Method for performing sarcoma high-frequency neoantigen function verification based on tumor organoids

Abstract

The invention discloses a method for performing sarcoma high-frequency neoantigen function verification based on tumor organoids, and the method for performing sarcoma high-frequency neoantigen function verification based on tumor organoids comprises the following steps: after separating PBMC, enriching CD8 + T cells, and stimulating the CD8 + T cells by using DC loaded with candidate neoantigen polypeptide to obtain neoantigen specific CTL; new antigen-specific CTL is tested through the kit, secretion of IFN-gamma is detected, and antigen-specific CTL with positive reaction is screened out; the method comprises the following steps: selecting a candidate new antigen capable of specifically stimulating CTL to generate an IFN-gamma positive reaction according to an experiment result of secreting IFN-gamma by the CTL, establishing a tumor organoid-CTL cell co-culture system, setting PBMC primary T cells of a patient which are not stimulated by DC loaded with the candidate new antigen as a control group, and measuring organoid light microscope radiography and tumor cell death conditions. The function of the sarcoma high-frequency neoantigen is evaluated based on tumor organs.

Description

Technical Field

[0001] The present invention relates to a method for functional detection of sarcoma neoantigens, and in particular to a method for functional verification of high-frequency sarcoma neoantigens based on tumor organoids. Background Art

[0002] Tumor neoantigens (neoantigens) are produced by somatic gene mutations and do not exist in normal tissues or cells. Compared with tumor-associated antigens, the main advantages of tumor neoantigens as targets for immunotherapy include no central tolerance, strong affinity with MHC molecules, accurate recognition and killing of tumor cells, and reduced off-target side effects in normal tissues. T cell therapy based on tumor neoantigens represents a new direction in the development of precision cell immunotherapy. Although it is theoretically feasible to search for neoantigens for each patient, it is time-consuming, labor-intensive, and costly in clinical practice, and is not suitable for patients who urgently need effective treatment when their disease progresses.

[0003] It is worth mentioning that a certain type of tumor often has a large number of high-frequency tumor gene mutations. The high-frequency neoantigens associated with these common mutations can cover more people, taking into account both specificity and representativeness, and are expected to be developed into ready-made immunotherapy products with strong clinical practical value. At present, a large number of studies have used bioinformatics methods to use gene sequencing data or mass spectrometry data to predict high-frequency tumor neoantigens with therapeutic effects.

[0004] It should be pointed out that it takes a complex biological process for tumor cells to undergo gene mutations and be recognized by mature T cell lineages. Even if the mutant gene produces abnormal proteins, only partially hydrolyzed peptides can be presented on the cell surface and recognized by immune cells. Therefore, the high-frequency tumor neoantigens predicted by various bioinformatics methods may not actually have the ability to stimulate the production of specific cytotoxic T cells (CTLs). The functional verification of candidate high-frequency neoantigens is an important issue that needs to be addressed in the field of immunotherapy research. Currently commonly used methods include enzyme-linked immunosorbent spot assay (ELISPOT) and flow cytometry to detect the expression of activated T cell markers IFN-γ and CD137 to evaluate the role of neoantigen presentation and induction of activated CTL, HLA-restricted tetramer staining detection, etc. However, the above methods cannot directly evaluate the actual killing effect of neoantigen presentation and induced activated CTL on tumors. Summary of the invention

[0005] The present invention provides a method for validating the function of high-frequency neoantigens in sarcoma based on tumor organoids, wherein the method can not only detect the activated T cell marker IFN-γ to evaluate the effect of neoantigen presentation and inducing activated CTL, but also evaluate the killing effect of the CTL on soft tissue sarcoma organoids, further verifying the function of high-frequency neoantigens in vitro, and providing a more reliable experimental basis for the development of high-frequency neoantigens for immunotherapy of soft tissue sarcoma.

[0006] In order to achieve at least one of the above advantages and solve the above technical problems, the present invention provides a method for functional verification of high-frequency neoantigens in sarcoma based on tumor organoids, comprising:

[0007] After PBMC isolation, CD8 + T cells, stimulate CD8 with DC loaded with candidate neoantigen peptides + T cells to obtain neoantigen-specific CTLs;

[0008] The new antigen-specific CTL was tested by the kit, the secretion of IFN-γ was detected, and the antigen-specific CTL with positive reaction was screened out;

[0009] According to the results of the CTL IFN-γ secretion experiment, candidate neoantigens that can specifically stimulate CTL to produce IFN-γ positive reactions were selected, and a tumor organoid-CTL cell co-culture system was established. Patient PBMC primary T cells that were not stimulated by DCs loaded with candidate neoantigens were set up as a control group. Organoid light microscopy and tumor cell death were measured to evaluate the function of sarcoma high-frequency neoantigens based on tumor organoids.

