Tumor-infiltrating T lymphocyte of salivary gland malignant tumor as well as culture method, culture medium and application thereof

Abstract

The invention discloses a salivary gland malignant tumor infiltrating T lymphocyte as well as a culture method, a culture medium and application of the salivary gland malignant tumor infiltrating T lymphocyte. The culture medium comprises an ImmunoCall-XF T cell amplification culture medium and the following components based on the ImmunoCall-XF T cell amplification culture medium: 3000 IU / mL of IL-2, 1500 IU / mL of IL7 and 1500 IU / mL of IL32. The culture medium and the culture method disclosed by the invention are particularly suitable for culturing the tumor-infiltrating T lymphocytes of the salivary gland malignant tumors, and the obtained tumor-infiltrating T lymphocytes have important meanings for researching the occurrence and growth mechanism of the salivary gland malignant tumors and preparing or screening medicines for treating the salivary gland malignant tumors, and have wide application prospects.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical biology, and particularly to a tumor-infiltrating T lymphocyte of salivary gland malignant tumor, a culture method thereof, a culture medium and an application thereof. Background Art

[0002] Salivary gland malignant tumor is a maxillofacial malignant tumor with a relatively low incidence rate and a lack of effective treatment means. The treatment of patients is mainly based on surgery, supplemented by radiotherapy and chemotherapy. Due to its high recurrence rate and metastasis rate, there is currently no effective treatment plan for cases that cannot be surgically treated again and those who have failed radiotherapy and chemotherapy. Immunotherapy has achieved curative effects in the treatment of many tumors, but there are few reports on its application in salivary gland malignant tumor.

[0003] Tumor-infiltrating lymphocytes (TIL) are at the forefront of immune response and regulation in the tumor immune mechanism, not only affecting the efficacy of immunotherapy, but also participating in immune resistance. The main effector cells in TIL are T cells, and among T cells, CD4 + and CD8 + are the main ones. There are literature reports that the infiltration degree of CD8 + TILS is positively correlated with the response to anti-PD-L1 inhibitor treatment. To achieve tumor regression using immunotherapy, the infiltration of CD8 + T lymphocytes is required as a prerequisite, so it can be used as a predictive indicator for immunotherapy. Existing reports have shown that there is a certain degree of TILs infiltration in different tumors (such as melanoma, gastric cancer, lung cancer, etc.). The in vitro culture and preparation of T lymphocytes have always been somewhat difficult, and there is currently no report on the culture method of TILs in salivary gland malignant tumor, which limits the treatment plan for salivary gland malignant tumor. Therefore, it is urgent to construct a stable culture system for tumor-infiltrating T lymphocytes of salivary gland malignant tumor to provide a reference plan for the establishment and application of a culture system for salivary gland malignant tumor derived from patients clinically. Summary of the Invention

[0004] Aiming at the defects of the prior art, the purpose of the present invention is to provide a tumor-infiltrating T lymphocyte of salivary gland malignant tumor, a culture method thereof, a culture medium and an application thereof.

[0005] The technical solution of the present invention is as follows: In the first aspect of the present invention, a culture medium for culturing tumor-infiltrating T lymphocytes of salivary gland malignant tumors is provided. The culture medium includes ImmunoCult-XF T cell expansion medium and the following components based on the ImmunoCult-XF T cell expansion medium: 3000 IU / mL IL-2, 1500 IU / mL IL7, and 1500 IU / mL IL32.

[0006] In the second aspect of the present invention, a kit for culturing tumor-infiltrating T lymphocytes of salivary gland malignant tumors is provided. The kit includes the above-mentioned culture medium for culturing tumor-infiltrating T lymphocytes of salivary gland malignant tumors.

[0007] In the third aspect of the present invention, a method for culturing tumor-infiltrating T lymphocytes of salivary gland malignant tumors is provided. The culturing method uses the above-mentioned culture medium for culturing tumor-infiltrating T lymphocytes of salivary gland malignant tumors or the above-mentioned kit for culturing tumor-infiltrating T lymphocytes of salivary gland malignant tumors for culturing to obtain the tumor-infiltrating T lymphocytes of salivary gland malignant tumors.

