Application of dry memory T cells in preparation of medicine for treating lung metastasis of colorectal cancer

Abstract

The invention relates to application of a dry memory T cell in preparation of a medicine for treating colorectal cancer pulmonary metastasis, and belongs to the technical field of biological medicine. The invention firstly proves that the capability of differentiating the Tscm cells into effector killer T cells can be effectively improved by knocking out the Erbin genes in the Tscm cells in a mouse body, lung metastasis of colorectal cancer is inhibited, and the treatment effect of CAR-T cells can be enhanced by the Tscm cells with the Erbin genes knocked out. In addition, xanthine is detected for many times in a mouse colorectal cancer lung metastasis focus after the Erbin gene in the Tscm cell is knocked out, hypoxanthine can promote generation of more Tscm cells in the tumor metastasis focus, and the Tscm cells are differentiated to generate killer T cells, so that colorectal cancer lung metastasis is inhibited. The metabolic pathway of hypoxanthine is further studied, it is found that allopurinol can inhibit conversion of hypoxanthine into xanthine and uric acid, and it is found that allopurinol can also inhibit colorectal cancer lung metastasis by injecting allopurinol into a colorectal cancer lung metastasis mouse model.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and particularly to the application of stem memory T cells in the preparation of drugs for the treatment of lung metastasis of colorectal cancer. Background Art

[0002] Colorectal cancer (CRC) is one of the most common cancers in the world. In recent years, immunotherapy has made remarkable progress in the treatment of CRC. It is a treatment method that uses the body's own immune system to fight tumors. It can activate the patient's immune system, thereby generating immune memory against tumors, preventing tumor recurrence, and compared with traditional treatment methods, immunotherapy has relatively fewer side effects and better patient tolerance. However, this therapy still faces some challenges. For example, the in vivo expansion amount of CAR-T cells is not high and the persistence of CAR-T cells is not good. In addition, about 85% of CRC patients are microsatellite stable / mismatch repair gene wild-type (MSS / pMMR CRC), and for these patients, the efficacy of immune checkpoint inhibitor therapy is relatively low. Therefore, discovering new targets, especially targeting the characteristics of the tumor immune microenvironment, expanding the scope of application of immunotherapy, improving the treatment effect, overcoming drug resistance, and promoting individualized treatment are of great significance for promoting the development of the immunotherapy field.

[0003] In the anti-tumor immune response, T cells play a key role. Memory T cells have the ability to recognize and remember tumor antigens and can prevent tumor recurrence. By regulating the stemness and memory state of T cells, the anti-tumor activity of T cells can be enhanced and the effect of tumor immunotherapy can be improved. Stem memory T cells (Tscm) are a type of memory T cells with self-renewal ability and pluripotency. They exist in blood and lymphoid tissues. Tscm cells have strong proliferative ability and can differentiate into effector memory T cells (Tem) and central memory T cells (Tcm). Tscm cells have a long survival time and can be maintained in the body for decades. However, in the tumor microenvironment (TME), T cells are affected by various factors and lose their effector functions, manifested as reduced cytokine secretion, decreased killing ability, and weakened proliferative ability, making it easier for tumor cells to escape. In recent years, studies have shown that the TME has a significant impact on the function and phenotype of memory T cells. There are various inhibitory immune cells and factors in the TME, such as myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), TGF-β, IL-10, etc., which can inhibit the proliferation and activation of memory T cells. There are stress factors such as hypoxia and nutrient deficiency in the TME, which can promote the apoptosis and exhaustion of memory T cells. The TME can induce memory T cells to express inhibitory receptors, such as PD-1, CTLA-4, etc., thereby inhibiting their anti-tumor activity. Therefore, researchers are urgently needed to develop some strategies to transform memory T cells to make them more anti-tumor active.

[0004] In enhancing the long-term efficacy of T cell therapy, Tscm cells have become a hot topic of concern. Stem memory T cells are more suitable for immunotherapy than other memory cells such as Tcm cells. For example, Tscm cells have stronger self-renewal ability and pluripotency, and can continuously differentiate into effector T cells to maintain the anti-tumor immune response. After re-exposure to antigens, Tscm cells have stronger proliferative ability, stronger renewal ability to maintain a stable number in the long term, stronger migratory ability to reach the tumor site, stronger ability to recognize tumor antigens, and more effectively kill tumor cells. However, there is currently no study showing how Tscm cells affect the pulmonary metastasis of colorectal cancer. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the problem in the prior art that there is a lack of a therapeutic drug for pulmonary metastasis of colorectal cancer targeting stem memory T cells.

