Application of Sirt6 in Establishing Tumor-Bearing Models of Animals with Normal Immune Systems and Tumor Immunotherapy

Abstract

The present invention belongs to the technical fields of biomedicine and molecular biology, and particularly relates to the application of Sirt6 in constructing an animal tumor-bearing model with a normal immune system and tumor immunotherapy. By increasing the activity and expression of Sirt6, the present invention enables human tumor cells to grow into human tumor tissues in mice with a normal immune system. Since the used mice have a perfect immune system, the human cancer cell tumor-bearing model and research results are closer to the actual situation of tumor clinical practice. Therefore, using this research model can be better used for the study of tumor pathogenesis, tumor immunity exploration, and tumor drugs. In addition, activating this gene allows tumor cells in immune-normal mice to escape immune surveillance and grow, indicating that this gene locus is a key point for immune escape during tumor, especially early tumorigenesis. Reverse inhibition of the activity of this gene should be beneficial to tumor prevention and treatment, thus having good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biomedicine and molecular biology, and particularly relates to the application of Sirt6 in constructing a tumor-bearing animal model with a normal immune system and tumor immunotherapy. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Sirtuins (Sirts) are class III histone deacetylases (HDACs), including 7 members, which are widely expressed in mammals and mainly participate in metabolic homeostasis, DNA damage repair, cell survival and differentiation. Sirtuins have been proven to be widely applied in tumor-related biological processes. More and more data confirm that sirtuins play an immunomodulatory role in various tumors.

[0004] Sirt6 is a member of the sirtuin family, and its expression increases in various tumor tissues, including ovarian, renal cell carcinoma, hepatocellular carcinoma, colon cancer and prostate cancer tissues. Studies have found that the decrease in Sirt6 expression is related to the improvement of overall survival (OS), and the silencing of Sirt6 slows down the growth of MDA-MB-231BC cell xenografts. Many studies have elaborated on the key role of Sirt6 in immunomodulation from the perspectives of immunosenescence, immunometabolism and tumor immunology. For example, Sirt6 can regulate the production of TNF-α and promote the progression of prostate cancer by inhibiting the RIPK3-mediated innate immune response. Therefore, Sirt6 is considered as an immunotherapy target for tumor treatment.

[0005] However, the inventors found that the prognostic role and biological function of Sirt6 in tumors are still unclear at present, and the molecular mechanism of the immunomodulatory properties of Sirt6 and its application value in tumor immune intervention still need to be further studied. Summary of the Invention

[0006] In view of the deficiencies in the prior art, the object of the present invention is to provide the application of Sirt6 in constructing a tumor-bearing model of animals with a normal immune system and tumor immunotherapy. By increasing the activity and expression of Sirt6, the present invention enables human tumor cells to grow into human tumor tissues in BALB / c mice with a normal immune system. Since the used mice have a perfect immune system, this human cancer cell tumor-bearing model and research results are closer to the actual situation of tumors in clinical practice. Therefore, this research model can be better used for studying tumor pathogenesis, tumor immunity exploration, and tumor drugs. In addition, since activating this gene allows tumor cells in immune-normal mice to escape immune surveillance and grow, it indicates that this gene locus is a key point for immune escape during tumor development, especially in the early stage of tumors. Inhibiting the activity of this gene in the opposite direction should be beneficial to tumor prevention and treatment. Therefore, based on the above research results, the present invention is completed.

[0007] In order to achieve the above technical object, the technical solution provided by the present invention is as follows:

[0008] In the first aspect of the present invention, there is provided the application of substances that promote the expression of Sirt6 or increase its activity in any one or more of the following:

[0009] a1) Constructing a tumor-bearing model of non-human animals with a normal immune system;

[0010] a2) Inhibiting the viability of tumor cells in vitro or preparing a product for inhibiting the viability of tumor cells in vitro;

[0011] a3) Inhibiting the glycolysis of tumor cells in vitro or preparing a product for inhibiting the glycolysis of tumor cells in vitro;

[0012] a4) Promoting the increase of CD38+NK-like T cells and Treg cells in the co-culture system of tumor cells and immune cells in vitro or preparing a product for promoting the increase of CD38+NK-like T cells and Treg cells in the co-culture system of tumor cells and immune cells in vitro;

[0013] a5) Promoting the expression of PD-1, PD-L1, and adenosine (ADO) in the co-culture system of tumor cells and immune cells in vitro or preparing a product for promoting the expression of PD-1, PD-L1, and ADO in the co-culture system of tumor cells and immune cells in vitro;

[0014] a6) Inhibiting the expression of monocyte chemoattractant protein-1 (MCP-1) in the co-culture system of tumor cells and immune cells in vitro or preparing a product for inhibiting the expression of MCP-1 in the co-culture system of tumor cells and immune cells in vitro.

[0015] The product can be a drug or an experimental reagent, and the experimental reagent can be used for basic research to further study the mechanism of tumor occurrence and development, etc.

[0016] In the second aspect of the present invention, there is provided the use of Sirt6 as a target in the preparation or screening of anti-tumor drugs.

[0017] Wherein, the anti-tumor treatment is anti-tumor immunotherapy.

[0018] The beneficial technical effects of the above one or more technical solutions:

[0019] By increasing the activity and expression of Sirt6, the above technical solution enables human tumor cells to grow into human tumor tissues in BALB / c mice with a normal immune system. Since the used mice have a perfect immune system, this human cancer cell tumor-bearing model and research results are closer to the actual situation of tumor clinical practice. Therefore, this research model can be better used for the study of tumor pathogenesis, tumor immunity exploration, and anti-tumor drugs.

[0020] Meanwhile, since activating this gene can enable tumor cells in immune-normal mice to escape immune surveillance and grow, it indicates that this gene locus is a key point for immune escape during tumor occurrence, especially in the early stage of tumor. And inhibiting the activity of this gene in reverse should be beneficial to tumor prevention and treatment. Therefore, Sirt6 is also a key gene for anti-tumor treatment, especially anti-tumor immunotherapy, and thus has good practical application value. Description of the Drawings

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0022] Figure 1 . Tumor growth after injection of UBCS039 in SMMC-7721 cells in normal BALB / c mice. (A) Tumor mass growth curve of mice. (B) Obvious tumor masses appear in the axilla of mice. (C) These tumor mass tissues are taken from these mouse models.

