Diet and PD-1 antibody-based microsatellite-stabilized colorectal cancer combination therapy

Abstract

The invention discloses a microsatellite stable colorectal cancer combination therapy based on diet and a PD-1 antibody, and belongs to the field of cancer treatment. The invention particularly relates to application of a methionine limited dietary therapy product and a PD-1 antibody in preparation of a medicine for treating microsatellite stable colorectal cancer. The invention discloses the combined use of a methionine limited diet therapy product and a PD-1 antibody, can promote CD8T cell infiltration and inhibit tumor growth, and also discloses a new mechanism of applying methionine limited diet to anti-tumor therapy, namely, the limited diet inhibits the expression of tumor cell immunometabolism checkpoint PCSK9 protein.

Description

Technical Field

[0001] The present invention relates to the technical field of cancer treatment, and specifically to a combination therapy for microsatellite stable colorectal cancer based on diet and PD-1 antibody, namely PD-1. Background Art

[0002] With the aging of the population, the number of new cancer cases and deaths in China has been continuously increasing, and the situation of cancer prevention and treatment is severe. Immunotherapy is an important means for treating malignant tumors after traditional treatment methods such as surgery, radiotherapy, and chemotherapy. Compared with traditional therapies, immunotherapy focuses on the body's own immune system and recognizes and eliminates tumor cells by restoring or reconstructing its own immune capacity, with characteristics such as significant curative effect and long-lasting effect (complete cure in some patients), which is a major progress in the field of tumor treatment in recent years. However, in colorectal cancer (CRC), which ranks third in terms of global incidence and mortality, the PD-1 / PD-L1 blockade therapy has little effect and is only recommended as the first-line treatment for highly microsatellite unstable (MSI-H) colorectal cancer, which accounts for about 5% of advanced colorectal cancer. Patients with microsatellite stable (MSS) colorectal cancer, which accounts for about 95%, do not benefit, and there is an urgent need to develop new treatment strategies to improve the efficacy of immunotherapy for MSS colorectal cancer.

[0003] The role of diet in the development and treatment of cancer has always been a topic of great concern and has made significant progress in recent years. In the tumor microenvironment, nutrient competition is a key factor affecting the function of immune cells and tumor progression. Both immune cells and tumor cells compete for essential nutrients required to maintain their metabolic activities and growth. As an important nutrient, the transport and metabolism of amino acids are crucial for the activation, differentiation, and effector functions of immune cells. However, tumor cells compete with immune cells for amino acids by highly expressing selective transporters, impairing the proliferation, survival, and effector functions of immune cells. In addition to taking in amino acids to maintain their own proliferation and invasion, tumor cells also produce immunosuppressive metabolites through amino acid catabolism, targeting and inhibiting the function of immune cells and creating an immunosuppressive tumor microenvironment, mediating immunosuppression and immune escape. Therefore, immunotherapy targeting amino acid metabolism from two perspectives of inhibiting tumor growth and changing the microenvironment to enhance immunotherapy has important clinical application potential.

[0004] Aiming at the unsatisfactory efficacy of existing anti-tumor therapies in microsatellite stable colorectal cancer, the purpose of the present invention is to provide an anti-tumor combination therapy that combines methionine-restricted diet with PD-1 antibody, effectively inhibiting tumor growth and promoting CD8 T cell infiltration. Summary of the Invention

[0005] Methionine is an essential amino acid for protein synthesis and the production of the methyl donor S-adenosylmethionine (SAM). Methionine metabolism is crucial for the occurrence and development of tumors. Deletion of the methionine transporter SLC7A5 leads to a decrease in methionine influx in CD4 T cells, inhibiting the activation and differentiation of T cells. Methionine deficiency also inhibits histone H3K4 methylation in Th17 cells. Tumor cells highly express the methionine transporter SLC43A2, competing with CD8 T cells for methionine in the microenvironment, disrupting methionine metabolism and SAM production in CD8 T cells, thereby inhibiting histone H3K79 methylation, resulting in low expression of STAT5 and impairing T cell survival and function.