[0006] To solve the above technical problem, the present invention provides an application of stem memory T cells in the preparation of a therapeutic drug for pulmonary metastasis of colorectal cancer. The present invention first proves that knocking out the Erbin gene in Tscm cells in mice can effectively improve the ability of Tscm cells to differentiate into effector cytotoxic CD8+ T cells, inhibit the pulmonary metastasis of colorectal cancer, and Tscm cells with the Erbin gene knocked out can enhance the therapeutic effect of CAR-T cells. In addition, the present invention further detects the differential metabolites in the pulmonary metastases of colorectal cancer lung metastasis mice with conditional knockout of Erbin in CD8+ Tscm cells and wild-type colorectal cancer lung metastasis mice by metabolomics, and finds that more hypoxanthine is produced in the pulmonary metastases after knocking out the Erbin gene in Tscm cells. Injecting hypoxanthine into the colorectal cancer lung metastasis mouse model, it is found that hypoxanthine can promote the generation of more Tscm cells in the tumor metastases, differentiate into cytotoxic CD8+ T cells, reduce the number of exhausted T cells in the tumor, and inhibit the pulmonary metastasis of colorectal cancer. Further exploring the hypoxanthine metabolic pathway, it is found that allopurinol can inhibit the conversion of hypoxanthine to xanthine and uric acid. Injecting allopurinol into the colorectal cancer lung metastasis mouse model, it is found that allopurinol can also inhibit the pulmonary metastasis of colorectal cancer, further verifying the role of hypoxanthine in inhibiting the pulmonary metastasis of colorectal cancer.

[0007] The first object of the present invention is to provide an application of stem memory T cells in the preparation of a therapeutic drug for pulmonary metastasis of colorectal cancer.

[0008] Further, the therapeutic drug for pulmonary metastasis of colorectal cancer targets to reduce the expression of the Erbin gene in stem memory T cells.

[0009] Further, the therapeutic drug for pulmonary metastasis of colorectal cancer promotes the proliferation of stem memory T cells in the pulmonary metastases.

[0010] Furthermore, the therapeutic drug for colorectal cancer lung metastasis promotes the differentiation of stem memory T cells in lung metastases into cytotoxic T cells.

[0011] Furthermore, the therapeutic drug for colorectal cancer lung metastasis reduces the number of lung metastases.

[0012] Furthermore, the therapeutic drug for colorectal cancer lung metastasis includes hypoxanthine.

[0013] Furthermore, the therapeutic drug for colorectal cancer lung metastasis includes allopurinol.

[0014] The second object of the present invention is to provide a therapeutic drug for colorectal cancer lung metastasis, which includes stem memory T cells containing a chimeric antigen receptor; wherein, the Erbin gene in the stem memory T cells is knocked out, and the chimeric antigen receptor targets colorectal cancer tumors.

[0015] Furthermore, the therapeutic drug for colorectal cancer lung metastasis inhibits the metastasis of colorectal cancer tumors.

[0016] Furthermore, the therapeutic drug for colorectal cancer lung metastasis reduces the number of lung metastases.

[0017] Advantages of the present invention:

[0018] The present invention first proves that knocking out the Erbin gene in Tscm cells in mice can effectively improve the ability of Tscm cells to differentiate into effector cytotoxic CD8+ T cells, inhibit colorectal cancer lung metastasis, and Tscm cells with the Erbin gene knocked out can enhance the therapeutic effect of CAR-T cells. In addition, the present invention further detects differential metabolites in lung metastases of colorectal cancer lung metastasis mice with conditional knockout of Erbin in CD8+ T cells and wild-type colorectal cancer lung metastasis mice by metabolomics, and finds that more hypoxanthine is detected in the lung metastases of mice after knocking out the Erbin gene in Tscm cells. By injecting hypoxanthine into the colorectal cancer lung metastasis mouse model, it is found that hypoxanthine can promote the generation of more Tscm cells in tumor metastases, differentiate into cytotoxic CD8+ T cells, reduce the number of exhausted T cells in tumors, and inhibit colorectal cancer lung metastasis. Further exploring the hypoxanthine metabolic pathway, it is found that allopurinol can inhibit the conversion of hypoxanthine into xanthine and uric acid. By injecting allopurinol into the colorectal cancer lung metastasis mouse model, it is found that allopurinol can also inhibit colorectal cancer lung metastasis, further verifying the role of hypoxanthine in inhibiting colorectal cancer lung metastasis. The present invention provides a new therapeutic target for colorectal cancer lung metastasis and has good medical value. Description of the Drawings