[0023] Figure 2 . Examination of tumor tissues and tumor-bearing mice. (A) Hematoxylin-eosin staining of tumor mass tissues, (B) Immunohistochemical staining of liver tumor biomarkers, (C) PET-CT scan of tumor-bearing mice, (D) Fluorescence imaging of tumor-bearing mice. Arrows indicate concave and convex areas.

[0024] Figure 3.Flow cytometry was used to detect various lymphocyte subtypes in experimental mice. Representative flow cytometry plots of NK cells (A), NK cells (B), CD38+NK (C), CD38+NK-like T cells (D), B cells (E), and CD38+B cells (F); quantification of Tregs (G), NK (H), CD38+NK (I), CD38+NK-like T cells (J), B cells (K), and CD38+B cells (L); Th1 / Th2 ratio (M), Th17 / Treg ratio (N). * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

[0025] Figure 4 .Effect of SMMC-7721 cells pretreated with UBCS039 on CD4+ T cell differentiation. SMMC-7721 cells were treated with UBCS039 or DMSO or left untreated. They were co-cultured with human CD4+ T cells. Flow cytometry was used to detect the proportions of CD4+ T cells (A), Tregs (B), NK-like T cells (C), CD38+NK-like T cells (D), CD8+ T cells (E), and Th17 cells (F). Quantification of the proportion of Treg cells (G), NK-like T cells (H), CD38+NK-like T cells (I), and Th7 cells (J). * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

[0026] Figure 5 .Key gene expression in co-cultures of SMMC-7721 cells pretreated with UBCS039 and CD4+ T cells. The mRNA levels of CD38 (A), Sirt1 (B), Sirt3 (C), Sirt6 (D), PD-1 (E) in SMMC-7721 cells and PD-L1 (F) in CD4+ T cells were detected using real-time PCR. The ADO level in the culture medium was determined by ELISA (G). * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

[0027] Figure 6 .Effect of UBCS039 on SMMC-7721 cells. (A) Cell proliferation was measured using the RTCADPlus station. (B) Cell apoptosis was detected by flow cytometry. (C) Cell migration was measured using the Transwells method. (D) Cell metabolism was determined using the Agilent Seahorse XFp system. OCR: oxygen consumption rate. ECAR: extracellular acidification rate. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

[0028] Figure 7. The effect of UBCS039 on gene expression in SMMC-7721 cells was studied using real-time PCR. Expression of CD38 (A), Sirt1 (B), Sirt3 (C), and Sirt6 (D). Cells treated with DMSO were used as the control. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

[0029] Figure 8 . The energy metabolism of experimental mice was detected using the Oxylet Pro animal metabolic system. (A) Respiratory quotient (PER) and (B) energy expenditure (EE).

[0030] Figure 9 . Flow cytometry was used to detect the cytokine levels in the peripheral blood of experimental mice. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

[0031] Figure 10 . Immunofluorescence immunohistochemistry was used to detect infiltrating Tregs in tumor tissues.

[0032] Figure 11 . The effect of UBCS039-pretreated SMMC-7721 cells on NK cell differentiation was studied using flow cytometry. SMMC-7721 cells were untreated (A), treated with DMSO (B), or treated with UBCS039 (C). They were co-cultured with human NK cells. After co-culture with tumor cells, the proportion of CD38+ NK cells (D) and the proportion of CD38+ NK-like T cells (E) in NK cells. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

[0033] Figure 12 . Flow cytometry was used to analyze the cytokine levels in the co-culture supernatant of UBCS039-pretreated SMMC-7721 cells and CD4+ T cells. MFI: Mean fluorescence intensity. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001. Detailed implementation

[0034] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which the present invention belongs.

[0035] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they specify the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0036] As mentioned above, the prognostic role and biological function of Sirt6 in tumors are still not clear at present. The molecular mechanism of the immunomodulatory properties of Sirt6 and its application value in tumor immunotherapy still need to be further studied.

[0037] In view of this, in a typical specific embodiment of the present invention, there is provided the use of a substance that promotes the expression of Sirt6 or increases its activity in any one or more of the following:

[0038] a1) Establishing a tumor-bearing model of non-human animals with a normal immune system;

[0039] a2) Inhibiting the viability of tumor cells in vitro or preparing a product for inhibiting the viability of tumor cells in vitro;

[0040] a3) Inhibiting the glycolysis of tumor cells in vitro or preparing a product for inhibiting the glycolysis of tumor cells in vitro;

[0041] a4) Promoting the increase of CD38+NK-like T cells and Treg cells in the co-culture system of tumor cells and immune cells in vitro or preparing a product for promoting the increase of CD38+NK-like T cells and Treg cells in the co-culture system of tumor cells and immune cells in vitro;

[0042] a5) Promoting the expression of PD-1, PD-L1 and adenosine (ADO) in the co-culture system of tumor cells and immune cells in vitro or preparing a product for promoting the expression of PD-1, PD-L1 and ADO in the co-culture system of tumor cells and immune cells in vitro;

[0043] a6) Inhibiting the expression of monocyte chemoattractant protein-1 (MCP-1) in the co-culture system of tumor cells and immune cells in vitro or preparing a product for inhibiting the expression of MCP-1 in the co-culture system of tumor cells and immune cells in vitro.

[0044] Among them, substances that promote Sirt6 expression or enhance its activity include, but are not limited to, substances that up-regulate Sirt6 expression based on gene-specific Mimics technology; such as artificially synthesized short hairpin RNA (shRNA) of Sirt6, or promoters that up-regulate Sirt6 expression, or lentiviruses; and also include compound promoters. In a specific embodiment of the present invention, the substance that promotes Sirt6 expression or enhances its activity can be UBCS039 (CAS: 358721-70-7), which is the first synthetic specific activator of SIRT6.

[0045] The product can be a drug or an experimental reagent, and the experimental reagent can be used for basic research, so as to be used for further studying the mechanisms of tumorigenesis and development, etc.

[0046] The above product can also be a drug or an experimental reagent, and the experimental reagent is for basic research. Preferably it is a drug.

[0047] According to the present invention, when the product is a drug, the drug further includes at least one pharmaceutically inactive ingredient.

[0048] The pharmaceutically inactive ingredient can be a carrier, excipient, diluent, etc. commonly used in pharmacy. Moreover, according to the usual methods, it can be made into dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc., for oral administration, external use, suppositories, and sterile injection solutions.

[0049] The non-drug active ingredients such as the carrier, excipient, and diluent that can be included are well-known in the art, and those of ordinary skill in the art can determine that they meet clinical standards.