[0006] In addition, methionine-restricted diet can not only inhibit tumor cell m6A methylation to regulate the translation of immune checkpoint PD-L1 and VISTA, but also block cGAS methylation to increase cGAS activity and delay tumor progression. The inventors further found that the combination of methionine-restricted diet and anti-PD-1 antibody significantly enhanced the therapeutic effect on tumor-bearing mice with microsatellite-stable colorectal cancer. The present invention discovers a new mechanism of methionine-restricted diet applied to anti-tumor therapy, and its combination with anti-PD-1 antibody provides a new strategy for the clinical treatment of microsatellite-stable colorectal cancer.

[0007] Aiming at the deficiencies of the prior art, the present invention proposes a combined therapy for microsatellite-stable colorectal cancer based on diet and anti-PD-1 antibody. The object of the present invention can be achieved by the following technical solutions:

[0008] In the first aspect of the present invention, it relates to the application of a methionine-restricted diet product and an anti-PD-1 antibody in the preparation of a drug for treating microsatellite-stable colorectal cancer.

[0009] In the second aspect of the present invention, it relates to the application of a methionine-restricted diet product and an anti-PD-1 antibody in the preparation of a drug for increasing the number of CD8 + T cell infiltration in microsatellite-stable colorectal cancer.

[0010] In the third aspect of the present invention, it relates to the application of a methionine-restricted diet product in the preparation of a drug for inhibiting the expression of tumor cell PCSK9.

[0011] In the fourth aspect of the present invention, it relates to a combined product for treating microsatellite-stable colorectal cancer, comprising a methionine-restricted diet product and an anti-PD-1 antibody.

[0012] Optionally, the PD-1 antibody includes one or more of Nivolumab, Pembrolizumab, Toripalimab, Sintilimab, Camrelizumab, Tislelizumab, Serplulimab, Penpulimab, Zimberelimab, Pucotenlimab, Cadonilimab, Ivonescimab.

[0013] The fifth aspect of the present invention relates to a method for constructing a mouse model, including any of the following steps:

[0014] Step 1: Construct a subcutaneous xenograft model of microsatellite-stable colorectal cancer, divided into four groups, and the treatment methods are respectively feeding with normal feed and intraperitoneal injection of normal saline, feeding with normal feed and intraperitoneal injection of anti-PD-1 antibody, feeding with methionine-restricted feed and intraperitoneal injection of normal saline, feeding with methionine-restricted feed and intraperitoneal injection of anti-PD-1 antibody; measure the size of the subcutaneous xenograft, and evaluate the effects of methionine-restricted diet and anti-PD-1 antibody on the tumor.

[0015] Step 2: Construct the subcutaneous xenograft model of microsatellite-stable colorectal cancer described in Step 1, using the same grouping and treatment methods as in Step 1; sacrifice the mice, take the subcutaneous tumor vesicles, and detect the infiltration of CD8 + T cells in the subcutaneous xenograft of microsatellite-stable colorectal cancer by immunohistochemistry.

[0016] Step 3: Construct the subcutaneous xenograft model of microsatellite-stable colorectal cancer described in Step 1, divided into four groups, and the treatment methods are respectively feeding with normal feed and intraperitoneal injection of PBS, feeding with methionine-restricted feed and intraperitoneal injection of anti-PD-1 antibody, feeding with normal feed and intraperitoneal injection of PBS and CD8 neutralizing antibody, feeding with methionine-restricted feed and intraperitoneal injection of anti-PD-1 antibody and CD8 neutralizing antibody; regularly measure the size of the subcutaneous xenograft, and evaluate the effects of methionine-restricted diet and anti-PD-1 antibody on the tumor.