[0019] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0020] Figure 1 This is a diagram of the identification of mice with conditional knockout of Erbin in CD4+Tscm cells / CD8+Tscm cells;

[0021] Figure 2 It is a schematic diagram of obtaining Tscm cells;

[0022] Figure 3 It is a method of adoptively transferring Tscm cells to tumor-bearing mice in vivo;

[0023] Figure 4 It's Erbin - / - The results of CarTscm inhibiting colorectal cancer lung metastasis, where Ctrl is NCG tumor-bearing mouse with only CART vector, WT+CarTscm is NCG tumor-bearing mouse with Car vector connected to wild-type Tscm cells, Erbin - / - +CarTscm connects Erbin to Car carrier - / - NCG tumor-bearing mice with Tscm cells, a is the in vivo imaging of three types of mice, b is the detection of lung metastasis at three time points, and the in vivo quantitative analysis of bioluminescence results by the in vivo imaging system (IVIS), c is the general view of the lung metastasis of mice on the 20th day, d is the HE staining result of the lung metastasis;

[0024] Figure 5 Figure 1 is the result of inhibiting colorectal cancer lung metastasis by targeting Erbin gene in Tscm cells, including the gross appearance of lung metastasis in CD4+Tscm cell conditional knockout Erbin mice (a), HE staining of lung metastasis (b), quantitative number of lung metastasis and ratio of lung weight to body weight (c); df is the gross appearance of lung metastasis in CD8+Tscm cell conditional knockout Erbin mice (d), HE staining of lung metastasis (e), quantitative number of lung metastasis and ratio of lung weight to body weight (f);

[0025] Figure 6 The effect of Erbin knockout in Tscm cells on the differentiation of multiple organ cells, including flow cytometry analysis of the ratio of CD4+ / CD8+CD62LhighCD44low (Tscm) cells in the axillary lymph nodes (a), inguinal lymph nodes (b), mesenteric lymph nodes (c), and lung metastases (d) of mice with Erbin knockout in CD4+T cells and CD8+T cells;

[0026] Figure 7They are differential metabolites in the lung metastases of CD8+ Tscm cell conditional knockout Erbin mice and wild-type mice detected by metabolomics;

[0027] Figure 8 Hypoxanthine (HY) inhibits the lung metastasis of colorectal cancer in wild-type mice. Among them, a. Gross appearance of lung metastases in CD8+ T cell conditional knockout Erbin mice, wild-type mice receiving hypoxanthine (HY), and wild-type mice; b. Ratio of lung weight to body weight in three groups of mice; c. HE staining of lung metastases in three groups of mice; d. Number of lung metastases in three groups of mice;

[0028] Figure 9 Hypoxanthine (HY) promotes the differentiation of Tscm. Among them, the results of quantitative flow cytometry analysis of the proportion of CD4+ / CD8+CD62LhighCD44low (Tscm) cells in the axillary lymph nodes (a), inguinal lymph nodes (b), and lung metastases (c) of CD8+ T cell conditional knockout Erbin mice, wild-type mice receiving hypoxanthine (HY), and wild-type mice;

[0029] Figure 10 It is a graph showing the proliferation of hypoxanthine on CD8+ Tscm cells analyzed and quantified by flow cytometry;

[0030] Figure 11 It is a graph showing the inhibition of lung metastasis of colorectal cancer in wild-type mice by allopurinol (Allo). Among them, a is the gross appearance of lung metastases in CD8+ T cell conditional knockout Erbin mice, wild-type colorectal cancer lung metastasis mice injected with allopurinol (Allo), and wild-type colorectal cancer lung metastasis mice; b is the ratio of lung weight to body weight in three groups of mice; c is the HE staining of lung metastases in three groups of mice; d is the number of lung metastases in three groups of mice;

[0031] Figure 12 It is a graph showing the effect of allopurinol on the differentiation of Tscm, including the proportion of CD4+ / CD8+CD62LhighCD44low (Tscm) cells in the axillary lymph nodes (a), inguinal lymph nodes (b), and lung metastases (c) of CD8+ T cell conditional knockout Erbin mice, wild-type mice treated with allopurinol (Allo), and wild-type mice;

[0032] Figure 13 Allopurinol (Allo) promotes the proliferation of Tscm. Detailed implementation manners

[0033] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the specific embodiments cited are not intended to limit the present invention.

