment groups described in (D) (n=6); (G-H) CD8 (G) and CD8 in each area (H) of LLC subcutaneous tumors + Representative images of T cell numbers, scale bar = 20 μm; (I-L) are flow cytometric analyses of T cell subsets in LLC tumors from mice in the corresponding treatment groups described in (D) (n = 6).

[0032] Figure 6Radiosensitivity, mutation level, and immunogenicity were examined. (A-B) Cell viability assays of SCC7-RS and SCC7-RR (A), B16-OVA-RS, and B16-OVA-CR (B) at different radiation doses (n=5); (C-D) Colony formation assays of B16-OVA-RS and B16-OVA-CR (C), LLC-OVA-RS, and LLC-OVA-RR (D) at different radiation doses (n=3); (E-F) WES bioinformatics analysis of Indel (E) and SV (F), normalized to RS cell line (n=3); (G) Representative flow cytometry dot plots of live cells and FDV; (H) SDS-PAGE and Coomassie blue staining of RS FDV and RR FDV proteins (n=3); (I) PCA score diagram of FDV proteomic analysis; (J) RS-FDV KEGG enrichment and RR-FDV proteomic analysis.

[0033] Figure 7 The application of RR-FDV in prevention models and memory T cell analysis. (A) Tumor growth curves for the corresponding treatment groups in the SCC7 tumor vaccine immunization experiment; (B-C) Tumor growth curves for the corresponding treatment groups in the B16 (B) or LLC (C) subcutaneous transplant models (n = 6-8); (D) Flow cytometry gating strategy for detecting memory T cells.

[0034] Figure 8 APCs internalize RR FDV and activate it. (A-B) Representative flow cytometry results and the ratio of phagocytic cells that internalize FDV in BMDCs (A) and BMDMs (B) (n = 3); (C-D) Flow cytometric analysis of CD86 and CD80 expression in BMDCs (C) and BMDMs (D) treated with PBS, SCC7-RS, SCC7-RR, SCC7-RS-FDV, or SCC7-RRFDV (n = 3); (E) Flow cytometric analysis of CD80 and CD86 expression in BMDMs treated with PBS, B16-OVA-RS FDV, or B16-OVA-RR FDV (n = 3).

[0035] Figure 9 Figure 2 shows the vaccine treatment plan and flow cytometric analysis of lymph node DCs and spleen T cells. (A) Schematic diagram of the B16 vaccine treatment plan and analysis (n=4); (B-C) Flow cytometric gating strategy for detecting DCs in the lymph nodes (B) and T cells in the spleen (C) of mice treated in (A); (D-E) CD4 T cells in the spleen of mice treated in (A) 48 hours later. + Flow cytometric analysis of T cell subsets, CD4 +Expression of CD69 on T cell subsets (D) and Th1 (CD4 + IFNγ + ) ratio (E); (F-G) is the ratio of CD4 in the spleen of mice treated in (A) after 120 hours. + Flow cytometric analysis of T cell subsets, CD4 + Expression of CD69 on T cell subsets (F) and Th1 (CD4 + IFNγ + ) ratio (G).

[0036] Figure 10 Figure 4: Efficacy and TME modulation of RR-FDV. (A) and (B) LLC tumor growth curves (A) and Kaplan-Meier survival plots (B) for the corresponding treatment groups in the vaccine immunization experiment (n = 8); (C) LLC tumors dissected from the corresponding treatment groups in the LLC vaccine adjuvant immunization experiment; (D) Flow cytometric gating strategy for detecting T cells in LLC subcutaneous tumors; (E) Flow cytometric analysis of T cell proportions. DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with specific research examples.

[0038] 1. Experimental Methods

[0039] 1.1 Cell Lines and Cell Culture: LLC mouse lung tumor cell line, SCC7 mouse head and neck squamous cell carcinoma cell line, and B16 mouse melanoma cell line were purchased from the American Type Culture Collection (ATCC). Cells were cultured in complete medium supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin solution (100 U / mL) at 37°C and 5% CO2. Dulbecco's modified Eagle's medium (DMEM) was used to maintain LLC and SCC7 cells, while RPMI 1640 medium was used to maintain B16 cells. LLC and B16 cell lines were transduced with the corresponding lentivirus and then selected with puromycin (2 μg / ml) to generate LLC-OVA, B16-OVA, and B16-LUC cell lines, which overexpress ovalbumin and luciferase, respectively. All cells were routinely tested for mycoplasma contamination.