[0017] Step 4: Pretreat human SW480, SW620, HCT116, LoVo cells, mouse MC38 cells and Colon26 cells with methionine-free medium for 6-8 hours, and then change to medium containing different amounts of methionine and continue to culture for 24 hours. Detect the content of PCSK9 in the cells by immunoblotting and detect the content of PCSK9 in the culture supernatant by ELISA; inoculate Colon26 cells or MC38 cells subcutaneously into the lower back of mice to establish a subcutaneous xenograft model of mouse colorectal cancer; detect the effect of methionine-restricted diet on the expression of PCSK9 in the subcutaneous xenograft of mouse colorectal cancer by immunohistochemistry.

[0018] Optionally, the immunohistochemistry method in the second step includes the following steps:

[0019] Take out the subcutaneous tumor and soak it in paraformaldehyde tissue fixative. Embed the fixed tissue in paraffin and cut it into sections. Place the sections in sodium citrate and heat them in a pressure cooker for antigen retrieval. Pre-drop the repaired tissue sections with goat serum blocking solution; then incubate them overnight in a wet box with the primary antibody against CD8, and use the secondary antibody rabbit anti-mouse IgG labeled with HRP.

[0020] Optionally, the immunohistochemistry method in the fourth step includes the following steps: Take out the subcutaneous tumor and soak it in paraformaldehyde tissue fixative. Embed the fixed tissue in paraffin and cut it into sections. Place the sections in sodium citrate and heat them in a pressure cooker for antigen retrieval. Pre-drop the repaired tissue sections with goat serum blocking solution; then incubate them overnight in a wet box with the primary antibody against PCSK9, and use the secondary antibody rabbit anti-mouse IgG labeled with HRP.

[0021] Optionally, the anti-PD-1 antibody includes one or more of Nivolumab, Pembrolizumab, Toripalimab, Sintilimab, Camrelizumab, Tislelizumab, Serplulimab, Penpulimab, Zimberelimab, Pucotenlimab, Cadonilimab, Ivonescimab.

[0022] Optionally, the methionine content in the methionine-restricted diet is 0.12% (w / w) or less.

[0023] Advantages of the present invention:

[0024] On the one hand, the present invention provides a combined anti-tumor therapy, which includes the combined treatment of methionine-restricted diet and PD-1 antibody for microsatellite stable colorectal cancer. Compared with the single use of PD-1 antibody and methionine-restricted diet, this combined therapy has better efficacy, significantly slows down tumor growth, and more CD8 T cells infiltrate into the tumor. After clearing CD8 T cells, the efficacy of the combined therapy of methionine-restricted diet and PD-1 monoclonal antibody disappears.

[0025] On the other hand, the present invention discovers a new mechanism of the application of methionine-restricted diet in anti-tumor therapy, that is, the restricted diet inhibits the expression of the tumor cell immune metabolic checkpoint PCSK9 protein.

[0026] In addition, the mouse model and experimental data constructed in the present invention can reflect the combined effect of methionine-restricted diet and PD-1 antibody, and also reflect the new mechanism of methionine-restricted diet applied to anti-tumor therapy, having potential value for academic research. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be further described below with reference to the accompanying drawings.

[0028] Figure 1 It is the tumor growth curve, tumor body photo and tumor weight statistical chart of subcutaneous transplanted tumors of the mouse Colon26 microsatellite stable (MSS) colorectal cancer (CRC) model under the conditions of methionine-restricted diet and PD-1 antibody alone or in combination;

[0029] Figure 2 It is the infiltration of CD8 + T cells in subcutaneous transplanted tumors of the mouse Colon26 microsatellite stable colorectal cancer model under the conditions of methionine-restricted diet and PD-1 antibody alone or in combination;

[0030] Figure 3 It is the effect of methionine-restricted diet and PD-1 antibody alone or in combination on the tumor growth of subcutaneous transplanted tumors of the mouse Colon26 microsatellite stable colorectal cancer model under the condition of depleting CD8 + T cells;

[0031] Figure 4 It is that in human and mouse colorectal cancer cells, supplementing methionine promotes the expression of PCSK9 protein;

[0032] Figure 5 It is that in the mouse colorectal cancer model, methionine-restricted diet inhibits the expression of tumor PCSK9 protein;

[0033] Figure 6 It is that in the mouse colorectal cancer model with knockdown of tumor PCSK9, methionine-restricted diet cannot further slow down tumor growth. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0035] Example 1: Detect the effect of the combined therapy of methionine-restricted diet and PD-1 antibody on the growth of subcutaneous transplanted tumors of mouse Colon26 microsatellite stable colorectal cancer.