[0034] The animal model symbols and their explanations involved in the following examples are as follows: (1) NCG mice are immunodeficient mice lacking the ability to synthesize T cells; (2) Erbin L / L ; CD8cre mice are mice with specific deletion of the Erbin gene in CD8 stem memory T cells; (4) Erbin L / L ; CD4cre mice are mice with deletion of the Erbin gene in CD4 stem memory T cells; (5) WT is a wild-type mouse model with colorectal cancer lung metastasis.

[0035] The methods for extracting lymphocytes from various organs in the following examples are as follows:

[0036] 1. Isolation of peripheral blood lymphocytes:

[0037] Collect peripheral blood (~500 μL) from mice and dilute it with PBS at a ratio of 1:1. Add the diluted blood onto the surface of Ficoll-Paque PLUS (GE Healthcare) and centrifuge at 450×g for 25 minutes. This step uses Ficoll-Paque density gradient centrifugation to separate mononuclear cells.

[0038] Collect the lymphocyte layer, wash it twice with PBS, and resuspend it in PBS containing 1% FBS. Ficoll-Paque density gradient centrifugation is a commonly used method for separating peripheral blood mononuclear cells (PBMCs) and can effectively remove red blood cells and granulocytes.

[0039] 2. Isolation of lymph node and spleen cells:

[0040] Excise the axillary lymph nodes (ALN), inguinal lymph nodes (ILN), and mesenteric lymph nodes (MLN), as well as the spleen, and rinse them with PBS. Mechanically dissociate the tissues using a syringe plunger. Filter the homogenate through a 100-μm filter mesh and centrifuge at 300×g for 7 minutes. Treat with ACK lysis buffer (for 5 minutes) to lyse red blood cells (RBCs). Wash the cells with PBS and resuspend them in PBS containing 1% FBS.

[0041] 3. Isolation of lymphocytes from lung metastases:

[0042] The lung metastases were dissected into 2 - 3 mm fragments and enzymatically digested for 30 minutes at 37°C in RPMI - 1640 medium containing 10% heat - inactivated fetal bovine serum (FBS; ABW), 1% penicillin - streptomycin (Beyotime, China), and 1% Collagenase III (Worthington Biochemical, NJ, USA). The digest was filtered through a 100 - μm cell strainer, centrifuged at 300×g for 7 minutes, and resuspended in 40% Percoll (GE Healthcare). Lymphocytes were isolated using 40% - 70% Percoll density gradient centrifugation, centrifuged at 800×g for 15 minutes. Percoll density gradient centrifugation can also be used to isolate PBMC and T lymphocytes. The lymphocyte - rich interface was collected and treated with ACK lysis buffer (5 minutes) to remove red blood cells. The cells were washed with PBS and resuspended in PBS containing 1% FBS.

[0043] Example 1: Obtaining donor - derived Tscm cells with Erbin gene knockout

[0044] (1) E L / L ; Obtaining CD4cre cells: Mice with conditional knockout of the Erbin gene in CD4 + T cells (E L / L ; CD4cre mice) were provided by Jiangsu Genscript Biogene Technology Co., Ltd., and their identification is as Figure 1 shown. To generate stem - cell - like memory T cells (Tscm cells), naive CD4 + T cells were isolated from the spleens of E L / L ; CD4cre mice using a CD4 + naive T cell isolation kit (BioLegend) and cultured on culture plates pre - coated with anti - CD3 (3 μg / mL) and anti - CD28 (1 μg / mL) antibodies. The medium was supplemented with IL - 7 (80 ng / mL) and IL - 15 (80 ng / mL) to induce the differentiation of Tscm cells. After culturing for 7 days, donor - derived E L / L ; CD4cre cells were obtained.