[0040] 1.2 Radioresistant Cell Lines and Generation of RR-FDV: To establish radioresistant tumor cell lines, SCC7, LLC-OVA, and B16-OVA cells underwent repeated cycles of radiation exposure and passage, followed by high-dose radiation selection. Radioresistance was confirmed by cell viability and colony formation assays. RR cells were cell-shocked in liquid nitrogen and then thawed at room temperature to eliminate tumorigenicity. RR-FDV was then subjected to freeze-thaw inactivation cycles to generate the RR-FDV.

[0041] 1.3 Cell Viability Assay: To measure cell viability, cells were seeded at a density of 2,000 cells per well in 96-well plates one day before treatment. Cells were then treated with radiation doses of 2, 4, 6, and 8 Gy. After a 72-hour incubation period, cell viability was assessed using a CCK-8 assay kit according to the manufacturer's instructions.

[0042] 1.4 Colony Formation Assay: Cells were seeded into six-well plates at 500 to 10,000 cells per well and exposed to various doses of radiation (2, 4, 6, and 8 Gy, respectively). Cells were maintained for 7 to 14 days until most individual colonies had more than 50 cells. Subsequently, cells were fixed with 4% formaldehyde for 30 minutes and stained with 2% crystal violet overnight. The stained cells were photographed, and the colony counts were recorded.

[0043] 1.5 4D-DIA quantitative proteomics: Total proteins were extracted from LLC-OVA RS FDV and LLC-OVA RR FDV. Briefly, proteins were incubated with DTT (5 mM) at 55°C for 30 minutes, iodoacetamide (10 mM) at room temperature for 15 minutes, and then incubated with 6 volumes of acetone (6 volumes) at -20°C for 4 hours. Protein precipitates were then obtained by centrifugation at 8000 g for 10 minutes. The protein precipitates were resuspended with NH4HCO3 (50 mM), digested with trypsin TPCK (1 mg / ml), and then lysed with SOLA. TM SPE 96-well plates were desalted. For DIA detection, peptides from all digested samples were separated using an EASY nLC 1200 liquid chromatograph and injected into a timsTOF Pro mass spectrometer (Bruker) for analysis. For quantitative and functional analysis, DIA raw data were analyzed using a Spectronaut Pulsar TM 18.4 (Biognosys, Switzerland). Differentially expressed proteins with P < 0.05 and FC > 1.2 or FC < 1 / 1.2 were considered significantly upregulated or downregulated, respectively. The Gene Ontology (GO) database was used to analyze the biological processes (BP), cellular components (CC), and molecular functions (MF) of the differentially expressed proteins based on their biological functions and classification.

[0044] 1.6 Generation of BMDMs and BMDCs: To generate BMDCs and BMDMs, bone marrow cells obtained from the femurs of C57BL / 6 mice were supplemented with granulocyte-macrophage colony-stimulating factor (GM-CSF, 20 ng / ml) or macrophage colony-stimulating factor (M-CSF, 20 ng / ml). Red blood cells (RBCs) were eliminated using erythrocyte lysis buffer. The culture medium was refreshed on days 3 and 5. Mature BMDCs and BMDMs were collected on day 7 for further experiments.

[0045] 1.7 In vitro cellular uptake assay: To quantitatively detect the internalization of FDV by APCs, BMDCs were stained with carboxyfluorescein diacetate succinimidyl ester (CFSE, 10 M) and seeded in 12-well plates (4 × 10 cells per well). 5 FDV was labeled with PKH26 red fluorescent cell membrane (PKH-26, 2×10 -6 M) Staining. APCs were incubated with PKH-26-labeled FDV for 3 or 24 hours and then collected for flow cytometric analysis.

[0046] 1.8RNA sequencing: Total RNA was extracted from FDV-treated DCs using TRIzol. DNA digestion was performed using DNaseI. Qubit2.0 Fluorometer was used to quantify RNA. To prepare the stranded RNA sequencing library, total RNA was processed using Illumina's KCTM stranded mRNA library preparation kit according to the manufacturer's protocol. The raw sequencing data were initially filtered using Trimmermatic to remove low-quality reads. The edgeR package was applied to identify genes differentially expressed between groups. The p-value threshold was set to 0.05 and the fold change threshold was set to 2 to determine the statistical significance of gene expression differences. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes were performed using KOBAS software. A p-value critical value of 0.05 was used to assess statistically significant enrichment.

[0047] 1.9 Real-time Quantitative PCR: Total RNA was extracted from FDV-treated DCs using TRIzol. The amount of RNA obtained was determined using a Thermo NanoDrop ND-1000. Purified RNA was reverse transcribed using reverse transcription reagents. RT-qPCR was performed on the Step One system using the ChamQ SYBR qPCR Master Mix. Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was used to normalize gene expression. Primer sequences are shown in Table S1.