[0008] The culturing method includes the following steps: S1. Tissue fragmentation Fragment the salivary gland malignant tumor tissue to obtain tissue fragments. S2. Tissue digestion Digest the tissue fragments with digestive enzymes to obtain a suspension of salivary gland malignant tumors. S3. Filtration Filter the suspension of salivary gland malignant tumors, centrifuge to discard the supernatant, resuspend the precipitate to obtain a single-cell suspension of salivary gland malignant tumors, inoculate and culture the single-cell suspension of salivary gland malignant tumors to obtain tumor-infiltrating T lymphocytes of salivary gland malignant tumors.

[0009] Further preferably, the digestive enzymes are dispase, collagenase type IV, and DNase I.

[0010] Further preferably, the concentrations of dispase, collagenase type IV, and DNase I are 0.5 - 2.0 mg / ml, 0.5 - 2.5 mg / ml, and 0.1 - 1.0 mg / ml, respectively.

[0011] Further preferably, in step S3, when resuspending the precipitate, a CD3 / CD28 T cell activator needs to be added.

[0012] Further preferably, the concentration of the CD3 / CD28 T cell activator is 10 - 30 μL / ml.

[0013] In the fourth aspect of the present invention, there is provided a tumor-infiltrating T lymphocyte of salivary gland malignant tumor, which is obtained by culturing the tumor-infiltrating T lymphocyte of salivary gland malignant tumor by the above-mentioned culture method.

[0014] In the fifth aspect of the present invention, there is provided an application of a tumor-infiltrating T lymphocyte of salivary gland malignant tumor in studying the pathogenesis of salivary gland malignant tumor, preparing or screening drugs for treating salivary gland malignant tumor.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The tumor-infiltrating T lymphocytes of salivary gland malignant tumor obtained by using the culture medium and culture method provided by the present invention have good growth status. Flow cytometry experiments show that the tumor-infiltrating T lymphocytes of salivary gland malignant tumor obtained by using the culture medium and culture method provided by the present invention have a relatively high purity of TILS and almost do not contain other cell types.

[0016] 2. The tumor-infiltrating T lymphocytes of salivary gland malignant tumor obtained by using the culture medium and culture method provided by the present invention can be used to study the occurrence / development mechanism of salivary gland malignant tumor, prepare or screen drugs for treating salivary gland malignant tumor, which not only meets the needs of scientific research, but also provides a beneficial choice in clinical drug guidance, and has a wide application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] By reading the following detailed description of non-limiting embodiments with reference to the accompanying drawings, other features, objects and advantages of the present invention will become more apparent: Figure 1 It is a schematic diagram of the morphological observation result of the tumor-infiltrating T lymphocyte of salivary gland malignant tumor provided by the embodiment of the present invention; Figure 2 It is a schematic diagram of the flow cytometry detection result of the tumor-infiltrating T lymphocyte of salivary gland malignant tumor provided by the embodiment of the present invention ( Figure 2 In A: a schematic diagram of the result that the proportion of immune cells in live cells reaches 99.2%; Figure 2 In B: a schematic diagram of the result that the proportion of T cells in immune cells reaches 93.5%; Figure 2 In C: CD4 in T cells + The proportion of T cells reaches 31.2%, and CD8 + The proportion of T cells reaches 49.4% of the result diagram). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0018] Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific embodiments; it should also be understood that the terms used in the embodiments of the present invention are for describing specific embodiments and not for limiting the protection scope of the present invention.

[0019] The present invention will be described in detail below with reference to specific embodiments.

[0020] First Embodiment This embodiment provides a culture medium for culturing tumor-infiltrating T lymphocytes of salivary gland malignant tumors. The culture medium includes ImmunoCult-XF T cell expansion medium and the following components based on ImmunoCult-XF T cell expansion medium: 3000 IU / mL IL-2, 1500 IU / mL IL7, 1500 IU / mL IL32.