[0045] (2) E L / L ; Obtaining CD8cre cells: Mice with conditional knockout of the Erbin gene in CD8 + T cells (E L / L ; CD8cre mice) were provided by Jiangsu Genscript Biogene Technology Co., Ltd., and their identification is as Figure 1 shown. To generate stem - cell - like memory T cells (Tscm cells), CD8 + naive T cells were isolated from E L / L; Naive CD8+ T cells were isolated from the spleens of CD8cre mice and cultured on plates pre-coated with anti-CD3 (3 μg / mL) and anti-CD28 (1 μg / mL) antibodies. The culture medium was supplemented with IL-7 (80 ng / mL) and IL-15 (80 ng / mL) to induce the differentiation of Tscm cells. After culturing for 7 days, donor-derived E L / L ; CD8cre cells were obtained.

[0046] Example 2: Effect of Erbin gene knockout Tscm cells on mice with colorectal cancer lung metastasis

[0047] Pretreatment: 2.5×10 5 mouse colorectal cancer cells (MC38 cells) were injected into wild-type mice via the tail vein. Five days after the implantation of MC38 cells, the recipient mice received 1.25 Gy of radiation. The purpose of radiation was to eliminate the recipient mice's own immune cells and create conditions for the implantation of exogenous Tscm cells.

[0048] Tscm cell injection: Within 24 hours after radiation, wild-type recipient mice received 2×10 6 donor-derived E L / L ; CD4cre cells and E L / L ; CD8cre Tscm cells.

[0049] Re-establishment of the lung metastasis model: Twenty-four hours after the infusion of Tscm cells, MC38 cells were injected via the tail vein again to establish a colorectal cancer lung metastasis model. This step was to observe the effect of Tscm cells on tumor metastasis.

[0050] The experimental results were as Figure 5 shown. It was found that targeting the Erbin gene in Tscm cells could effectively inhibit the lung metastasis of colorectal cancer. Promote the proportion of Tscm cells infiltrated in the circulating organs of T cells in the host (multiple circulating lymph nodes, such as axillary, inguinal, and mesenteric lymph nodes), as well as in the lung tumors of tumor metastasis sites. Tscm cells targeting Erbin had strong self-renewal and the ability to differentiate into effector cytotoxic CD8+ T cells.

[0051] Example 3: Construction of Car-Tscm cells

[0052] The CAR molecule consists of an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain. The CAR gene in this example was provided by Zhenjiang Weigen Biotechnology Company.

[0053] Method for constructing CarTscm:

[0054] (1) Isolation of T cells from mouse spleen: Female mice at 6 weeks of age were euthanized, and their whole bodies were disinfected with 75% alcohol and then placed in a sterile operating table. After the mouse spleen was removed, it was ground with a 45-μm filter sieve to make a single-cell suspension. After centrifugation, the supernatant was discarded, and the precipitate was resuspended with ACK red blood cell lysate. After 3 minutes, 2 volumes of NCS neutralizing medium were added. After centrifugation, there was no visible red precipitate to the naked eye. At the same time, the precipitate was resuspended with PBS and counted. Finally, the mouse T cell medium was used to inoculate the cells into a culture dish at a density of 1×10 7 cells / mL and cultured in an incubator with 5% CO2 at 37°C for 24 h.

[0055] (2) Activation of mouse T cells: The mouse T cells cultured for 24 h after spleen isolation were re-counted. The mouse T cell medium was used to resuspend the cells at a density of 1×10 7 cells / mL and inoculated into a culture dish, and concanavalin (ConA) and human interleukin-2 (IL-2) at 100 U / mL were added. Then it was cultured in an incubator with 5% CO2 at 37°C for 24 h.

[0056] (3) Viral transduction of mouse Tscm cells: The mouse spleen cells after 24 h of activation were changed to fresh medium and transduced with the supernatant of Rv-EGFRvIII·CAR-T2A-GFP virus. The RetroNectin-coated 24-well plate was placed in a 4°C refrigerator overnight in advance. Then the coating solution was discarded, 1 mL of virus supernatant, 1×10 6 cells and 10 μg / mL of Polybrene were added. After mixing, it was centrifuged at 2000 g at room temperature for 60 min. After centrifugation, it was cultured in an incubator with 5% CO2 at 37°C. At 24-48 h after transduction, the GFP expression of CAR-T cells was observed by an inverted fluorescence microscope.