[0048]

[0049] 1.10 In vitro flow cytometry analysis: To analyze the function of APC cells, 4 × 10 5 BMDM and BMDC cells were seeded in 12-well plates and treated with PBS, RR-FDV, and RS-FDV for 24 hours. APC cells were then collected and stained with F4 / 80, CD11c, CD86, and CD80 antibodies for 30 minutes at 4°C.

[0050] 1.11 T cell specific lysis assay: On day 15 after injection, CD8 T cells were isolated from the spleen of mice treated with LLC-OVA-RR FDV and LLC-OVA-RS FDV using the MojoSortTM Mouse CD8 T Cell Isolation Kit. + T cells. T cells were activated with CD3 / CD28 antibodies (5 μg / ml) for 4 hours and labeled with calcein AM (10 μM). Subsequently, calcein AM-labeled T cells were co-cultured with LLC or LLC-OVA cells and B16 or B16-OVA cells in 96-well plates at a specific efficacy target ratio of 50:1, 25:1 or 12.5:1 for 4 hours. The fluorescence intensity of the supernatant was measured using a fluorescence microplate reader with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Spontaneous release wells were set up using complete culture medium. Maximum release wells were set up using 2% Triton X-100 to achieve complete lysis. The specific lysis rate was calculated by (experimental release-spontaneous release) / (maximum release-spontaneous drug release)×100%.

[0051] 1.12 Model animal experiments and efficacy evaluation: C57BL / 6 and C3H mice were purchased from SLB Laboratory Animal Company (Hunan, China). All animals were housed in individually ventilated cages (IVC) at the Cancer Center of Wuhan Union Hospital. All animal studies were performed in accordance with the protocols approved by the Hubei Provincial Animal Care and Use Committee and in compliance with the experimental guidelines of the Animal Experiment Ethics Committee of Huazhong University of Science and Technology (Wuhan, China). In the animal experiments, mice aged 6 to 8 weeks were used. A subcutaneous tumor model was established, and tumor growth was monitored to evaluate the therapeutic effect. Before all surgeries, mice were anesthetized with 1% sodium pentobarbital. The length (L) and width (W) of the tumor were measured every two days using a vernier caliper, and the tumor volume (V) was calculated according to the formula V = (L*W2) / 2. The observation endpoint was when the tumor volume reached 1000mm. 3 For tumor vaccine immunization experiments, PBS or different vaccines were injected intradermally in the tail root area on days -15, -12, and -9. Then, on day 0, 1×10 6 In the vaccine therapy study, B16-LUC and LLC cells were injected subcutaneously into the right flank of mice. 3Mice were randomly divided into corresponding groups. On days 11, 12, and 13 after tumor inoculation, RR-FDV, RS-FDV, or PBS were administered.

[0052] 1.13 Bioluminescence Imaging: Mice were anesthetized and intraperitoneally injected with 150 mg / kg of D-luciferin sodium. Ten to 15 minutes later, the mice were imaged using a Bruker In Vivo MS FX PRO imager. The exposure time for luminescence images was set to 3 minutes, while the exposure time for X-ray images was 30 seconds.

[0053] 1.14 Immunofluorescence: Cells were fixed with 4% paraformaldehyde for 20 minutes, permeabilized with 1% Triton X-100 for 10 minutes, and then blocked with 5% BSA for 1 hour at room temperature. Subsequently, cells were incubated with mouse CD8 antibody overnight at 4°C for CD8. + T cells were stained. Cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Images were captured using a fluorescence microscope (Olympus FV3000).

[0054] 1.15 In vivo flow cytometric analysis: To analyze immune cells at the inoculation site and peripheral immune organs, inoculated nodules, lymph nodes, and spleens from different treatment groups were collected, minced, and filtered to obtain single-cell suspensions. To analyze immune cells in the TME, subcutaneous tumors from different treatment groups were harvested, minced, digested with collagenase and hyaluronidase, and then filtered to obtain single-cell suspensions. RBC lysis buffer was used to remove RBCs. To exclude dead cells, single-cell suspensions were stained with a zombie purple fixative activity kit. To analyze DCs, single-cell suspensions were stained with CD45, CD11c, CD86, CD80, MHC II, CCR7, and SIINFEKL-H-2Kb antibodies for flow cytometric analysis. To analyze T cells, cell suspensions were stimulated with phorbol 12-myristate 13-acetate (100 ng / mL), monensin sodium salt (1.5 mg / mL), and ionomycin calcium salt (1 mg / mL) for 6 hours. Single-cell suspensions were then stained with antibodies against CD45, CD3, CD4, CD8, CD44, CD62L, CD69, and SIINFEKL-h-2Kb. Cells were then fixed and permeabilized before staining with antibodies against IFNγ, GrzmB, and FoxP3 for flow cytometric analysis.