[0050] In another specific embodiment of the present invention, the carrier, excipient, and diluent include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, arabic gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl paraben, propyl paraben, talc, magnesium stearate, and mineral oil, etc.

[0051] In another specific embodiment of the present invention, the drug of the present invention can be administered into the body by known means. For example, it can be delivered systemically via intravenous injection or locally injected into the tissue of interest. Optionally, it can be administered via intravenous, transdermal, intranasal, mucosal, or other delivery methods. Such administration can be carried out via a single dose or multiple doses. Those skilled in the art understand that the actual dose to be administered in the present invention can vary to a large extent depending on various factors, such as the target cells, biological type or its tissue, the general condition of the subject to be treated, the route of administration, the mode of administration, and so on.

[0052] Among them, in the above a1), the non-human animal can specifically be a non-human mammal, including but not limited to rats, mice, guinea pigs, rabbits, monkeys, orangutans, etc. The above non-human mammals are all animals with a normal immune system, rather than immunodeficient animals. Taking mice as an example, the mouse tumor-bearing model is an important means for tumor mechanism research and screening of anti-tumor drugs. Therefore, researchers often inoculate human tumor cells into immunodeficient nude mice to avoid immune rejection of human tumor cells by mice. Due to the immunodeficiency of nude mice, although the human tumor cells successfully form tumors, the results are often inconsistent with the actual situation, especially in the research on tumor immune mechanisms and tumor drug screening. Sometimes, it even brings opposite results, leading to misunderstandings in drug R & D and mechanism research, which is also one of the reasons for the slow progress of tumor research. In the present invention, by increasing the activity and expression of Sirt6, human tumor cells can grow into human tumor tissues in BALB / c mice with a normal immune system. Since the used mice have a perfect immune system, this human cancer cell tumor-bearing model and research results are closer to the actual situation of tumor clinics. Therefore, using this research model can be better used for the research on tumor pathogenesis, tumor immunity exploration, and tumor drugs.

[0053] Therefore,

[0054] In the above a1), the specific method for constructing a tumor-bearing model of a non-human animal with a normal immune system includes: transplanting the exogenous tumor cells treated with UBCS039 into a mouse with a normal immune system;

[0055] Specifically, the treatment concentration of UBCS039 is controlled at 1 - 200 μM, such as 10, 50, 100 μM. In a specific embodiment of the present invention, the treatment concentration of UBCS039 is 100 μM, and the treatment time of UBCS039 is 12 - 84 h, such as 12 h, 24 h, 48 h, 72 h, 84 h. In a specific embodiment of the present invention, the treatment time is 72 h.

[0056] The exogenous tumor cells can be human tumor cells, and the human tumor cells can be any human solid tumor or human hematological tumor cells. In a specific embodiment of the present invention, the human tumor cells are human liver cancer cells, such as SMMC - 7721 cells.

[0057] The mouse is specifically a BALB / c mouse.

[0058] In the above a2) - a6), the tumor can be any human solid tumor or human hematological tumor cells. In a specific embodiment of the present invention, the human tumor cells are human liver cancer cells, such as SMMC - 7721 cells.

[0059] And in one or more specific embodiments of the present invention, in the above applications, UBCS039 is used to pretreat tumor cells.

[0060] In the above a2), inhibiting the viability of tumor cells includes, but is not limited to, inhibiting the proliferation of tumor cells, inhibiting the migration or invasion of tumor cells, and promoting the apoptosis of tumor cells.

[0061] In the above a4)-a6), the immune cells are CD4+ T cells or NK cells;

[0062] In a4), when the immune cells are CD4+ T cells, in the co-culture system of SMMC-7721 cells pretreated with UBCS039 and CD4+ T cells, the proportion of Treg cells in CD4+ T cells increases significantly, and the proportion of CD38+ NK-like T cells also increases significantly, but the pretreatment of tumor cells with UBCS039 has no effect on the differentiation of NK cells.

[0063] In a5), when the immune cells are CD4+ T cells, the expression of PD-1 in tumor cells increases, the expression of PD-L1 in CD4+ T cells increases, and the level of ADO in the culture system increases significantly;

[0064] In another specific embodiment of the present invention, the application of Sirt6 as a target in the preparation or screening of anti-tumor drugs is provided.

[0065] Among them, the anti-tumor treatment is anti-tumor immunotherapy.

[0066] The tumors include solid tumors and hematological tumors, especially those tumors overexpressing Sirt6, such as ovarian, renal cell carcinoma, hepatocellular carcinoma, colon cancer, and prostate cancer, etc., which are not specifically limited herein.

[0067] The present invention will be further described below in conjunction with examples. The present invention will be further illustrated by the following examples, but the present invention is not limited to the scope of the examples. Based on the examples of the present invention, any changes to the present invention made by those skilled in the art without creative efforts fall within the protection scope of the present invention.

[0068] Examples

[0069] Materials and Methods

[0070] Cell Culture

[0071] Human hepatocellular carcinoma cells (SMMC-7721 cells) were obtained from ATCC. The tumor cells were maintained in RPMI 1640 medium (HyClone, USA) containing 10% (v / v) FBS (GIBCO) and 1% (v / v) pen / strep (GIBCO) at 37 °C with 5% CO2. UBCS039 (MCE, China) was dissolved in dimethyl sulfoxide (DMSO, Solarbio, China). SMMC-7721 cells were treated with 100 μM UBCS039 for 72 h. Cells treated with DMSO or untreated were used as controls.

[0072] Cell proliferation assay

[0073] Cell proliferation was measured using an RTCA DPlus workstation (Agilent, USA). Cultured cells were collected in RPMI 1640 and added to the wells of an E-plate 16 and cultured in the RTCA workstation for 24 h to monitor cell proliferation in real time.

[0074] Flow cytometry for apoptosis detection

[0075] Apoptosis was analyzed using an Annexin V-Fluorescein Isothiocyanate (FITC) / Propidine iodide (PI) apoptosis detection kit (Elabscience, USA). Cultured cells were washed and resuspended in 1× Annexin V Binding Buffer. Annexin V-FITC and PI staining solution were added and incubated in the dark for 20 min. Stained cells were analyzed using a flow cytometer (Apogee A50, NovoCyte D2040R, UK).

[0076] Cell migration and invasion analysis using Transwell chambers

[0077] Cultured cells were collected and resuspended in serum-free medium. The serum-free cell suspension was added to the upper chamber of Transwell chambers (Corning, USA), and medium containing 20% FBS was added to the lower chamber. After incubation for 24 h, the cells were fixed with methanol, and the cells in the lower chamber were stained with 0.1% crystal violet (Solarbio, China) and photographed and counted using an EVOS M5000 cell imaging system (Thermo, USA).