[0057] (4) Flow cytometry detection of the transduction rate of CAR-Tscm cells: At 4 days after transduction and culture, 2×10 5 cells were resuspended with 100 μL of FACS fixative, and 1.5 μL of anti-mouse Fab-APC antibody was added. After mixing, it was placed on ice for 30 min. After the ice bath, it was first washed once with 900 μL of PBS, the supernatant was discarded after centrifugation, then the precipitate was resuspended with 1 mL of PBS, the supernatant was discarded after centrifugation, and finally the precipitate was fixed to a volume of 300 μL for preparation of detection on the machine, and the data was exported for analysis.

[0058] Example 4: Effect of CAR-loaded Tscm cells on lung metastasis of colorectal cancer

[0059] (1) Construction of NCG tumor-bearing mice with only CAR-Tscm cells

[0060] To establish a CRC lung metastasis mouse model for CAR-Tscm immunotherapy, 5×10 5 MC38-LUC-RFP+EGFRvⅢ (human) cells were intravenously injected into immunodeficient mice (NCG mice) to obtain a mouse model of colorectal cancer lung metastasis (NCG tumor-bearing mice). On the 7th day, 2×10 6 CAR-Tscm cells constructed in vitro in Example 3 were adoptively transferred into NCG tumor-bearing mice via the tail vein to construct NCG tumor-bearing mice with only the CART vector.

[0061] (2) NCG tumor-bearing mice with the Car vector linked to wild-type Tscm cells

[0062] 5×10 5 MC38-LUC-RFP+EGFRvⅢ (human) cells were intravenously injected into immunodeficient mice (NCG mice) to obtain a mouse model of colorectal cancer lung metastasis (NCG tumor-bearing mice). On the 7th day, 2×10 6 CAR-Tscm cells constructed in vitro (where the Tscm cells were from wild-type mice) were adoptively transferred into NCG tumor-bearing mice via the tail vein to construct NCG tumor-bearing mice with the Car vector linked to wild-type Tscm cells.

[0063] (3) NCG tumor-bearing mice with the Car vector linked to Erbin - / - Tscm cells

[0064] To establish a CRC lung metastasis mouse model for CAR-Tscm immunotherapy, 5×10 5 MC38-LUC-RFP+EGFRvⅢ (human) cells were intravenously injected into immunodeficient mice (NCG mice) to obtain a mouse model of colorectal cancer lung metastasis (NCG tumor-bearing mice). On the 7th day, 2×10 6 CAR-E L / L ; CD8cre cells (i.e., Erbin - / - Tscm cells) were adoptively transferred into NCG tumor-bearing mice via the tail vein to construct a mouse model of colorectal cancer lung metastasis with Erbin gene deletion in Tscm cells.

[0065] Bioluminescence imaging was performed on tumor-bearing NCG mice on days 0, 7, and 14 respectively. All groups were euthanized on the 26th day. The above three types of NCG tumor-bearing mice were subjected to in vivo imaging detection and HE staining of lung metastases to observe their colorectal cancer lung metastasis conditions. The experimental results are as Figure 4As shown, the Erbin gene was targeted and knocked out in CD8+ Tscm cells (CD8+ stem memory T cells), and then presented to the Car vector. It was found that the CarTscm cells with the Erbin gene knocked out could more effectively target solid tumors and inhibit the metastasis of colorectal cancer tumors.

[0066] Example 5: Effect of Hypoxanthine on Mice with Colorectal Cancer Lung Metastasis

[0067] By metabolomics detection of differential metabolites in the lung metastases of mice with conditional knockout of Erbin in CD8+ T cells and wild-type mice, it was found that the content of hypoxanthine was higher in the lung metastases of mice with conditional knockout of Erbin in CD8+ T cells. Hypoxanthine (abbreviated as HY): an intermediate product of purine metabolism, which may affect cell growth and differentiation. In the tumor microenvironment, the change in the content of hypoxanthine may affect the behavior of tumor cells.