[0055] 1.16 Statistical Analysis: Statistical analyses were performed using Prism software. Comparisons between groups were assessed using one-way analysis of variance (ANOVA) with Student's t-test (two-sided) and Tukey's multiple comparison test. Quantitative data are presented as mean ± SEM. Statistical significance was set at a threshold of P < 0.05, with P < 0.05 indicated as *, P < 0.01 as **, and P < 0.001 as ***. NS indicates not significant.

[0056] 2. Experimental Results

[0057] 2.1 Generation and characterization of radiation-resistant FDV

[0058] To establish radioresistant tumor cell lines, three different tumor cell models were used. The mouse head and neck squamous cell carcinoma line SCC7, as well as two modified cell lines expressing ovalbumin (OVA, a well-characterized epitope for cancer vaccine efficacy evaluation)—mouse Lewis lung carcinoma (LLC-OVA) and mouse melanoma (B16-OVA)—underwent repeated cycles of radiation exposure and subculture, followed by high-dose radiation selection ( Figure 1 Cells irradiated with a cumulative 70 Gy (SCC7-RR and LLC-OVA-RR) and 80 Gy (B16-OVA-RR) showed radioresistance, as confirmed by cell survival and colony formation assays ( Figure 1 Middle B and Figure 6 Whole exome sequencing (WES) of LLC-OVA-RR and parental radiosensitive cells (LLC-OVA-RS) showed that TMB was significantly increased in the resistant cell lines ( Figure 1 As TMB increased, single nucleotide polymorphism (SNP) counts also increased ( Figure 1 Insertion / deletion (Indel) and structural variation (SV) frequencies remained almost unchanged ( Figure 6 These mutational signatures suggest the accumulation of neoantigens during radiation adaptation.

[0059] To exploit radiation-induced TMB for vaccine development, tumor cells were cell-shocked in liquid nitrogen and then thawed at room temperature to eliminate tumorigenicity ( Figure 1 Middle E). Frozen fragment vaccines (FDV) produced by freeze-thaw inactivation cycles lose their proliferation capacity and normal cell size, but maintain the integrity of intracellular components and proteins ( Figure 1 Medium F and Figure 6 To further characterize their protein properties, quantitative proteomics studies were performed on LLC-OVA-RS FDV and LLC-OVA-RR FDV. RS FDV and RR FDV showed different protein profiles ( Figure 6 RR-FDV upregulated proteins involved in innate immune responses, interferon-β signaling, and cytokine-mediated pathways, many of which had immunogenic properties and were associated with antitumor immune responses ( Figure 1 KEGG enrichment analysis further confirmed the activation of RIG-I and NOD signaling pathways ( Figure 6 These findings suggest that RR-FDV has the potential to initiate activation of innate immune cells, present vaccine antigens to T cells, and enhance immune activation.

[0060] RR-FDV induces tumor-specific immunity and immune memory

[0061] The upregulation of biological processes and key immune-related signaling pathways in RR-FDV suggests that they may have higher immunogenicity and enhance immune responses. To test this hypothesis, we evaluated the efficacy of RR-FDV in multiple prevention models. In the prevention experiment of head and neck squamous cell carcinoma in mice, mice were randomly assigned to receive intradermal injection of phosphate-buffered saline (PBS), SCC7-RS FDV, or SCC7-RR FDV ( Figure 2 Fifteen days after the initial vaccination, we injected SCC7 tumor cells subcutaneously and recorded the presence of measurable tumor nodules (tumor volume exceeding 50 mm 3 By day 25, only 1 of 8 mice in the SCC7-RR FDV-treated group had a tumor, while 5 of 8 mice in the SCC7-RS FDV-treated group had a tumor ( Figure 2 Furthermore, SCC7-RR FDV prolonged survival, whereas none of the tumor-bearing mice treated with SCC7-RS FDV or PBS survived beyond 40 days (tumor volume less than 1000 mm 3 )( Figure 2 Middle C and Figure 7 (A). SCC7-RR FDV showed superior preventive efficacy to SCC7-RS FDV, significantly inhibiting tumor formation and prolonging survival. We further implemented the same in vivo treatment plan using B16-OVA-RR FDV in B16 and LLC tumor animal models, respectively, to investigate whether the RR FDV vaccination regimen exhibited antigen specificity. Representative images showed that B16-OVA-RR FDV treatment had a significant growth inhibitory effect on B16 tumors, while all PBS-treated B16 tumor-bearing mice reached the endpoint within 20 days after vaccination ( Figure 2 D~E and Figure 7 In contrast, B16-OVA-RR FDV treatment had no preventive or therapeutic effect on LLC tumors ( Figure 2 Medium F and Figure 7Middle C), indicating that RR-FDV acts in an antigen-specific manner.