[0021] Second Embodiment This embodiment provides a kit for culturing tumor-infiltrating T lymphocytes of salivary gland malignant tumors. The kit provided in this embodiment contains the culture medium for culturing tumor-infiltrating T lymphocytes of salivary gland malignant tumors provided in the first embodiment. Based on the culture medium for culturing tumor-infiltrating T lymphocytes of salivary gland malignant tumors provided in the first embodiment and combined with a scientific R & D process, it ensures the successful R & D and market launch of the kit for culturing tumor-infiltrating T lymphocytes of salivary gland malignant tumors.

[0022] Third Embodiment This embodiment provides a tumor-infiltrating T lymphocyte of salivary gland malignant tumor and its culture method. In this culture method, the culture medium used is the culture medium for culturing tumor-infiltrating T lymphocytes of salivary gland malignant tumors provided in the first embodiment.

[0023] The culture method of tumor-infiltrating T lymphocytes of salivary gland malignant tumors includes the following steps: S1. Tissue fragmentation Fragment the salivary gland malignant tumor tissue to obtain tissue fragments; S2. Tissue digestion Digest the tissue fragments with digestive enzymes to obtain a suspension of salivary gland malignant tumors; S3. Filtration Filter the suspension of salivary gland malignant tumors, centrifuge to discard the supernatant, resuspend the precipitate to obtain a single-cell suspension of salivary gland malignant tumors, inoculate and culture the single-cell suspension of salivary gland malignant tumors to obtain tumor-infiltrating T lymphocytes of salivary gland malignant tumors.

[0024] The digestive enzymes are dispase, collagenase type IV, and DNase I.

[0025] The concentrations of dispase, collagenase type IV, and DNase I are 0.5 - 2.0 mg / ml, 0.5 - 2.5 mg / ml, and 0.1 - 1.0 mg / ml, respectively.

[0026] In step S3, when resuspending the precipitate, a CD3 / CD28 T cell activator also needs to be added.

[0027] The concentration of the CD3 / CD28 T cell activator is 10 - 30 μL / ml.

[0028] In this embodiment, the culture method is specifically as follows: S1. Tissue fragmentation The salivary gland malignant tumor tissue is fragmented to obtain tissue fragments: On the ultra-clean bench, the salivary gland malignant tumor tissue is immersed in PBS solution containing 5% double antibiotics, and the tumor tissue is cut into pieces of 1 mm 3 size with ophthalmic scissors. Change to a clean small dish and continue to wash 3 times in PBS solution containing 5% double antibiotics.

[0029] S2. Tissue digestion The tissue fragments are digested with digestive enzymes to obtain a suspension of salivary gland malignant tumors: Use dispase (1 mg / ml), collagenase type IV (1 mg / ml), and DNase I (0.5 mg / ml) to digest the tumor tissue at 37°C for 20 min to remove most of the extracellular matrix components of the tumor cells (such as collagen, mucinous substances, etc.). After the digestion is completed, terminate the digestion with DMEM high-glucose medium containing 10% FBS, centrifuge at 1300 rpm for 3 min, discard the supernatant, and wash the precipitate once with PBS containing 5% double antibiotics.

[0030] Transfer the tissue fragments after PBS washing to a 1.5 ml EP tube, continue to cut the tissue blocks in the EP tube with ophthalmic scissors, centrifuge at 1300 rpm for 5 min, discard the supernatant (to remove impurities such as fat), add 1 ml of medium to resuspend the tissue fragments, and then add a CD3 / CD28 T cell activator (from Stemcell) with a concentration of 25 μg / ml, gently pipette evenly (do not pipette too hard to prevent tissue overflow) to obtain a tissue block suspension, and then transfer the tissue block suspension to a 12-well plate. Note: The 12-well plate can ensure that CAF cells and cancer cells can adhere to the wall to facilitate the isolation of T cells, and when using the medium, it is also very difficult for CAF cells and cancer cells to grow.