[0010] According to an embodiment of the present invention, the DC loaded with the candidate neoantigen polypeptide is prepared by the following method:

[0011] PBMCs were collected and isolated from patients with advanced soft tissue sarcoma using an automated blood cell harvester;

[0012] Enrichment of CD14 + The cells were cultured in CellGenixTM DC medium supplemented with 2% human serum albumin, GM-CSF and IL-4 for 5 days. On the 6th day, TNF-α, IL-6, IL-1β, prostaglandin E2 and Poly were added and cultured for another 2 days to obtain mature DCs. After harvesting, DCs were incubated with candidate neoantigen polypeptides in serum-free RPMI-1640 medium at 37°C for 4 hours to obtain DCs loaded with candidate neoantigen polypeptides.

[0013] According to an embodiment of the present invention, before testing the culture fluid after mixed culture by a kit to detect the secretion of IFN-γ and screen out positive antigen-specific CTL, the method further includes:

[0014] The new antigen-specific CTLs are amplified and then co-cultured with T2 cells in a predetermined ratio to obtain a mixed culture medium of the cultured antigen-specific CTLs.

[0015] According to one embodiment of the present invention, two rounds of amplification are performed.

[0016] According to an embodiment of the present invention, the method for functional verification of high-frequency neoantigens in sarcoma based on tumor organoids includes:

[0017] Minced sarcoma tissue, preferably, the size of the sarcoma tissue is about 1 mm3;

[0018] Then they were cultured for a certain period of time in digestion medium containing 1% fetal bovine serum, 2% penicillin / streptomycin, 1.5 mg / mL collagenase II, 500 U / mL collagenase IV, 200 μg / mL hyaluronidase, 0.1 mg / mL DNase-I, 100 μg / mL DispaseII, and 10 μM Y-27632;

[0019] After digestion, the cells were treated with 0.1 mg / mL DNase-I at 37°C for 3-5 minutes; and red blood cell lysis buffer on ice for 1-3 minutes. Between each step, the cells were washed with phosphate-buffered saline containing 5% FBS and then filtered with a 100 μm cell strainer to obtain a single-cell suspension. After aspirating the supernatant, the cells were added to 15-20 μL Matrigel in a 24-well or 48-well plate, allowed to solidify for 5-10 minutes, and then covered with organoid culture medium. The culture medium was replaced every 3-4 days. Organoids were passaged every 15-20 days, first digested with 250-500 μL TrypLE Express at 37°C for about 10 minutes, and then washed with ADMEM / F12 medium. The organoids and their primary tissue samples were paraffin-embedded and sectioned. HE staining and immunohistochemistry identified that the histopathological characteristics of the organoids were consistent with those of the primary tissues.

[0020] According to an embodiment of the present invention, the digestion medium is DMEM / F12.

[0021] According to one embodiment of the present invention, the kit is a Human IFN-γ ELISA Kit.

[0022] According to an embodiment of the present invention, CD8 + The ratio of T:DC is 4:1.

[0023] According to one embodiment of the present invention, T2 cells have been loaded with 10 μg / mL polypeptide / are not loaded with polypeptide.

[0024] According to an embodiment of the present invention, the absorbance is read at a wavelength of 630 nm and calculated to detect the secretion of IFN-γ. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A schematic diagram showing an example of an ELISA assay for CTL secretion of IFN-γ to validate the immunogenicity of a candidate neoantigen.

[0026] Figure 2 A schematic diagram showing an example of the Calcein-AM / PI double staining method for evaluating the killing effect of candidate neoantigen-specific CTLs on soft tissue sarcoma organoids.

[0027] Figure 3 A schematic diagram showing the growth inhibition rate of soft tissue sarcoma organoids by candidate neoantigen-specific CTLs in one example.

[0028] Figure 4 A schematic diagram showing another example of ELISA assay for CTL secretion of IFN-γ to verify the immunogenicity of candidate neoantigens.

[0029] Figure 5 Schematic diagram showing organoid light microscopy to evaluate the killing effect of drug-sensitive candidate neoantigen-specific CTL on soft tissue sarcoma organoids.