[0062] In preventive experiments using the SCC7 animal model, vaccination resulted in a significant number of mice with no detectable tumor formation before day 60: 3 of 8 mice in the SCC7-RS FDV-treated group and 7 of 8 mice in the SCC7-RR FDV-treated group. Next, we sought to explore whether these tumor-free mice had developed a memory response. Three mice each in the SCC7-RS FDV- and SCC7-RRFDV-treated groups were re-challenged with SCC7 cells ( Figure 2 None of the mice previously treated with SCC7-RR FDV developed tumors, whereas two weeks after re-inoculation of tumor-free mice, all mice previously treated with SCC7-RS FDV developed measurable tumors ( Figure 2 This suggests that SCC7-RR FDV triggered a stronger immune memory response. Immune memory is a hallmark of adaptive immunity. Immune memory against cancer is primarily mediated by memory T cells, which, after initial exposure to antigens, are able to generate faster and more effective responses upon re-exposure. Further analysis of memory T cells supported our hypothesis ( Figure 7 D), because CD4 + and CD8 + Effector memory T lymphocytes (TEM; CD44 + CD62Low) is higher, although the proportion of central memory T lymphocytes (Tcm; CD44 + There was no significant difference in CCD62Low Figure 2 Collectively, these results highlight the important role of RR-FDV in tumor prevention and inducing long-term antitumor immunity.

[0063] 2.3RR-FDV promotes APCs activation and antigen presentation at the vaccination site

[0064] Antigen presentation is the first critical step and prerequisite for initiating adaptive immune responses. We first used a flow cytometry-based phagocytosis assay to demonstrate that FDV can be rapidly internalized by APCs ( Figure 8To elucidate the mechanisms underlying the antitumor efficacy of RR-FDV, we next focused on the effects of FDV vaccination on dendritic cells (DCs) and macrophages, the primary and potent types of APCs. We performed transcriptome sequencing on bone marrow-derived dendritic cells (BMDCs) treated with PBS, LLC-OVA-RS FDV, or LLC-OVA-RR FDV. Gene set enrichment analysis (GSEA) pathway analysis indicated that RR-FDV could effectively activate DCs ( Figure 3 Compared with RS FDV treatment, key immune pathways, including inflammatory response, INFα response, INFγ response, and TNFα signaling, were more effectively triggered ( Figure 3 Real-time quantitative polymerase chain reaction (RT-qPCR) analysis confirmed the sequencing results. Genes related to DCs maturation, activation, and migration, such as Ifng, Il1b, Tnfa, and Ccr7, were significantly upregulated in RRFDV-treated DCs ( Figure 3 (F) The results showed that when APCs were co-cultured with LLC-OVA-RR FDV, a significant increase in the co-stimulatory signals CD80 and CD86 was observed compared to APCs treated with PBS or LLC-OVA-RS FDV. This indicates that APCs have acquired the ability to present antigens to T cells, provide the second signal required for T cell activation, and initiate immune responses ( Figure 3 Consistent results were obtained using SCC7 RR FDV or B16-OVA-RR FDV. RR FDV exhibited immune activation comparable to that of living cells and was far superior to its RS counterpart ( Figure 8 Middle C to E) indicate that the freeze-thaw vaccine preparation method does not impair the immune activation effect of radiation-resistant cells and does not affect protein expression.

[0065] Given the strong activation of APCs by RR-FDV in vitro, we further investigated whether DCs could be recruited and activated in vivo at the vaccination site, where they could present specific antigens to initiate anti-tumor adaptive immune responses. Figure 3 24 hours after vaccination, we collected the small nodules formed at the injection site and analyzed the immune response using flow cytometry ( Figure 3 Compared with LLC-OVA-RS FDV, LLC-OVA-RS FDV recruited more dendritic cells at the vaccination site ( Figure 3 The increased expression of CD86, CD80 and MHC II indicated the strong activation of DCs ( Figure 3L~N), while the significant increase of SIINFEKL-H-2Kb (OVA 257-264 peptide and MHC I complex) indicated that DCs antigen uptake and presentation were enhanced ( Figure 3 In summary, RR-FDV can effectively activate APCs both in vitro and in vivo, thereby increasing the recruitment and activation of DCs and improving antigen presentation.