[0036] 1. Research subjects

[0037] BALB / c mice at 6 - 8 weeks of age were purchased from Guangdong Zhiyuan Biomedical Technology Co., Ltd.; mouse Colon26 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd.

[0038] 2. Experimental methods

[0039] 1*10 6 Colon26 cells were inoculated subcutaneously into the lower dorsal side of BALB / c mice to establish a subcutaneous xenograft model of microsatellite - stable colorectal cancer in mice. After inoculating the tumor cells, the mice were randomly assigned to the following four groups for treatment:

[0040] ① Normal diet (ND) + saline, the mice were fed with normal diet on the day of subcutaneous tumor inoculation, and saline was administered by intraperitoneal injection on the 8th day after subcutaneous tumor inoculation. The volume and frequency of intraperitoneal injection of saline were the same as those of anti - PD - 1 antibody.

[0041] ② Normal diet (ND) + anti - PD - 1 antibody (α - PD - 1), the mice were fed with normal diet on the day of subcutaneous tumor inoculation, and 100 μg of anti - PD - 1 antibody (1 μg / μl) was administered by intraperitoneal injection on the 8th day after subcutaneous tumor inoculation, once every other day for a total of 6 times.

[0042] ③ Low - methionine diet (DMR) + saline, the mice were fed with low - methionine diet on the day of subcutaneous tumor inoculation, and saline was administered by intraperitoneal injection on the 8th day after subcutaneous tumor inoculation.

[0043] ④ Low - methionine diet (DMR) + anti - PD - 1 antibody (α - PD - 1), the mice were fed with low - methionine diet on the day of subcutaneous tumor inoculation, and anti - PD - 1 antibody was administered by intraperitoneal injection on the 8th day after subcutaneous tumor inoculation.

[0044] The size of the subcutaneous tumor was measured every other day, and the tumor volume was calculated using the formula: Volume = 0.52 * length * width 2 . When the subcutaneous tumor volume reached the ethical endpoint, all mice were euthanized, the subcutaneous tumors were dissected and weighed. Two - way ANOVA and t - test were used to evaluate the differences between the two groups. When *P < 0.05, **P < 0.01, ***P < 0.001, the differences were considered statistically significant.

[0045] 3. Experimental conclusions

[0046] Figure 1 In [reference], A is the specific administration time of the combination therapy of methionine - restricted diet and PD - 1 antibody.Figure 1 In B, it is the tumor growth curve of subcutaneous transplanted tumors in the mouse Colon26 microsatellite stable colorectal cancer model under the conditions of methionine-restricted diet alone or in combination with PD-1 antibody. Figure 1 In C, it is the photo of the tumor body of subcutaneous transplanted tumors in the mouse Colon26 microsatellite stable colorectal cancer model under the conditions of methionine-restricted diet alone or in combination with PD-1 antibody. Figure 1 In D, it is the statistical chart of the weight of subcutaneous transplanted tumors in the mouse Colon26 microsatellite stable colorectal cancer model under the conditions of methionine-restricted diet alone or in combination with PD-1 antibody.

[0047] The above results show that compared with the control group of mice, methionine-restricted diet alone or PD-1 antibody alone can slow down the growth rate of subcutaneous colorectal cancer transplanted tumors in mice and reduce the weight of transplanted tumors. While the combination of methionine-restricted diet and PD-1 antibody can further slow down the growth rate of subcutaneous colorectal cancer transplanted tumors in mice and reduce the weight of transplanted tumors, exerting a better therapeutic effect.