[0078] Cell energy metabolism analysis

[0079] The cell density was 1×104 SMMC-7721 cells at 1 cell / well were placed in an XFp cell culture plate (Agilent, USA) and cultured overnight for 24 h at 37 °C in a CO2-free cell incubator. The culture medium was discarded and the detection solution (including RPMI1640, pyruvate, glucose, and glutamine) was added. The XFp cell culture plate was placed in a CO2-free cell culture chamber and cultured at 37 °C for 1 h. A test solution containing rotenone (Rot, 5 μM) / antimycin A (AA, 5 μM) (Agilent, USA) was added to well A of the probe plate, and a test solution containing oligomycin (Agilent, USA) was added to well B. After calibrating the probe plate in the Agilent Seahorse XFp system (Agilent, USA), the energy metabolism of the treated cells was detected. Data were analyzed on the website (https: / / seahorsaanalytics.agilent.com).

[0080] Real-time quantitative PCR

[0081] Total RNA was extracted from cultured cells or animal tissues using RNAiso Plus (Takara, Japan). The isolated RNA was reverse transcribed into complementary deoxyribonucleic acid (cDNA) using HiScript III RT SuperMix (Vazyme, China). The cDNA was added to ChamQ Universal SYBR qPCR Master Mix (Vazyme, China), and the mRNA expression level of the target gene was detected by fluorescence-based real-time quantitative PCR using LightCycle96 (Switzerland). The relative mRNA expression level was analyzed using the 2 -ΔΔ -CT calculation method. β-actin mRNA was used as an internal control to quantify the expression level of the target gene.

[0082] Tumor-bearing mice were established using BALB / c mice

[0083] Six-week-old BALB / c mice were purchased from Vital River Laboratory (China). SMMC-7721 cells were treated with UBCS039 at a final concentration of 100 μM for 72 h, then the culture medium was removed, washed with PBS, fresh medium was added, and cultured for 48 h. The cells were resuspended in the medium and seeded at 1×10 7Injected subcutaneously into the right axilla of mice at a concentration of 100 μl. Cells treated with DMSO were also injected into mice used as controls. There were 5 mice in each group. All animals were housed under specific pathogen-free conditions and treated in accordance with the Helsinki Convention for the Protection of Animals. Detection of cytokine levels in mouse serum and culture medium

[0084] Blood samples were collected from the experimental mice. The supernatant was collected by centrifugation. Various cytokine levels in mouse blood were detected using the mouse inflammation panel (13-plex) (BioLegend, USA). The cytokine concentration was determined using a flow cytometer (Apogee A50, novo cell d2040r, UK). The concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-β (IFN-β), interferon-γ (IFN-γ), interleukin-1α (IL-1α), interleukin (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12p70 (IL-12p70), interleukin-17A (IL-17A), interleukin-23 (IL-23), and interleukin-16 in mouse serum were analyzed using Legend_plex_v8.0 software (BioLegend, USA).

[0085] The culture supernatant was collected from the co-culture of SMMC7721 cells with CD4+ T cells or NK cells. The cytokine levels were determined by a similar method as above using the 13-plex Human Inflammation Panel (BioLegend, USA) according to the manufacturer's instructions.

[0086] Detection of subtypes of mouse peripheral blood lymphocytes

[0087] Peripheral blood was collected from experimental mice and placed into EDTA blood collection tubes, and then lysed with red blood cell lysate (Solarbio, China) on ice. White blood cells were collected by centrifugation, and various flow cytometry antibodies were added to the samples and incubated. CD3+ T cells were detected with APC-conjugated anti-Mouse CD3e (eBioscience, USA), CD3+CD4+ T cells were detected with APC-conjugated anti-Moouse CD3e and FITC-conjugated anti-Mouse CD4 (eBioscience, USA), CD3-CD19+ B cells were detected with APC-conjugated anti-Mouse CD3e and PE-conjugated anti-Mouse CD19 (eBioscience, USA), and CD38+ NK cells and CD38+ NK-like T cells were detected with APC-conjugated anti-Mouse CD3e, FITC-conjugated anti-mouse CD38 (BioLegend, USA) and PE-conjugated anti-mouse CD49b (BioLegend, USA).

[0088] Treg cells were detected according to the following protocol. APC anti-mouse CD4 and FITC anti-mouse CD25 antibodies (BioLegend, USA) (BioLegend, USA) were added to blood samples or CD4+ T cells. After centrifugation, the cell pellet was fixed with fixation buffer (BioLegend, USA). After centrifugation, the fixed cells were resuspended in permeabilization wash buffer (BioLegend, USA), and Perkin-Elmer (PE, CA, USA) anti-mouse FOXP3 antibody (BioLegend, USA) (BioLegend, USA) was added and incubated. The mixture was centrifuged and resuspended in low cytometry staining buffer.

[0089] The Th1 subset was detected using fluorochrome-conjugated antibodies specific for mouse CD4 and IFN-γ (BioLegend, USA), the Th2 subset was detected using fluorochrome-conjugated antibodies specific for mouse CD4 and IL-4 (BioLegend, USA), and the Th17 subset was detected using fluorochrome-conjugated antibodies specific for mouse CD4 and IL-17 (BioLegend, USA). The Th1 / Th2 ratio = the percentage of CD4+IFN-γ+ / CD4+IL4+. Cell surface antigens CD3 and CD4 were labeled for 30 minutes in the dark. Then, the cells were fixed and permeabilized in the fixation / permeabilization mixture for 1 hour. Finally, anti-mouse IFN-γ, IL-4, and IL-17 antibodies against intracellular antigens were added and incubated for 30 minutes in the dark.

[0090] The above lymphocytes were detected by flow cytometry (ACEA Biosciences, NovoCyte D2040 R), and the data were analyzed using FlowJo software BD (Franklin Lake, USA).

[0091] Mouse metabolic detection

[0092] These treated mice were placed in a metabolic cage for 24 hours, and their rates of VO2 (oxygen consumption) and VCO2 (carbon dioxide production) were measured using the Oxylet system (Panlab, Spain), which has O2 and CO2 sensors coupled to the SEDACOM infrared system. The respiratory quotient (RQ) is defined as the ratio of CO2 production to O2 consumption, and the energy expenditure (EE) was calculated using the Weir equation (EE = 1.44 x VO2 x (3.815 + 1.23 x RQ)).