[0068] To explore the effect of hypoxanthine on mice with colorectal cancer lung metastasis, wild-type mice with colorectal cancer lung metastasis were intraperitoneally injected with 1 mg of hypoxanthine (Sigma) every two days (WT+HY) until the animals were sacrificed and further experimental analysis was performed. Wild-type mice with colorectal cancer lung metastasis without hypoxanthine injection (WT) and mice with specific knockout of the Erbin gene (E L / L ) were used as controls. It was found that the gross appearance of the lung metastases in mice with specific knockout of the Erbin gene was similar to that of wild-type mice with colorectal cancer lung metastasis that received hypoxanthine, and the number of lung metastases in wild-type mice after injection of hypoxanthine decreased significantly ( Figure 8 and Figure 9 ). Quantitative flow cytometry results showed that knocking out the Erbin gene in Tscm cells caused the cells to produce more hypoxanthine, and hypoxanthine could further promote the activation, proliferation of Tscm cells and the ability to effectively kill tumor cells. The method for detecting the proliferation ability of Tscm cells by CFSE: Splenocytes (1×10 6 ) were labeled with 10 μM CFSE (37 °C, 20 minutes), cultured for 5 days, and then analyzed by flow cytometry (FlowJo v10.8). All centrifugation steps: 300 g, 4 °C. By detecting the proliferation ability of Tscm cells by the CFSE method, it was found that knocking out the Erbin gene and injecting hypoxanthine could both improve the proliferation ability of Tscm cells ( Figure 10 ).

[0069] Example 6: Effect of Allopurinol on Mice with Colorectal Cancer Lung Metastasis

[0070] In the study of the hypoxanthine metabolic pathway, it was found that allopurinol (Allo) plays an important role in hypoxanthine metabolism. Allopurinol is a xanthine oxidase inhibitor and is commonly used to treat hyperuricemia and gout. It acts by inhibiting the conversion of hypoxanthine to xanthine and uric acid, that is, allopurinol can inhibit the further conversion of hypoxanthine and increase the content of hypoxanthine.

[0071] To further explore the effect of allopurinol on the lung metastasis of colorectal cancer, wild-type mice with lung metastasis of colorectal cancer were gavaged with 50 mg / kg of allopurinol (WT+Allo) for about 20 days until the animals were sacrificed for further experimental analysis. Wild-type mice with lung metastasis of colorectal cancer injected with allopurinol (WT) and Erbin gene-specific knockout mice (E L / L ) were used as controls. It was found that the gross appearance of the lung metastases in Erbin gene-specific knockout mice was similar to that in wild-type mice with lung metastasis of colorectal cancer treated with allopurinol, and the number of lung metastases in wild-type mice injected with allopurinol decreased significantly. Allopurinol can also improve the differentiation ability and proliferation ability of Tscm, increase the number of Tscm cells and promote their differentiation into cytotoxic CD8+ cells, further inhibiting the lung metastasis of colorectal cancer ( Figures 11 - 13 ).

[0072] Obviously, the above embodiments are only examples for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. Application of stem memory T cells in the preparation of therapeutic drugs for colorectal cancer lung metastasis.

2. The use according to claim 1, characterized in that: The colorectal cancer lung metastasis therapeutic drug targets and reduces the expression of the Erbin gene in stem memory T cells.

3. The use according to claim 1, characterized in that: The colorectal cancer lung metastasis therapeutic drug promotes the proliferation of stem memory T cells in lung metastases.

4. The use according to claim 3, characterized in that: The colorectal cancer lung metastasis therapeutic drug promotes the differentiation of stem memory T cells in lung metastases into killer T cells.

5. The use according to claim 4, characterized in that: The colorectal cancer lung metastasis therapeutic drug reduces the number of lung metastatic foci.

6. The use according to claim 5, characterized in that: The colorectal cancer lung metastasis treatment drug includes hypoxanthine.

7. The use according to claim 5, characterized in that: The colorectal cancer lung metastasis treatment drug includes allopurinol.

8. A drug for treating lung metastasis of colorectal cancer, characterized in that: The colorectal cancer lung metastasis therapeutic drug includes stem memory T cells containing chimeric antigen receptors; wherein the Erbin gene in the stem memory T cells is knocked out, and the chimeric antigen receptor targets colorectal cancer tumors.

9. The drug for treating colorectal cancer lung metastasis according to claim 8, characterized in that: The colorectal cancer lung metastasis therapeutic drug inhibits the metastasis of colorectal cancer tumors.

10. The drug for treating colorectal cancer lung metastasis according to claim 8, characterized in that: The colorectal cancer lung metastasis therapeutic drug reduces the number of lung metastatic foci.

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