[0048] Example 2: Detect the effect of the combination therapy of methionine-restricted diet and PD-1 antibody on CD8 + T cell infiltration in subcutaneous transplanted tumors of mouse Colon26 microsatellite stable colorectal cancer.

[0049] 1. Research objects

[0050] BALB / c mice are the same as in Example 1, and Colon26 is the same as in Example 1.

[0051] 2. Experimental methods

[0052] The effect of methionine-restricted diet alone or in combination with PD-1 antibody on CD8 + T cell infiltration in subcutaneous transplanted tumors of mouse Colon26 microsatellite stable colorectal cancer was detected by immunohistochemistry. The specific steps are as follows: The establishment of the subcutaneous transplanted tumor model of mouse Colon26 microsatellite stable colorectal cancer and the grouping and treatment methods are the same as in Example 1. When the tumor growth reaches the end point, the mice are sacrificed, and the subcutaneous tumors are taken out and soaked in 4% paraformaldehyde tissue fixative, fixed at 4°C for 24 hours, the fixed tissues are paraffin-embedded and cut into 2.5 μm sections, the sections are placed in 10 mM sodium citrate, heated in a pressure cooker for 30 minutes for antigen repair, and the repaired tissue sections are pre-dropped with goat serum blocking solution. Then the primary antibody against CD8 (Cell Signaling Technology) is incubated overnight in a wet box at 4°C, and the secondary antibody is rabbit anti-mouse IgG labeled with HRP. The slices are scanned with a scanner (Hamamatsu) and the data are analyzed and statistically processed.

[0053] 3. Experimental conclusions

[0054] Figure 2 In which, A is the infiltration of CD8 + T cells in the subcutaneous transplanted tumors of the Colon26 microsatellite stable colorectal cancer model in mice under the conditions of immunohistochemical detection of methionine-restricted diet alone or in combination with PD-1 antibody. Figure 2 In which, B is the statistical chart of the number of CD8 + T cell infiltration per unit area in the subcutaneous transplanted tumors of the Colon26 microsatellite stable colorectal cancer model in mice under the conditions of immunohistochemical detection of methionine-restricted diet alone or in combination with PD-1 antibody.

[0055] The above results show that, compared with the control group, methionine-restricted diet or PD-1 antibody alone promotes the infiltration of CD8 + T cells in the tumor, while under the condition of combined use of methionine-restricted diet and PD-1 antibody, the infiltrated CD8 + T cells in the tumor increase significantly, indicating that the combined use of methionine-restricted diet and PD-1 antibody can effectively improve the current situation of low infiltration of CD8 + T cells in the subcutaneous transplanted tumors of the microsatellite stable colorectal cancer model, thereby enhancing the anti-tumor immune ability of the body.

[0056] Example 3: Under the condition of using CD8 neutralizing antibody to deplete CD8 + T cells, the tumor growth of the subcutaneous transplanted tumors of the Colon26 microsatellite stable colorectal cancer model in mice when methionine-restricted diet and PD-1 antibody are used alone or in combination.

[0057] 1. Research objects

[0058] BALB / c mice are the same as in Example 1, and Colon26 is the same as in Example 1.

[0059] 2. Experimental methods

[0060] The establishment of the subcutaneous transplanted tumor model of murine Colon26 microsatellite stable colorectal cancer was the same as that in Example 1. Mice were randomly assigned to the following four groups for treatment: ND + PBS group, DMR + α-PD-1 group, ND + PBS + α-CD8 group, DMR + α-PD-1 + α-CD8 group. The treatment methods of methionine-restricted diet and anti-PD-1 antibody were the same as those in Example 1. The ND + PBS + α-CD8 group and the DMR + α-PD-1 + α-CD8 group were intraperitoneally injected with 200 μg of CD8 neutralizing antibody (2 μg / μl) respectively, and the corresponding ND + PBS group and DMR + α-PD-1 group were intraperitoneally injected with isotype control IgG. The injection time points were 2 days before tumor inoculation, on the same day, and on the 4th, 8th, 12th, 16th days. The size of the subcutaneous tumor was measured every other day, and the tumor volume was calculated using the formula: Volume = 0.52 * length * width 2 . Two-way ANOVA was used to evaluate the differences between the two groups. When *P < 0.05, **P < 0.01, ***P < 0.001, the differences were considered statistically significant.