[0093] Biochemical examination of mouse peripheral blood

[0094] The peripheral blood of the experimental mice was collected in heparin blood collection tubes. The concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), calcium (Ca), alkaline phosphatase (ALP), triglyceride (TG), creatinine (CREA), uric acid (UA), urea, lactate dehydrogenase (LDH), phosphorus (P), and glucose (GLU) in the mouse plasma were measured using an automatic biochemical analyzer (Mindray, China).

[0095] Mouse blood routine examination

[0096] Collect the peripheral blood of experimental mice in EDTA blood collection tubes. Use an automatic blood analyzer (Mindray) to measure the concentrations of white blood cells (WBCs), neutrophils (Neu), lymphocytes (Lym), monocytes (Mon), eosinophils (Eos), basophils (Bas), hemoglobin (HGB), and platelets (PLTs) in mouse blood. Computed tomography (PET-CT) examination of tumor-bearing mice.

[0097] Inject the imaging agent 18 F-fluorodeoxyglucose ( 18 F-FDG) into the tail vein of mice at a dose of 12 MBq per mouse. PET-CT projection scanning is performed using a PET-CT device (MadicLab PSA094, Madic Technology, China). PET-CT fusion of the images is performed using Pmod software (Madic Technology, China). The standardized uptake value (SUV) of tumor tissue is calculated by the following formula: SUV = activity concentration in the region of interest (MBq / ml) / injected activity concentration (MBq / ml) / body weight (kg).

[0098] Mouse bioluminescence imaging detection

[0099] Luciferase-positive SMMC-7721 cells are prepared conventionally using a lentiviral vector system (GeneChem, China). The cells are infected according to the manufacturer's protocol. Before injection, the luciferase-positive tumor cells are observed under NightOWL LB 983 (Berthold Technologies, Germany) using the D-luciferin (MCE, China) substrate. Luciferase-positive cells (1×10 7 ) in PBS are subcutaneously injected into BALB / c mice. After intraperitoneal injection of D-luciferin (150 mg / kg), tumor growth is monitored by bioluminescence imaging using NightOWL LB 983.

[0100] Histopathological observation of tumor tissues in tumor-bearing mice

[0101] Collect the tumor tissues and fix them with 4% paraformaldehyde. Dehydrate the tissues with ethanol, embed them in paraffin, section them with a microtome, and stain them with hematoxylin and eosin (HE). Observe the histopathological changes under a light microscope and analyze them by a hospital pathologist.

[0102] Immunohistochemical detection

[0103] The paraffin sections of tumor tissues were incubated overnight at 4°C with polyclonal antibodies against HepPar-1 antibody (CST, USA), cytokeratin 19 (CK19, CST, USA), glypican-3 (GPC-3, CST, USA), and arginase-1 (CST, USA). Then the tissue sections were treated with diaminobenzidine and stained with hematoxylin.

[0104] Immunofluorescence detection

[0105] The tumor tissues were fixed overnight at 4°C in periodate-lysine-paraformaldehyde solution (containing 0.05 M phosphate buffer containing 0.1 M L-lysine (pH 7.4), 2 mg / ml NaIO4, and 10 mg / ml paraformaldehyde), then the tissues were embedded in 5% low-gelling-temperature agarose (type VII-A, Sigma-Aldrich) and cut into 400-μm thick sections. The sections were incubated in Biodewax and clearing solution for 10 minutes, dehydrated in absolute ethanol for 5 minutes, and washed in distilled water. The slides with tissue sections were immersed in EDTA antigen retrieval buffer (pH 8.0) and kept at sub-boiling temperature for 8 minutes, allowed to stand for 8 minutes, and then kept at another sub-boiling temperature for 7 minutes. After incubation with 10% donkey serum as a blocking solution, the sections were incubated overnight at 4°C with FITC-labeled anti-CD25 antibody, and then incubated with Cy3-labeled antibody anti-Foxp3 (Abcam, USA) for 50 minutes at room temperature in the dark. After washing with PBS, the sections were covered with DAPI solution. Images were taken under an inverted confocal microscope (IXplore, Olympus). DAPI emits blue light at an ultraviolet excitation wavelength of 330 - 380 nm and an emission wavelength of 420 nm, FITC emits green light at an excitation wavelength of 465 - 495 nm and an emission wavelength of 515 - 555 nm, and CY3 emits red light at an excitation wavelength of 510 - 560 nm and an emission wavelength of 590 nm.

[0106] Isolation of monocytes

[0107] Peripheral blood was collected from healthy volunteers (n = 30). Ficoll cell separation solution (TBD, China) was added to the diluted blood. After centrifugation, monocytes (MNCs) were extracted by conventional methods and randomly mixed for every 10 samples.

[0108] Enrichment of NK cells

[0109] Add NK-Cell Biotin-Antibody Cocktail (Miltenyi Biotec, Germany) to the above-mentioned MNCs, which includes biotin-conjugated monoclonal anti-human antibodies that are expressed in all lymphocytes, monocytes, and plasma cells except NK cells. Also add Anti-biotinantibody-conjugated microbeads to the mixture. After centrifugation, apply the cell suspension to a MACS column (Miltenyi Biotec). The unlabeled cells representing NK cells are eluted and collected in the flow-through fraction. Add anti-CD56 antibody-conjugated microbeads from the kit to the enriched NK cells, and then load the mixture onto the MACS column. Remove the column from the separator and place it on a suitable collection tube. Add an appropriate amount of buffer to the column, and the immediately eluted fraction is enriched in CD3-CD56+ NK cells.

[0110] Enrichment of CD4+ T cells

[0111] Resuspend the above-mentioned MNCs in PBS containing 2 mM EDTA and 0.5% BSA. Add Naive CD4+T-Cell Biotin-Antibody Cocktail II (Miltenyi Biotec, Germany) to the MNCs, which reagent includes biotin-conjugated monoclonal antibodies against CD8, CD14, CD16, CD19, CD36, CD45RO, CD56, CD123, TCRγ / δ, and glycophorin A. Microbeads conjugated to a monoclonal antibiotin antibody are also added to the mixture. Place the MACS column in the magnetic field of a suitable MACS separator (Miltenyi Biotec), and add the cell suspension to the column. The labeled cells representing the enriched naive CD4+ T cells are eluted and collected in the flow-through fraction.