[0066] 2.4RR-FDV enhances DC homing to peripheral immune organs and T cell priming

[0067] After demonstrating the encouraging local immune response elicited by RR-FDV at the vaccination site, we delved deeper into the immune characteristics of peripheral immune organs. First, we explored whether dendritic cells could localize lymph nodes and present antigens to T cells. Then, we investigated whether splenic T cells could effectively initiate an immune response against tumor antigens. Based on our previous in vivo studies, mice were intradermally injected with B16-OVA-RS FDV or B16-OVA-RR FDV. We then harvested peripheral immune organs and performed flow cytometric staining with CD80, CD86, MHC-II, SIINFEKL-H-2Kb, IFNγ, and GrzmB antibodies to monitor the activation status of DCs and T cells ( Figure 9 In lymph nodes, 48 ​​hours after injection, the expression of CD80 and CD86 in DCs and the presentation of ovalbumin peptide increased, with the increase being more pronounced with B16-OVA-RR FDV. This suggests that B16-OVA-RR FDV has a greater potential to activate DCs ( Figure 4 (A-D). CD11c + MHC II + The upregulation of cell ratio and the increase of SIINFEKL-H-2Kb expression indicated that the maturation and activation of DCs continued until day 5 after injection ( Figure 4 Higher levels of CCR7, a signaling molecule that directs DC migration to tumor-draining lymph nodes, were observed in the B16-OVA-RRFDV-treated group ( Figure 4 Furthermore, 48 hours after vaccination, B16-OVA-RRFDV led to a significant increase in CD69 (one of the earliest markers of activated lymphocytes) as well as IFNγ and GrzmB (classical intracellular effectors of cytotoxic T lymphocytes), indicating that splenic T cells had initiated an immune response ( Figure 4 (H-J) On day 5, quantitative analysis showed that CD8 + T cell activity exceeded that of the RS FDV-treated group, indicating that effector cells expanded significantly after vaccination ( Figure 4In CD4 + Similar immune activation trends were also observed in T cells ( Figure 9 Middle D~G).

[0068] After evaluating T cell activation and expansion, we directly assessed their cytotoxic efficacy. Splenic T cells from mice treated with LLC-OVA-RR FDV and LLC-OVA-RS FDV were harvested on day 15 after injection, activated with CD3 / CD28 antibodies, and subsequently tested for their effects on target cells, LLC or LLC-OVA cells ( Figure 4 N) and specific lysis of B16 or B16-OVA cells ( Figure 4 For the same effector T cells, the levels of lysis of LLC and LLC-OVA target cells were comparable, indicating that the T cells can recognize the tumor-associated antigen of LLC. However, when B16 target cells were examined, a significant difference emerged: cells expressing ovalbumin (OVA) and cells lacking OVA showed a clear difference, with B16 cells showing almost no lysis, reflecting the specific recognition of the OVA antigen by T cells ( Figure 4 In the cytotoxicity assay, T cells from LLC-OVA-RR FDV-treated mice showed enhanced cytotoxic activity against various target cells ( Figure 4 Combined with our in vivo flow cytometric assessment, these findings confirm that LLC-OVA-RR FDV promotes the migration of dendritic cells to lymph nodes and further activates T cells, laying the foundation for its application in anti-tumor immunity.

[0069] 2.5 RR-FDV-based immunotherapy inhibits tumor progression and reshapes the TME

[0070] RR-FDV can effectively activate DCs at the inoculation site, further promoting DCs homing in peripheral immune organs and T cell priming. To promote the clinical translation of RR-FDV, we studied the therapeutic effect of RR-FDV. Mice bearing B16-LUC tumors were randomly divided into three treatment groups: PBS, B16-OVA-RS FDV, and B16-OVA-RR FDV ( Figure 5 Compared with the other treatment groups, the bioluminescent signals of mice treated with B16-OVA-RRFDV on days 14 and 17 indicated a significant reduction in tumor growth ( Figure 5 Furthermore, in the LLC tumor model, RR-FDV treatment again outperformed other treatments, limiting tumor growth and prolonging survival ( Figure 10 (A~B).

[0071] To further enhance immunogenicity and boost immune responses for better tumor control, we included Freund's Adjuvant in our animal studies. LLC tumor-bearing mice received PBS, Freund's Adjuvant (sigma lot# SLCF1289), LLC-OVA-RR FDV, or a combination of LLC-OVA-RR FDV and adjuvant ( Figure 5 Combined therapy showed the most effective inhibition of tumor proliferation ( Figure 5 Middle E~F and Figure 10 The most prominent T cell infiltration further confirmed the strong therapeutic effect, as immunofluorescence staining showed that CD8 + T cells (red) were clearly present, exceeding the levels in other treatment groups ( Figure 5 (G~H).