[0061] 3. Experimental conclusions

[0062] Figure 3 In A, it is the specific administration time of the combination therapy of methionine-restricted diet and PD-1 antibody under the condition that CD8 T cells are depleted by CD8 neutralizing antibody. + In B, it is the tumor growth curve of the subcutaneous transplanted tumor of the murine Colon26 microsatellite stable colorectal cancer model when methionine-restricted diet and PD-1 antibody are used in combination under the condition that CD8 T cells are depleted by CD8 neutralizing antibody. Figure 3 In A, CD8 refers to the depletion of CD8 + T cells.

[0063] The above results indicate that after depleting CD8 + T cells, the efficacy of the combination of methionine-restricted diet and PD-1 antibody disappeared. Together with the results of Example 2, it shows that methionine-restricted diet enhances the efficacy of immunotherapy for microsatellite stable colorectal cancer by increasing CD8 + T cell infiltration.

[0064] Example 4: In human and murine colorectal cancer cells, supplementing methionine promotes the expression of PCSK9 protein; in the murine colorectal cancer model, methionine-restricted diet inhibits the expression of tumor PCSK9 protein.

[0065] 1. Research objects

[0066] C57BL / 6 and BALB / c mice at 6 - 8 weeks of age were purchased from Guangdong Zhiyuan Biomedical Technology Co., Ltd.; human SW480, SW620, HCT116, LoVo cells and mouse MC38 cells were preserved in our laboratory, and mouse colon26 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd.

[0067] 2. Experimental methods

[0068] Human SW480, SW620, HCT116, LoVo cells and mouse MC38 cells and Colon26 cells were pretreated with methionine - free medium for 6 - 8 hours, and then changed to medium supplemented with different amounts of methionine and cultured for another 24 hours. The content of PCSK9 in cells was detected by immunoblotting, and the content of PCSK9 in the culture supernatant was detected by ELISA. The specific steps were as follows: Add PCSK9 monoclonal antibody (SEK10594, Sino Biological) to a 96 - well plate and incubate overnight at 4°C; Wash the plate and add PCSK9 standard or cell culture supernatant, and incubate at room temperature for 2 hours; Wash the plate and add biotin - labeled PCSK9 polyclonal antibody (SEK50251, Sino Biological), and incubate at room temperature for 1 hour; Wash the plate and add horseradish peroxidase - labeled avidin (Avidin - HRP), and incubate at room temperature for 30 minutes; Wash the plate and add TMB substrate to react for 15 minutes, and then terminate the reaction with 2N H2SO4. Read the absorbance at 450 nm and 570 nm with an enzyme - linked immunosorbent assay reader (Synergy H1, Agilent).

[0069] 1×10 6 Colon26 cells or MC38 cells were respectively inoculated subcutaneously into the lower dorsal side of BALB / c or C57BL / 6 mice to establish a subcutaneous xenograft model of mouse colorectal cancer. The effect of methionine - restricted diet on the expression of PCSK9 in subcutaneous xenografts of mouse colorectal cancer was detected by immunohistochemistry. The specific steps were as follows: Establish a subcutaneous xenograft model of mouse colorectal cancer and divide it into two groups, with normal diet or methionine - restricted diet. When the tumor growth reached the end point, the mice were sacrificed, and the subcutaneous tumors were removed and soaked in 4% paraformaldehyde tissue fixative and fixed at 4°C for 24 hours. The fixed tissues were paraffin - embedded and cut into 2.5 - μm sections. The sections were placed in 10 mM sodium citrate and heated in a pressure cooker for 30 minutes for antigen retrieval. The repaired tissue sections were pre - dropped with goat serum blocking solution. Then, the primary antibody against PCSK9 (Sino Biological) was incubated overnight in a wet box at 4°C, and the secondary antibody was HRP - labeled rabbit anti - mouse IgG. The slices were scanned with a scanner (Hamamatsu) and the data were analyzed and statistically processed.