[0030] Figure 6 A schematic diagram showing the growth inhibition rate of soft tissue sarcoma organoids by candidate neoantigen-specific CTLs in another example.

[0031] Figure 7 A schematic diagram showing another example of using ELISA to measure CTL secretion of IFN-γ to verify the immunogenicity of candidate neoantigens.

[0032] Figure 8 A schematic diagram showing another example of the Calcein-AM / PI double staining method for evaluating the killing effect of candidate neoantigen-specific CTLs on soft tissue sarcoma organoids.

[0033] Fig. 9 A schematic diagram showing the growth inhibition rate of soft tissue sarcoma organoids by candidate neoantigen-specific CTLs in yet another example. DETAILED DESCRIPTION

[0034] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are only examples, and those skilled in the art can think of other obvious variations. The basic principles of the present invention defined in the following description can be applied to other embodiments, variations, improvements, equivalents, and other technical solutions that do not deviate from the spirit and scope of the present invention.

[0035] In order to solve the above technical problems, the present invention provides a method for functional verification of high-frequency neoantigens in sarcoma based on tumor organoids. Preferably, the method comprises:

[0036] S1001, preparing DCs (dendritic cells) loaded with candidate neoantigen polypeptides.

[0037] Specifically, step S1001 includes: collecting 2 ml of peripheral blood from patients with advanced soft tissue sarcoma. Referring to the method used in the MASCT-I clinical trial, PBMCs are collected and separated from patients with advanced soft tissue sarcoma by a fully automatic blood cell collection instrument, one part of which is used to prepare DC cells, and the other part is used to prepare CTL (cytotoxic T lymphocytes);

[0038] Subsequently, CD14 + The cells were cultured in CellGenixTM DC medium (CellGenix) supplemented with 2% human serum albumin, GM-CSF and IL-4 for 5 days. On the 6th day, TNF-α, IL-6, IL-1β, prostaglandin E2 and Poly (I:C) were added and cultured for another 2 days to obtain mature DC. After harvesting, DC were incubated with candidate neoantigen peptides in serum-free RPMI-1640 medium at 37°C for 4 hours to obtain DC loaded with candidate neoantigen peptides.

[0039] Furthermore, the method for functional verification of high-frequency neoantigens in sarcoma based on tumor organoids includes:

[0040] S1002, after isolating PBMC, use CD8 MicroBeads to enrich CD8 + T cells, and then stimulate CD8 + T cells, preferably CD8 + The ratio of T:DC is 4:1.

[0041] Specifically, using HIPP TM -T009 lymphocyte serum-free medium, 10% fetal bovine serum and interleukins, such as IL-21, were co-cultured in a 37°C, 5% CO2 incubator for 12 days. On the third day of co-culture, interleukins, such as IL-2, IL-7 and IL-15, were added to the culture medium, and the culture medium was replaced every 2-3 days. After 12 days of culture, DCs loaded with the same candidate neoantigen peptides stimulated CD8 + T cells were then co-cultured for 12 days to finally obtain new antigen-specific CTLs.

[0042] Preferably, the method for functional verification of high-frequency neoantigens in sarcoma based on tumor organoids comprises:

[0043] S1003, amplify and obtain new antigen-specific CTL, preferably obtain new antigen-specific CTL by 2 rounds of amplification. Then, the amplified CTL cells are replaced with a medium containing 10% fetal bovine serum and cultured statically, such as culturing for 24 hours. Then, they are mixed and co-cultured with T2 cells (loaded with 10 μg / mL polypeptide / unloaded polypeptide) in a predetermined ratio, such as a 3:1 ratio, such as mixed culture for 24.

[0044] The method for functional verification of high-frequency neoantigens in sarcoma based on tumor organoids includes:

[0045] S1004, the culture fluid after mixed culture was tested by Human IFN-γ ELISA Kit (97024ES) to detect the secretion of IFN-γ, and the absorbance was read and calculated at 630nm. The test value of the experimental group was more than 3 times that of the negative control group, and the culture fluid after mixed culture with positive reaction was screened. The schematic diagram of the test is shown in Figure 1 shown.