[0072] To assess T cell function within the TME, we analyzed CD8 + T cell activity was quantitatively assessed ( Figure 10 Middle D). Combination therapy enhanced CD3 + Infiltration and accumulation of T cells, including cytotoxic T lymphocytes (CTL) and T helper type 1 cells (Th1) ( Figure 5 I~K and Figure 10 E), resulting in an increase in the number and activity of tumor-infiltrating T cells, consistent with our earlier observations of activated DCs and T cells in peripheral immune organs. In addition, the combination treatment reduced the percentage of regulatory T cells (Tregs), a known immunosuppressive component of the TME ( Figure 5 Together, these encouraging results from animal models reinforce the clinical potential of our RR-FDV as a personalized immunotherapy against cancer.

[0073] 3. Summary and Analysis

[0074] Radioresistant tumor cells generated by repeated radiation exposure exhibit higher TMB, which in turn elicits a more robust immune response. Here, we developed a personalized vaccine by freeze-thaw-inactivating radioresistant tumor cells to obtain a frozen fragment vaccine (RR-FDV). Compared with its traditional radiosensitive counterpart, RR-FDV exhibited enhanced immunogenicity. In vitro, RR-FDV demonstrated superior activation of antigen-presenting cells (APCs), while in vivo, it recruited more dendritic cells (DCs) to the vaccination site, promoted DC-mediated antigen presentation, and induced stronger antigen-specific T cell cytotoxicity. In mouse models, RR-FDV activated long-lasting anti-tumor immune memory, effectively reshaped the immunosuppressive tumor microenvironment, reduced tumor burden, and prolonged survival. Our results suggest that radiation-driven enrichment of neoantigens is a useful approach to overcome the immunogenicity limitations of autologous cancer vaccines and provide potential for the clinical translation of personalized immunotherapy.

[0075] Due to their low immunogenicity, whole tumor cell vaccines have long faced significant obstacles in their clinical application. In this study, we innovatively exploited the genotoxic effects of radiotherapy. Through repeated radiation screening, we obtained radioresistant tumor cells with a high tumor mutation burden (TMB). Based on this, we developed a personalized frozen fragment vaccine, the radiation-resistant frozen fragment vaccine (RR-FDV). Radiation-induced DNA damage significantly increased the diversity of tumor neoantigens. The freeze-thaw inactivation process eliminated tumorigenicity while fully preserving the tumor antigen spectrum. Compared with conventional radiosensitive vaccines, RR-FDV exhibited stronger antigen-presenting cell activation in vitro. In vivo, it effectively recruited dendritic cells to the vaccination site, where the presented neoantigens delivered co-stimulatory signals to T cells, subsequently activating antigen-specific cytotoxic T lymphocyte responses. Animal experiments further confirmed that RR-FDV not only induced long-lasting immune memory but also reshaped the immunosuppressive tumor microenvironment, significantly inhibiting tumor growth and prolonging the survival of tumor-bearing mice. This study combines radiobiology with vaccine engineering technology to overcome the immunogenicity bottleneck of traditional autologous tumor vaccines and provide a new strategy for personalized immunotherapy of cancer.

[0076] The RR-FDV vaccine is a personalized vaccine engineered through iterative radiation cycles to amplify tumor mutational burden and enrich for neoantigens, followed by freeze-thaw inactivation to ensure biosafety. RR-FDV exhibits potent immunogenicity in mouse models, driving DC activation, antigen cross-presentation, and cytotoxic T lymphocyte-mediated tumor eradication. Prophylactic vaccination with RR-FDV induced long-lasting immune memory, while therapeutic administration remodeled the immunosuppressive TME and significantly prolonged survival. These findings position radiation-driven neoantigen accumulation as a scalable strategy to overcome the limitations of autologous cancer vaccines.

[0077] In this study, WES confirmed that radiation-resistant cells established through high radiation dose accumulation exhibited increased TMB, indicating the accumulation of neoantigens. This finding is consistent with emerging evidence showing greater clonal diversity of T cell receptors after radiation. Our work repositions radiation therapy as a catalyst for the generation of immunogenic neoantigens and leverages this traditional cancer treatment approach as an innovative approach for the preparation of cancer vaccines.