[0031] S3. Filtration Filter the tissue mass suspension of salivary gland malignant tumor, centrifuge and discard the supernatant, resuspend the precipitate to obtain a single cell suspension of salivary gland malignant tumor, inoculate and culture the single cell suspension of salivary gland malignant tumor to obtain tumor infiltrating T lymphocytes of salivary gland malignant tumor. The specific operations are as follows: After continuously culturing the tissue mass suspension of salivary gland malignant tumor for 24 - 48 h, observe it under an optical microscope. When the cell mass occupies 70% of the field of view area, filter the tissue suspension through a 70 μm filter mesh, centrifuge at 1300 rpm for 5 min, and discard the supernatant; treat the cell precipitate with red blood cell lysate (Beyotime Biotechnology Co., Ltd.) for 3 min, centrifuge at 1300 rpm for 5 min, and discard the supernatant. Resuspend and wash it once with PBS; centrifuge again at 1300 rpm for 5 min, discard the supernatant, add 1 ml of culture medium to resuspend the cell precipitate to obtain a suspension of tumor infiltrating T lymphocytes of salivary gland malignant tumor, and then add CD3 / CD28 T cell activator (25 μL / 1 ml); after culturing for 24 h, change the culture medium (centrifuge at 1300 rpm for 5 min and discard the original culture medium), add 1 ml of culture medium to resuspend the cell precipitate, and then add CD3 / CD28 T cell activator (25 μL / 1 ml), and continue to culture. During the subsequent culture period, change the culture medium once every 3 days, and add CD3 / CD28 T cell activator (25 μL / 1 ml) each time after changing the culture medium. Note: IL7 and IL32 can promote T cell proliferation; to ensure the best effect, the CD3 / CD28 T cell activator should be freshly prepared before use to avoid a decrease in activity.

[0032] The morphological observation of tumor infiltrating T lymphocytes of salivary gland malignant tumor is as Figure 1 shown. At each stage of different growth densities, observe the general morphology of the cultured tumor infiltrating T lymphocytes of salivary gland malignant tumor under an optical microscope and take pictures for record. The tumor infiltrating T lymphocytes of salivary gland malignant tumor are similar to spherical in shape, with continuous edges, and the cells show good proliferation activity and growth state. It indicates that the culture medium and its culture method provided by the present invention can effectively support the in vitro culture of tumor infiltrating T lymphocytes.

[0033] Application Example In this application example, the tumor infiltrating T lymphocytes of salivary gland malignant tumor provided in the third embodiment are used in the study of the pathogenesis of salivary gland malignant tumor and the preparation or screening of drugs for treating salivary gland malignant tumor.

[0034] Experimental Example In this experimental example, a flow cytometry detection experiment is carried out on the tumor infiltrating T lymphocytes of salivary gland malignant tumor provided in the third embodiment to detect the purity of the cells.

[0035] The operation process of flow cytometry detection is as follows: 1) Pre-cool PBS at 4°C for later use; 2) Resuspend the cells with 1 mL of pre-cooled PBS, centrifuge at 300 rpm for 5 min, discard the supernatant, and repeat the operation once; 3) Count the cells, resuspend the cells with PBS to form a cell suspension of 1x10 7 / mL, and take 100 μL of the cell suspension for later use; 4) Add the fluorescently labeled primary antibody (CD4) according to the instructions (BD Biosciences), mix well, and incubate in the dark at 4°C for 30 min; 5) Resuspend the cells with 1 mL of PBS, centrifuge at 300 rpm for 5 min, discard the supernatant, and repeat the operation once; 6) Resuspend the cells with 200 μL of 4% paraformaldehyde (fixative, Biosharp), and incubate in the dark at room temperature for 20 min; 7) Resuspend the cells with 1 mL of PBS, centrifuge at 300 rpm for 5 min, discard the supernatant, and repeat the operation once; 8) Add the fluorescently labeled primary antibody (CD8) according to the instructions (BD Biosciences), mix well, and incubate in the dark at 4°C for 30 min; 9) Resuspend the cells with 1 mL of PBS, centrifuge at 300 rpm for 5 min, discard the supernatant, and repeat the operation once; 10) Resuspend the cells with 100 μL of PBS into a flow cytometry tube; 11) Detect the sample on the flow cytometer.