[0046] The method for functional verification of high-frequency neoantigens in sarcoma based on tumor organoids includes:

[0047] S1005, mincing the sarcoma tissue, preferably, the size of the sarcoma tissue is about 1 mm 3 ;

[0048] Then, the cells are cultured in a digestion medium for a certain period of time, preferably DMEM / F12, containing 1% fetal bovine serum (FBS), 2% penicillin / streptomycin, 1.5 mg / mL collagenase II, 500 U / mL collagenase IV, 200 μg / mL hyaluronidase, 0.1 mg / mL DNase-I, 100 μg / mL Dispase II and 10 μM Y-27632 (ROCK inhibitor, Sigma Aldrich, Y0503). Preferably, the cells are cultured for 0.5-1 h at 37° C. with continuous shaking.

[0049] After digestion, cells were treated with 0.1 mg / mL DNase-I at 37°C for 3-5 minutes, and red blood cell (RBC) lysis buffer on ice for 1-3 minutes. Between each step, cells were washed with phosphate-buffered saline (PBS) containing 5% FBS. Then filtered with a 100 μm cell strainer to obtain a single cell suspension. After aspirating the supernatant, the cells were added to 15-20 μL Matrigel (Corning, 356,231) in a 24-well or 48-well plate, allowed to solidify for 5-10 minutes, and then covered with organoid culture medium (500 μL and 250 μL for 24 wells and 48 wells, respectively). The culture medium was replaced every 3-4 days, and the organoids were passaged every 15-20 days, first digested with 250-500 μL TrypLE Express (Gibco, 12,604-021) at 37°C for about 10 minutes, and then washed with ADMEM / F12 culture medium. The organoids and their primary tissue samples were paraffin-embedded and sectioned, and HE staining and immunohistochemistry were used to identify that the histopathological characteristics of the organoids were consistent with those of the primary tissues.

[0050] The method for functional verification of high-frequency neoantigens in sarcoma based on tumor organoids includes:

[0051] S1006. Based on the results of the CTL IFN-γ secretion experiment, select candidate new antigens that can specifically stimulate CTL to produce IFN-γ positive response, and conduct subsequent organoid killing experiments.

[0052] Specifically, the killing effect of candidate high-frequency neoantigen-specific CTL on HLA-A*1101-restricted soft tissue sarcoma organoids was verified, further confirming the immune efficacy of the neoantigen.

[0053] First, a tumor organoid-CTL cell co-culture system is established, such as by the method described in [Cattaneo CM, Dijkstra KK, Fanchi LF, Kelderman S, Kaing S, van Rooij N, et al. Tumor organoid-T-cell coculture systems. Nat Protoc 2020; 15: 15-39], and primary T cells from patient PBMCs that have not been stimulated with DCs loaded with candidate neoantigens are set as a control group.

[0054] Use one or both of the two methods to evaluate the killing effect of neoantigen-specific CTL on organoids: (1) Organoid light microscopy to directly evaluate the growth inhibition rate; (2) Calcein-AM / PI live cell / dead cell double staining kit to detect tumor cell death and evaluate drug sensitivity. Calcein-AM is a cell staining reagent that fluorescently labels live cells and emits green fluorescence; Propidium iodide (PI) cannot pass through the cell membrane of live cells, but can only pass through the disordered area of ​​the dead cell membrane to reach the cell nucleus and embed into the cell's DNA double helix to produce red fluorescence. Therefore, PI only stains dead cells. Subsequent evaluation of fluorescence intensity using fluorescence microscopy can evaluate the anti-tumor activity of the drug. Live cell / dead cell imaging system is used to evaluate the killing effect of T cells.

[0055] It is worth mentioning that neoantigens, DC cells, and CTL cells from patients with soft tissue sarcoma are immunogenic stimuli derived from xenobiotics for any animal model, and there is currently no suitable animal model to evaluate their pharmacodynamics. Tumor organoids have become an ideal research platform for cellular immunotherapy in recent years because they better retain characteristics such as the tumor immune microenvironment. This patent is based on the soft tissue sarcoma organoid platform to further evaluate the specific killing effect of CTL activated by candidate neoantigens on sarcoma organoids, which can better simulate the in vivo immune effects of neoantigens.