[0070] 3. Experimental conclusions

[0071] Figure 4 In which, A is the content of PCSK9 protein in human SW480, SW620, HCT116, and LoVo cells after supplementing methionine. Figure 4 In which, B is the content of PCSK9 protein in the culture supernatant of human SW480, SW620, HCT116, and LoVo cells after supplementing methionine. Figure 4 In which, C is the content of PCSK9 protein in mouse MC38 and Colon26 cells after supplementing methionine. Figure 4 In which, D is the content of PCSK9 protein in the culture supernatant of mouse MC38 cells after supplementing methionine. Figure 5 A and B in which are that in the mouse MC38 colorectal cancer model, methionine-restricted diet inhibits the expression of tumor PCSK9 protein. Figure 5 C and D in which are that in the mouse Colon26 colorectal cancer model, methionine-restricted diet inhibits the expression of tumor PCSK9 protein.

[0072] The above results indicate that methionine deficiency significantly inhibits the expression of PCSK9 in colorectal cancer cells. The methionine-restricted diet experiment shows that methionine deprivation inhibits the expression of PCSK9 in colorectal cancer.

[0073] Example 5:

[0074] 1. Research subjects

[0075] BALB / c and C57BL / 6 mice are the same as in Example 4, and Colon26 and MC38 cells are the same as in Example 4.

[0076] 2. Experimental methods

[0077] The establishment of the subcutaneous xenograft tumor model of mouse colorectal cancer is the same as in Example 4. The methionine-restricted diet method is the same as in Example 1.

[0078] 3. Experimental conclusions

[0079] Figure 6 In which, A is the tumor growth curve of the subcutaneous xenograft tumor of the mouse MC38 colorectal cancer model under methionine-restricted diet conditions. Figure 6 In which, B is the weight statistical chart of the subcutaneous xenograft tumor of the mouse MC38 colorectal cancer model under methionine-restricted diet conditions. Figure 6 In which, C is the tumor growth curve of the subcutaneous xenograft tumor of the mouse Colon26 colorectal cancer model under methionine-restricted diet conditions. Figure 6 In which, D is the weight statistical chart of the subcutaneous xenograft tumor of the mouse Colon26 colorectal cancer model under methionine-restricted diet conditions. Figure 6In the figure, E and F are the tumor growth curves of subcutaneous xenografts of the MC38 colorectal cancer model with knockdown or knockout of PCSK9 by subcutaneous injection under the condition of methionine-restricted diet.

[0080] The above results indicate that methionine-restricted diet significantly inhibits the growth of MC38 and Colon26 tumors. However, after knockdown or knockout of PCSK9, methionine-restricted diet cannot further inhibit tumor growth. Combining the results of Examples 1-5, methionine-restricted diet inhibits tumor immune escape by suppressing the expression of PCSK9 in tumor cells.

[0081] In the description of this specification, the descriptions referring to terms such as "one embodiment", "example", "specific example", etc. mean that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0082] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments, and what is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed.

Claims

1. Application of methionine-restricted diet products and PD-1 antibodies in the preparation of drugs for the treatment of microsatellite-stable colorectal cancer.

2. Methionine-restricted diet products and PD-1 antibodies in the preparation of CD8 in microsatellite-stable colorectal cancer + T cell infiltration number in drug application.

3. Application of methionine-restricted dietary therapy products in the preparation of drugs for inhibiting PCSK9 expression in tumor cells.

4. A combination product for treating microsatellite stable colorectal cancer, characterized in that: Including methionine-restricted dietary therapy products and PD-1 antibodies.

5. The combination product for treating microsatellite stable colorectal cancer according to claim 4, characterized in that: The PD-1 antibody includes one or more of Nivolumab, Pembrolizumab, Toripalimab, Sintilimab, Camrelizumab, Tislelizumab, Serplulimab, Penpulimab, Zimberelimab, Pucotenlimab, Cadonilimab, and Ivonescimab.