[0112] Co-culture of SMMC-7721 cells with human NK cells or CD4+ T cells

[0113] Treat SMMC-7721 cells with UBCS039 at a final concentration of 10 nM for 24 hours. Remove UBCS039 by centrifugation and dilute the cells with fresh medium. Place the treated tumor cells at a concentration of 3×10 4 / 100 μl in the upper chamber of a Transwell (Corning Incorporated, Costar, USA) with a pore size of 0.4 μm. Place NK cells at a concentration of 3×10 4 / 600 μl or CD4+ T cells at a concentration of 3×10 4 / 600 μl in the lower chamber of the Transwell device. Co-culture these cells for 48 hours.

[0114] Detection of lymphocyte subtypes in cell co-culture

[0115] Collect NK or CD4+ T cells in the lower chamber of the above Transwell device and resuspend them in PBS. Add AAPC anti-human CD38 (BioLegend, USA), FITC anti-human CD3, and PE anti-human CD56 (BioLegend, USA) to the samples and incubate at 4°C for 30 min. Detect the proportions of CD38+ NK cells (CD38+CD3-CD56+) and CD38+ NK-like T cells (CD38+CD3+CD56+) using a flow cytometer (novo cell d 2040r). Analyze Treg cells and Th17 cells using a human Th17 / Treg staining kit (MULTI SCIENCES, China) according to the manufacturer's protocol.

[0116] Determination of adenosine (ADO) concentration

[0117] Collect the culture supernatant of co-cultured SMMC 7721 cells and CD4+ T cells. Determine the adenosine (ADO) level using an Adenosine Assay Kit (Fluorometric) (Abcam, USA) according to the manufacturer's protocol. Immediately measure the fluorescence on a microplate reader (Infinite M Plex, TECAN, Switzerland).

[0118] Data statistical analysis

[0119] Test the normality and homogeneity of variances using GraphPad Prism software (version 7). Analyze two groups using the LSD method, evaluate the significance of differences among multiple groups using one-way ANOVA, and perform correlation analysis using the Pearson correlation method.

[0120] Results

[0121] Effect of UBCS039 on SMMC-7721 cells

[0122] In this study, SMMC-7721 cells were treated with 100 μM UBCS039. Cell proliferation assays showed that compared with tumor cells treated with DMSO, UBCS039 treatment significantly inhibited the proliferation of SMMC-7721 cells. Cell apoptosis assays showed that UBCS039 treatment stimulated cell apoptosis ( Figure 6 A and B). Transwell analysis did not detect significant changes in cell migration or invasion between UBCS039-treated cells and the control group ( Figure 6 C). In addition, compared with tumor cells treated with DMSO, UBCS039 treatment significantly increased the OCR (oxygen consumption rate) and decreased the ECAR (extracellular acidification rate) in SMMC-7721 cells ( Figure 6 D). The above observations indicate that UBCS039 can inhibit the viability of tumor cells and inhibit cell glycolysis. In addition, UBCS039 treatment increased the expression level of Sirt6 mRNA, but did not change the expression levels of CD38, Sirt1, and Sirt3 ( Figure 7 ).

[0123] Effect of UBCS039-treated SMMC-7721 cells on tumor-bearing BALB / c mice

[0124] In this study, SMMC-7721 cells pretreated with UBCS039 were subcutaneously injected into BALB / c mice. Two weeks after injection, small masses were observed in the axilla of all 5 mice injected with UBCS039-pretreated cells. Eight weeks later, these masses grew into a prominent mass ( Figure 1 ). None of the 5 mice injected with SMMC-7721 cells containing DMSO or untreated cells had any swelling. HE staining showed that many cells in the mass tissue grew diffusely in lamellae, with obvious heterogeneity, were spindle-shaped, had a high rate of nuclear pyknosis, large and darkly stained nuclei, sparse cytoplasm, and mitosis was observed in some cells ( Figure 2 A). Immunohistochemistry detected significant expression of HepPar-1, CK19, GPC-3, and arginase-1 ( Figure 2 B). PET-CT detected an obvious area with high metabolic activity and high SUV in the mass ( Figure 2 C). We also injected fluorescent-labeled SMMC-7721 cells pretreated with UBCS039 into the mouse model. Significant fluorescent signals were detected in the mass area, indicating that the mass grew from exogenous human tumor cells rather than mouse-derived cells (Figure 2 D). The above examinations showed that the axillary mass in the mouse presented obvious human tumor characteristics.

[0125] In this study, the Oxylet Pro animal metabolic system was used to examine the energy consumption of these experimental mice. Compared with the normal mice injected with SMMC-7721 cells treated with DMSO, the tumor-bearing mice injected with cells treated with UBSC039 showed an increase in PER and a decrease in EE ( Figure 8 ). The peripheral blood of the experimental mice was detected by routine blood test indexes, biochemical examination and flow cytometry. Compared with the mice injected with SMMC-7721 cells treated with DMSO, the concentrations of GM-CSF, IL-6, IL-12p70, IL-23, and MCP-1 in the serum of the tumor-bearing mice were significantly decreased, and IL-1a was increased ( Figure 9 ), while the routine blood test indexes and biochemical indexes showed no significant differences between the two groups of mice (Tables 1 and 2). These measurement results indicated that tumor-bearing damaged the health of the mice and affected their immunity.

[0126] Table 1. Routine blood analysis of the peripheral blood of experimental mice

[0127]

[0128] Table 2. Biochemical analysis of the peripheral blood of experimental mice

[0129]

[0130] In this study, flow cytometry was used to measure the proportions of various lymphocyte subtypes in the experimental mice. Compared with the mice injected with SMMC-7721 cells treated with DMSO, in the peripheral blood of the tumor-bearing mice injected with SMMC-7721 cells pretreated with UBCS039, CD38+ B cells were significantly increased, but the proportions of NK, B, and CD38+ NK cells did not change significantly. Most importantly, compared with the control group, the proportion of Tregs in the tumor-bearing mice increased by 2.5 times, and the proportion of CD38+ NK-TT cells increased by 5 times. In addition, the proportion of Th1 / Th2 cells increased significantly in the tumor-bearing mice, while the proportion of Th17 / Treg cells showed almost no difference ( Figure 3 ). Immunofluorescence immunohistochemistry detected significant Foxp3 and CD25 immune signals in the mouse tumor tissue, indicating that many Treg cells expressing CD25 and Foxp3 infiltrated into the mouse tumor tissue ( Figure 10 ).