[0056] like Figure 2 and Figure 3 As shown in the figure, in a case of soft tissue sarcoma, based on the results of ELISA to determine the secretion of IFN-γ by CTL, we found that the USH2A_A1546D 1543-1551 、ADGRV1_D4403H 4298-4305 、P53_R248W 240-249 、APOB_S1723I 1721-1730 、FAT3_N2515K 2513-2522 、DMD_R1459Q 1454-1463 DCs with candidate neoantigen peptides can significantly and specifically stimulate CTLs to produce IFN-γ, which is 3 times higher than that of the negative control group. The ability of the remaining peptides to stimulate CTLs to produce IFN-γ is not significantly different from that of the negative control group. Figure 1As shown. Overall, among the 21 predicted candidate neoantigen peptides, 6 are immunogenic and can specifically stimulate CTL to secrete IFN-γ. The above 6 immunogenic candidate neoantigen peptides were further used to load DC to stimulate CTL and conduct soft tissue sarcoma organoid killing experiments. The growth inhibition rates of organoids were 5.2%, 8.2%, 9.8%, 23.3%, 11.1%, and 25.7%, respectively. The above results show that P53_R248W240-249 and DMD_R1459Q1454-1463 candidate neoantigen peptides can significantly activate CTL and induce specific organoid killing.

[0057] In another case, based on the results of ELISA assay for CTL secretion of IFN-γ, the RB1_I724S 722-729 、TTN_G2619D 2615-2623 、SCN2A_F1677Y 1675-1684 、P53_R248W 240-249 RYR1_P4713L 4713-4722 、FAT3_N2515K 2513-2522 、SCN9A_L231P 226-235 、DMD_R1459Q 1454-1463 DCs with candidate neoantigen peptides can significantly and specifically stimulate CTLs to produce IFN-γ, which is 3 times higher than that of the negative control group. The ability of the remaining peptides to stimulate CTLs to produce IFN-γ is not significantly different from that of the negative control group. Figure 4 . Overall, among the 21 predicted candidate neoantigen peptides, 8 were immunogenic and could specifically stimulate CTL to secrete IFN-γ. The above 8 immunogenic candidate neoantigen peptides were further used to load DC to stimulate CTL and conduct soft tissue sarcoma organoid killing experiments. The growth inhibition rates of organoids were 79.9%, 27.1%, 3.5%, 25.0%, 1.2%, 47.5%, 11.9% and 8.0%, respectively. The above results show that RB1_I724S 722-729 、FAT3_N2515K 2513-2522 、TTN_G2619D 2615-2623 and P53_R248W 240-249 The candidate new antigen peptides can significantly activate CTL and induce specific organoid killing effects, such as Figure 5 and Figure 6 shown.

[0058] In a case of soft tissue sarcoma with HLA-A*1101 typing, the results of IFN-γ secretion by CTLs were determined by ELISA, and the USH2A_A1546D 1543-1551 、TTN_G2619D 2615-2623、MACF1_Q6537H 6537-6545 、APOB_S1723I 1721-1730 、P53_R248W 240-249 、SCN2A_F1677Y 1675-1684 and RB1_I724S 722-729 DCs with candidate neoantigen peptides can significantly and specifically stimulate CTLs to produce IFN-γ, which is 3 times higher than that of the negative control group. The ability of the remaining peptides to stimulate CTLs to produce IFN-γ is not significantly different from that of the negative control group ( Figure 7 ). Overall, among the 21 predicted candidate neoantigen peptides, 7 were immunogenic and could specifically stimulate CTL to secrete IFN-γ. The above 7 immunogenic candidate neoantigen peptides were further used to load DC to stimulate CTL, and soft tissue sarcoma organoid killing experiments were performed. The Calcein-AM / PI live cell / dead cell double staining kit was used to detect tumor cell death and evaluate drug sensitivity. The growth inhibition rates of organoids were 1.2%, 1.8%, 2.3%, 5.5%, 35.7%, 41.0% and 60.0%, respectively. The above results show that RB1_I724S 722-729 、SCN2A_F1677Y 1675-1684 and P53_R248W 240-249 The candidate new antigen peptides can significantly activate CTL and induce specific organoid killing effects, such as Figure 8 and Fig. 9 shown.

[0059] It should be understood by those skilled in the art that the embodiments of the present invention described above and shown in the accompanying drawings are only examples and do not limit the present invention. The advantages of the present invention have been fully and effectively achieved. The functional and structural principles of the present invention have been demonstrated and explained in the embodiments, and the embodiments of the present invention may be deformed or modified in any way without departing from the principles.