6. A method for constructing a mouse model, characterized in that: This includes any of the following steps: Step 1: Construct a subcutaneous transplant tumor model of microsatellite stable colorectal cancer and divide it into four groups. The treatment methods are feeding with normal diet and intraperitoneal injection of normal saline, feeding with normal diet and intraperitoneal injection of anti-PD-1 antibody, feeding with methionine-restricted diet and intraperitoneal injection of normal saline, and feeding with methionine-restricted diet and intraperitoneal injection of anti-PD-1 antibody. The size of subcutaneous transplant tumors was measured to evaluate the effects of methionine-restricted diet and anti-PD-1 antibody on tumors. Step 2: Construct the microsatellite stable colorectal cancer subcutaneous transplant tumor model described in step 1, and use the same grouping and treatment methods as step 1; kill the mice, take the subcutaneous tumors, and detect the CD8 + T cell infiltration; Step 3: construct the subcutaneous transplant tumor model of microsatellite stable colorectal cancer described in step 1, and divide it into four groups. The treatment methods are normal feed feeding and intraperitoneal injection of PBS, methionine restricted feed feeding and intraperitoneal injection of anti-PD-1 antibody, normal feed feeding and intraperitoneal injection of PBS and CD8 neutralizing antibody, methionine restricted feed feeding and intraperitoneal injection of anti-PD-1 antibody and CD8 neutralizing antibody; regularly measure the size of subcutaneous transplant tumors to evaluate the effects of methionine restricted diet and anti-PD-1 antibody on tumors; Step 4: Pretreat human SW480, SW620, HCT116, LoVo cells and mouse MC38 cells and Colon26 cells with methionine-deficient culture medium for 6-8 hours, then switch to culture medium with different methionine content and continue culturing for 24 hours. Detect the intracellular PCSK9 content by Western blotting and the PCSK9 content in the culture supernatant by ELISA. Inoculate Colon26 cells or MC38 cells subcutaneously on the lower dorsal side of mice to establish a mouse colorectal cancer subcutaneous transplant tumor model. Detect the effect of methionine-restricted diet on the expression of PCSK9 in mouse colorectal cancer subcutaneous transplant tumors by immunohistochemistry.

7. The method for constructing a mouse model according to claim 6, characterized in that: The immunohistochemistry method in step 2 comprises the following steps: The subcutaneous tumor was removed and soaked in paraformaldehyde tissue fixative. The fixed tissue was embedded in paraffin and cut into slices. The slices were placed in sodium citrate and heated in a pressure cooker for antigen repair. Goat serum blocking solution was added to the repaired tissue slices in advance; then the primary antibody against CD8 was incubated in a wet box overnight, and the secondary antibody was rabbit anti-mouse IgG labeled with HRP.

8. The method for constructing a mouse model according to claim 6, characterized in that: Immunohistochemistry in step 4 The method comprises the following steps: taking out the subcutaneous tumor and soaking it in a paraformaldehyde tissue fixative, embedding the fixed tissue in paraffin and cutting it into slices, placing the slices in sodium citrate, heating them in a pressure cooker for antigen repair, and pre-adding goat serum blocking solution to the repaired tissue slices; then incubating them overnight in a wet box with an anti-PCSK9 primary antibody, and using a rabbit anti-mouse IgG labeled with HRP as the secondary antibody.

9. The method for constructing a mouse model according to claim 6, characterized in that: The anti-PD-1 antibodies include one or more of Nivolumab, Pembrolizumab, Toripalimab, Sintilimab, Camrelizumab, Tislelizumab, Serplulimab, Penpulimab, Zimberelimab, Pucotenlimab, Cadonilimab, and Ivonescimab.

10. The method for constructing a mouse model according to claim 6, characterized in that: The methionine content in the methionine-restricted feed is below 0.12% (w / w).

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