[0131] The effects of SMMC-7721 cells pretreated with UBCS039 on NK cells and CD4+ T cells.

[0132] SMMC-7721 cells pretreated with UBCS039 were co-cultured with NK cells or CD4+ T cells in transwell. Since UBCS039 is soluble in DMSO, SMMC-7721 cells in co-culture or SMMC-7721 cells pretreated with DMSO were used as controls. Whether the SMMC-7721 cells co-cultured with NK cells were pretreated with UBCS039 or DMSO, flow cytometry did not detect significant changes in the proportions of CD38+ NK cells and CD38+ NK-like T cells among the three groups of NK cells ( Figure 11 ). On the other hand, compared with CD4+ T cells co-cultured with untreated or DMSO-pretreated SMMC-7721 cells, the proportion of Treg cells in CD4+ T cells co-cultured with SMMC-7721 cells pretreated with UBCS039 increased significantly, although DMSO pretreatment also increased the proportion of Treg cells. Compared with CD4+ T cells co-cultured with untreated SMMC-7721 cells, the proportion of CD38+ NK-like T cells in CD4+ T cells co-cultured with SMMC-7721 cells pretreated with DMSO decreased, but the proportion of CD38+ NK-like T cells in CD4+ T cells co-cultured with SMMC-7721 cells pretreated with UBCS039 showed a significant increase. The proportion of Th17 cells in CD4+ T cells co-cultured with SMMC-7721 cells pretreated with DMSO or UBCS039 decreased significantly, but there was no difference in the proportion of Th17 cells in CD4+ T cells co-cultured with SMMC-7721 cells pretreated with DMSO or UBCS039. There was no statistically significant difference in the proportions of NK-like T and CD8+ T cells among the three groups ( Figure 4 ). The above results indicate that SMMC-7721 cells pretreated with DMSO have a certain inhibitory effect on the differentiation of CD4+ T cells into CD38+ NK-like T cells and a certain stimulatory effect on the differentiation into Treg cells. However, SMMC-7721 cells pretreated with UBCS039 itself have a greater stimulatory effect on the differentiation of CD4+ T cells into Treg cells and CD38+ NK-like T cells. Tumor cells pretreated with UBCS039 have no effect on NK cell differentiation.

[0133] In this study, real-time PCR was used to detect the expression of some important genes related to Sirt6 and immune surveillance in co-cultured cells. In SMMC-7721 cells, the mRNA levels of CD38, Sirt1, and Sirt3 did not change significantly whether treated with UBCS039 or DMSO. However, in SMMC-7721 cells pretreated with UBS039, the mRNA levels of Sirt6 and PD-1 increased significantly. Meanwhile, after co-culture with SMMC-7721 cells pretreated with UBS039, the mRNA level of PD-L1 in CD4+ T cells increased significantly. In addition, when CD4+ T cells were cultured with tumor cells pretreated with UBS039, the level of adenosine (ADO) in the culture medium increased significantly ( Figure 5 ). In this study, flow cytometry was also used to detect the concentration of each cytokine in the co-culture medium. Compared with the culture medium of co-culture of SMMC-7721 cells or SMMC-7721 cells pretreated with DMSO and CD4+ T cells, the concentration of MCP-1 in the culture medium of co-culture of SMMC-7721 cells pretreated with UBS039 and CD4+ T cells decreased significantly ( Figure 12 ).

[0134] In this study, the human hepatocellular carcinoma cell line SMMC-7721 was treated with UBCS039 and then injected into BALB / C mice. These pretreated human tumor cells were not immunologically rejected by the mouse immune system and grew into masses in BALB / C mice. Cells with typical tumor characteristics were detected in the masses by HE staining, and tumors with tumor image characteristics and active metabolism were detected by PET-CT. The expressions of CK19, arginase-1, GPC-3, and HepPar-1 were significantly increased in the masses detected by immunohistochemistry. CK19, arginase-1, GPC-3, and HepPar-1 are known liver tumor biomarkers, and these markers are commonly used in hospitals for the pathological diagnosis of liver cancer. We also injected fluorescently labeled SMMC-7721 cells pretreated with UBCS039 into mice, and obvious fluorescent signals were detected in the mass area, indicating that the tumor tissue was derived from the growth of exogenous human tumor cells rather than the malignant proliferation of mouse cells. In addition, the PER of these tumor-bearing mice decreased and the EE value increased, indicating that tumor growth affected the energy consumption of mice, which is often observed in tumor-bearing mouse models. The above observations indicate that human liver tumor cells with high Sirt6 activity can grow into human liver tumor tissues in immunocompetent BALB / C mice. We successfully established a tumor-bearing model derived from human tumors using immunocompetent mice instead of immunodeficient mice.

[0135] In the tumor microenvironment, tumor-infiltrating Tregs interact with tumor cells, stromal cells, and extracellular matrix components to establish an immunosuppressive phenotype. The activation or expansion of Tregs impedes tumor immunosurveillance and suppresses the anti-tumor immune response. This study found that the proportion of Tregs in peripheral blood increased in a mouse model, and the infiltration of Tregs in tumor-bearing tissues also increased significantly. In addition, when CD4+ T cells were co-cultured with tumor cells pretreated with UBSC039, the proportion of Tregs in CD4+ T cells increased significantly. Tregs are mainly developed from CD4+ T cells. Our observations showed that tumor cells with increased Sirt6 activity stimulated the differentiation of CD4+ T cells into Tregs, thereby inhibiting immunosurveillance and enhancing the immune tolerance of BALB / c mice to exogenous human tumor cells, which confirmed the importance of Sirt6 in establishing immune tolerance and immunosurveillance.

[0136] In this study, the proportion of CD38+ NK-like T cells in the peripheral blood of tumor-bearing mice was relatively high, but the proportion of CD38+ NK cells changed little. In addition, after co-culture with SMMC-7721 cells pretreated with UBCS039, CD38+ NK-like T cells increased significantly in CD4+ T cells. When NK cells were cultured with SMMC-7721 cells, whether or not the tumor cells were treated with UBCS039, the proportions of CD38+ NK cells and CD38+ NK-like T cells did not change significantly, indicating that tumor cells with increased Sirt6 activity did not affect the differentiation of NK cells into CD38+ NK cells or CD38+ NK-like T cells. We believe that tumor cells with increased Sirt6 activity can induce more CD4+ T cells to differentiate into Tregs by increasing the proportion of CD38+ NK-like T cells, which can explain the increase in Tregs in tumor-bearing mice after injection of human tumor cells pretreated with UBCS039. In our study, the mRNA expression of Sirt6 increased in SMMC-7721 cells treated with UBCS039 or in cells after co-culture with CD4+ T cells, but the expression levels of CD38, Sirt1, and Sirt3 did not change significantly. This result supports that UBCS039 enhanced the activity and expression level of Sirt6. Exogenous human tumor cells with increased Sirt6 activity hinder their immune surveillance of tumor growth and suppress the anti-tumor immune response by increasing Tregs in BALB / C mice.