Claims

1. A method for functional verification of high-frequency neoantigens in sarcoma based on tumor organoids, characterized in that: The method for functional verification of high-frequency neoantigens in sarcoma based on tumor organoids includes: After PBMC isolation, CD8 + T cells, stimulate CD8 with DC loaded with candidate neoantigen peptides + T cells to obtain neoantigen-specific CTLs; The new antigen-specific CTL was tested by the kit, the secretion of IFN-γ was detected, and the antigen-specific CTL with positive reaction was screened out; According to the results of the CTL IFN-γ secretion experiment, candidate neoantigens that can specifically stimulate CTL to produce IFN-γ positive reactions were selected, and a tumor organoid-CTL cell co-culture system was established. Patient PBMC primary T cells that were not stimulated by DCs loaded with candidate neoantigens were set up as a control group. Organoid light microscopy and tumor cell death were measured to evaluate the function of sarcoma high-frequency neoantigens based on tumor organoids.

2. The method for functional verification of high-frequency neoantigens in sarcoma based on tumor organoids according to claim 1, characterized in that: The DC loaded with candidate neoantigen polypeptides is prepared by the following method: PBMCs were collected and isolated from patients with advanced soft tissue sarcoma using an automated blood cell harvester; Enrichment of CD14 + The cells were cultured in CellGenixTM DC medium supplemented with 2% human serum albumin, GM-CSF and IL-4 for 5 days. On the 6th day, TNF-α, IL-6, IL-1β, prostaglandin E2 and Poly were added and cultured for another 2 days to obtain mature DCs. After harvesting, DCs were incubated with candidate neoantigen polypeptides in serum-free RPMI-1640 medium at 37°C for 4 hours to obtain DCs loaded with candidate neoantigen polypeptides.

3. The method for functional verification of high-frequency neoantigens in sarcoma based on tumor organoids according to claim 1, characterized in that: Before testing the culture fluid after mixed culture by a kit to detect the secretion of IFN-γ and screen out positive antigen-specific CTL, the method further comprises: The new antigen-specific CTLs are amplified and then co-cultured with T2 cells in a predetermined ratio to obtain a mixed culture medium of the cultured antigen-specific CTLs.

4. The method for functional verification of high-frequency neoantigens in sarcoma based on tumor organoids according to claim 2, characterized in that: Amplify for 2 rounds.

5. The method for functional verification of high-frequency neoantigens in sarcoma based on tumor organoids according to claim 1, characterized in that: The method for functional verification of high-frequency neoantigens in sarcoma based on tumor organoids includes: The sarcoma tissue is minced, preferably, the size of the sarcoma tissue is about 1 mm. 3 ; Then they were cultured for a certain period of time in digestion medium containing 1% fetal bovine serum, 2% penicillin / streptomycin, 1.5 mg / mL collagenase II, 500 U / mL collagenase IV, 200 μg / mL hyaluronidase, 0.1 mg / mL DNase-I, 100 μg / mL Dispase II, and 10 μM Y-27632; After digestion, the cells were treated with 0.1 mg / mL DNase-I at 37°C for 3-5 minutes; and red blood cell lysis buffer on ice for 1-3 minutes. Between each step, the cells were washed with phosphate-buffered saline containing 5% FBS and then filtered with a 100 μm cell strainer to obtain a single-cell suspension. After aspirating the supernatant, the cells were added to 15-20 μL Matrigel in a 24-well or 48-well plate, allowed to solidify for 5-10 minutes, and then covered with organoid culture medium. The culture medium was replaced every 3-4 days. Organoids were passaged every 15-20 days, first digested with 250-500 μL TrypLE Express at 37°C for about 10 minutes, and then washed with ADMEM / F12 medium. The organoids and their primary tissue samples were paraffin-embedded and sectioned. HE staining and immunohistochemistry identified that the histopathological characteristics of the organoids were consistent with those of the primary tissues.

6. The method for functional verification of high-frequency neoantigens in sarcoma based on tumor organoids according to claim 5, characterized in that: The digestion medium was DMEM / F12.

7. The method for functional verification of high-frequency neoantigens in sarcoma based on tumor organoids according to claim 1, characterized in that: The kit is Human IFN-γ ELISA Kit.

8. The method for functional verification of high-frequency neoantigens in sarcoma based on tumor organoids according to claim 1, characterized in that: CD8 + The ratio of T:DC is 4:

1.

9. The method for functional verification of high-frequency neoantigens in sarcoma based on tumor organoids according to claim 3, characterized in that: T2 cells have been loaded with 10 μg / mL peptide / not loaded with peptide.

10. The method for functional verification of high-frequency neoantigens in sarcoma based on tumor organoids according to claim 1, characterized in that: The absorbance was read at a wavelength of 630 nm and calculated to detect the secretion of IFN-γ.

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