[0036] The results of flow cytometry are as Figure 2 shown. The proportion of immune cells in live cells is 99.2% ( Figure 2 A); the proportion of T cells in immune cells is 93.5% ( Figure 2 B); the proportion of CD4 + T cells in T cells is 31.2%, and the proportion of CD8 + T cells in T cells is 49.4% ( Figure 2 C). The results show that the purity of this group of TILs cultured is relatively high, and almost no other cell types are contained. Further subgroup analysis found that among this group of T cells, 31.2% are CD4 + T cells, 49.4% are CD8 + T cells. The proportion of CD8 + T cells is significantly higher than that of CD4 + T cells, indicating that the cultured TILs are mainly effector CD8 + T cells and have potential tumor-killing ability.

[0037] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. Without conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

Claims

1. A culture medium for culturing tumor-infiltrating T lymphocytes of salivary gland malignant tumors, characterized in that, The culture medium includes ImmunoCult-XF T cell expansion medium, and the following components based on the ImmunoCult-XF T cell expansion medium: 3000 IU / mL IL-2, 1500 IU / mL IL7, 1500 IU / mL IL32.

2. A kit for culturing tumor-infiltrating T lymphocytes of salivary gland malignant tumor, characterized in that, The kit includes the culture medium for culturing tumor-infiltrating T lymphocytes of salivary gland malignant tumor described in claim 1.

3. A method for culturing tumor-infiltrating T lymphocytes in salivary gland malignant tumor, characterized in that, The tumor-infiltrating T lymphocytes of salivary gland malignant tumor are obtained by culturing using the culture medium for culturing tumor-infiltrating T lymphocytes of salivary gland malignant tumor described in claim 1 or the kit for culturing tumor-infiltrating T lymphocytes of salivary gland malignant tumor described in claim 2.

4. The culturing method of tumor-infiltrating T lymphocytes in salivary gland malignant tumor according to claim 3, characterized in that, It includes the following steps: S1. Tissue fragmentation The salivary gland malignant tumor tissue is fragmented to obtain tissue fragments. S2. Tissue digestion The tissue fragments are digested with digestive enzymes to obtain a suspension of salivary gland malignant tumor. S3. Filtration The suspension of salivary gland malignant tumor is filtered, the supernatant is discarded by centrifugation, and the precipitate is resuspended to obtain a single-cell suspension of salivary gland malignant tumor. The single-cell suspension of salivary gland malignant tumor is inoculated and cultured to obtain tumor-infiltrating T lymphocytes of salivary gland malignant tumor.

5. The culturing method of tumor infiltrating T lymphocytes of salivary gland malignant tumor according to claim 4, wherein, The digestive enzymes are dispase, collagenase type IV, and DNase I.

6. The culturing method of tumor-infiltrating T lymphocytes of salivary gland malignant tumor according to claim 5, wherein The concentrations of the dispase, collagenase type IV, and DNase I are 0.5 - 2.0 mg / ml, 0.5 - 2.5 mg / ml, and 0.1 - 1.0 mg / ml respectively.

7. The culturing method of tumor-infiltrating T lymphocytes in salivary gland malignant tumor according to claim 4, wherein, In step S3, when resuspending the precipitate, a CD3 / CD28 T cell activator also needs to be added.

8. The culturing method of tumor-infiltrating T lymphocytes in salivary gland malignant tumor according to claim 7, characterized in that The concentration of the CD3 / CD28 T cell activator is 10 - 30 μL / ml.

9. A tumor-infiltrating T lymphocyte of salivary gland malignant tumor, characterized in that, It is obtained by culturing according to the method for culturing tumor-infiltrating T lymphocytes of salivary gland malignant tumor described in any one of claims 3 - 8.

10. Use of the tumor-infiltrating T lymphocytes of salivary gland malignant tumor described in claim 9 in studying the pathogenesis of salivary gland malignant tumor, preparing or screening drugs for treating salivary gland malignant tumor.

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