[0137] In this study, UBCS039 treatment inhibited the proliferation of SMMC-7721 cells and stimulated apoptosis. In addition, UBCS039 treatment increased the oxygen consumption rate (OCR) and decreased the extracellular acidification rate (ECAR), thus reversing the metabolic balance of these tumor cells. This in vitro cell culture model showed that increased Sirt6 activity could inhibit tumor cell activity. Some studies have suggested that Sirt6 is a negative regulator of the Warburg effect. The increase in glycolysis within tumor cells is called the Warburg effect. Therefore, UBCS039 has completely different effects in vivo and in vitro. In vivo, UBSC039-pretreated tumor cells can stimulate immune escape of cells and promote tumor growth. Therefore, Sirt6 has two sides in tumor pathogenesis and progression.

[0138] Adenosine (ADO) emerged as one of the immune checkpoints, participating in the escape in the tumor-host immune system and inhibiting the anti-tumor immune response. The adenosine pathway is currently considered an important barrier to the effectiveness of immunotherapy and has become an important therapeutic target for cancer. Tregs expressing CD39 and CD73 can hydrolyze ATP into 5'-AMP and ADO, thereby mediating the inhibition of these immune cells. Based on the current results, we believe that after co-culture with tumor cells with enhanced Sirt6 activity, the increase in Tregs in CD4+ T cells led to a high level of ADO.

[0139] PD-L1 binds to PD-1 on the surface of T cells, initiating the PD-1 / PD-L1 immunosuppressive mechanism. This signaling pathway is currently one of the most promising immunotherapy targets. Cell types expressing PD-1 include CD8+ T cells, CD4+ T cells, and Tregs. PD-L1 is the natural receptor of PD-1 and is mainly expressed in tumor cells. The interaction between PD-1 and PD-L1 is one of the important mechanisms for immune escape in human tumors. In this study, we detected an increase in PD-L1 expression in UBCS039-pretreated SMMC-7721 cells, and an increase in PD-1 expression in CD4+ T cells after co-culture with UBCS039-pretreated tumor cells, indicating that tumor cells with increased Sirt6 activity can increase the expression of PD-1 and PD-L1, thereby inhibiting the anti-tumor immune response and facilitating successful tumor escape.

[0140] Tumor cells release various chemokines to attract Tregs to the tumor site. IL-1α secreted by tumor cells was identified as the main inducer of the Treg-attracting chemokine CCL22. An increase in the level of IL-1α was detected in the tumor-bearing mice in this study, which could explain the large number of infiltrating Tregs in the tumor-bearing tissue.

[0141] In summary, this study found that human tumor cells pretreated with the Sirt6 activator UBCS039 could grow into human tumor tissues in BALB / c mice under normal immune surveillance. Human tumor cells treated with UBCS039 could increase CD38+ NK-like T cells, stimulate CD4+ T cells to differentiate into more Tregs, and produce more ADO, PD-L1, and PD-1, which hindered immune surveillance and made the tumor microenvironment conducive to tumor growth.

[0142] The current findings may contribute to the understanding of immune escape in tumorigenesis. Increased Tregs are one of the most important reasons for suppressing the anti-tumor immune response, immune surveillance damage, and immune escape of tumor immune cells, but the reason has been unclear. This study used animal models and cell co-culture models to find that tumor cells with increased Sirt6 activity or expression could stimulate the differentiation of CD4+ T cells into Tregs, and at the same time increase the expression of ADO, PD-L1, and PD-1, resulting in the disruption of normal immune surveillance and the rapid growth of tumor cells escaping immune surveillance. Tumor cells with increased Sirt6 activity or expression may promote Treg differentiation by stimulating the differentiation of CD4+ T cells into CD4+ NK-like T cells. Tumor cells with increased Sirt6 activity or expression are an important reason for tumor escape from immune surveillance. Inhibiting the activity or expression of Sirt6 in tumor cells should be a new strategy for tumor immunotherapy.

[0143] The mouse tumor-bearing model is an important means for studying tumor mechanisms and screening anti-tumor drugs. Researchers often inoculate human tumor cells into immunodeficient nude mice to avoid immune rejection of human cells by mice. Due to the immune deficiency of nude mice, although human tumor cells are successfully tumor-bearing, the results often do not match the actual situation, especially in the research of tumor immune mechanisms and tumor drug screening, and sometimes even bring opposite results, leading to drug R & D and mechanism research falling into misunderstandings, which is also one of the reasons for the slow progress of tumor research. In this study, by increasing the activity and expression of Sirt6, human tumor cells were allowed to grow into human tumor tissues in BALB / c mice with a normal immune system. Since the mice used have a perfect immune system, this human cancer cell tumor-bearing model and research results are closer to the actual situation of tumor clinical practice. Therefore, using this research model can be better used for the study of tumor pathogenesis, tumor immunity exploration, and tumor drugs. In addition, since activating this gene can allow tumor cells in immune-normal mice to escape immune surveillance and grow, it shows that this gene locus is a key point for immune escape in tumors, especially in the early stage of tumorigenesis, and reverse inhibition of the activity of this gene should be beneficial to tumor prevention and treatment. Therefore, Sirt6 is also a key gene for tumor treatment, especially tumor immunotherapy.

[0144] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the given examples, those of ordinary skill in the art can modify or equivalently replace the technical solution of the present invention as needed, without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. Use of a substance that promotes Sirt6 expression or increases its activity in the following: a1) Establishing a tumor-bearing model of a non-human animal with a normal immune system; The substance that promotes Sirt6 expression or increases its activity is UBCS039; In the above a1), the specific method for constructing a non-human animal tumor-bearing model with a normal immune system includes: Transplanting exogenous tumor cells treated with UBCS039 into a mouse with a normal immune system, specifically a BALB / c mouse, and the exogenous tumor cells are human liver cancer cells; Among them, the treatment concentration of UBCS039 is controlled at 1 - 200 μM, and the treatment time is 12 - 84 h.

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