[0078] The clinical potential of RR-FDV stems from its unique advantages. As an autologous tumor cell vaccine, it retains the complete antigenic profile of the original tumor, and this engineering enhances immunogenicity. Compared to unirradiated RS FDV, RR-FDV exhibits remarkable ability to recruit and activate APCs. It effectively triggers immune responses in peripheral immune organs and significantly enhances the specific anti-tumor function of T cells. In multiple animal models, RR-FDV, alone or in combination with adjuvants, has demonstrated superior prophylactic and therapeutic efficacy compared to conventional tumor cell vaccines. Furthermore, unlike vaccines with predefined antigens, RR-FDV eliminates the need for laborious neoantigen identification, reducing costs and accelerating the personalized treatment workflow. RR-FDV addresses the dual challenges of immunogenicity and convenience inherent in personalized tumor vaccines. These features make RR-FDV a practical platform for clinical translation, particularly in malignancies with low baseline TMB or resistance to checkpoint inhibitors.

[0079] In summary, this work establishes radiation as a transformative tool for engineering neoantigen-rich tumor vaccines. By utilizing radiation mutagenesis, RR-FDV overcomes the immunogenicity limitations of traditional tumor cell vaccines while maintaining clinical feasibility. Future efforts should focus on deciphering the mutation-immunogenicity axis and advancing RR-FDV into combination regimens with immune adjuvants or checkpoint inhibitors, ultimately paving the way for personalized cancer vaccines in clinical oncology.

[0080] It will be apparent to those skilled in the art that various modifications to the above embodiments may be made without departing from the overall spirit and concept of the present invention. Where details are not fully detailed herein, reference may be made to the prior art and the information shown in the accompanying drawings. Such modifications fall within the scope of protection of the present invention. The protection scheme of the present invention shall be subject to the claims appended hereto.

Claims

1. Application of radioresistant tumor cell fragments in the preparation of anti-tumor vaccines; wherein, The radioresistant tumor cell fragments are screened at least once by passage of primary tumor cells through several cycles of radiation exposure at doses not exceeding 6±0.6 Gy and by high-dose radiation at doses not exceeding 10±1 Gy.

2. Use of the radiation-resistant tumor cell fragments according to claim 1 in the preparation of anti-tumor vaccines; wherein, The radioresistant tumor cell fragments are prepared by the following method: Obtaining ex vivo primary tumor cells, subjecting them to initial screening by X-ray irradiation, performing multiple cycles of X-ray irradiation and subculture, and then performing secondary screening by X-ray irradiation to obtain radioresistant tumor cell fragments; Preferably, 6 cycles are used when performing multiple cycles of X-ray irradiation and subculture.

3. Use of the radiation-resistant tumor cell fragments according to claim 1 in the preparation of anti-tumor vaccines; characterized in that: The radiation exposure during screening is 6Gy < dose ≤ 10Gy, and the radiation exposure during radiation exposure and passaging is 4Gy < dose ≤ 6Gy; the in vitro primary tumor cells are obtained by culturing primary tumor cells from in vitro tumor tissues.

4. Use of the radiation-resistant tumor cell fragments according to any one of claims 1 to 3 in the preparation of anti-tumor vaccines; characterized in that: The anti-tumor vaccine is an anti-solid tumor vaccine; Preferably, the anti-solid tumor vaccine is an anti-lung adenocarcinoma vaccine, a head and neck squamous cell carcinoma vaccine, or a melanoma vaccine.

5. The radioresistant tumor cell fragments according to any one of claims 1 to 3.

6. A method for preparing an anti-solid tumor vaccine, characterized in that: The radioresistant tumor cell fragments according to any one of claims 1 to 5 are inactivated by freeze-thawing several times to prepare a vaccine; Preferably, the anti-tumor vaccine is an anti-solid tumor vaccine, more preferably an anti-lung adenocarcinoma vaccine, an anti-head and neck squamous cell carcinoma vaccine, or an anti-melanoma vaccine; Preferably, Freund's adjuvant is also added.

7. An anti-solid tumor vaccine, characterized in that The invention comprises the radioresistant tumor cell fragments according to any one of claims 1 to 5 which have been subjected to multiple freeze-thaw cycles.

8. The anti-solid tumor vaccine according to claim 7, characterized in that It also includes Freund's adjuvant; the anti-solid tumor vaccine is an anti-lung adenocarcinoma vaccine or a head and neck squamous cell carcinoma vaccine or a melanoma vaccine.

9. Use of the radioresistant tumor cell fragments according to any one of claims 1 to 3 in combination with Freund's adjuvant in the preparation of an anti-solid tumor vaccine.

10. The use according to claim 9; wherein The anti-solid tumor vaccine is at least one of an anti-lung adenocarcinoma vaccine, a head and neck squamous cell carcinoma vaccine, or a melanoma vaccine.

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