Application of microbial metabolite in tumor immune response and microbial metabolite screening method

Abstract

The invention provides application of microbial metabolites in tumor immune response and an intestinal microbial metabolite screening method. A screening system is constructed by combining firefly luciferase with PD-1 / SHP-2, various intestinal microbial metabolites are independently co-cultured with the screening system, the screened microbial metabolites are glycodeoxycholic acid and taurocholic acid, and the influence of various metabolites on PD-1 pathway signal transduction is reflected by measuring the activity of luciferase. The invention constructs a reliable, rapid and effective screening method for discovering intestinal microbial metabolites influencing a PD-1 signal channel and evaluating the influence efficacy of the metabolites, and screening out metabolites having effects on intestinal cancer immune response, thereby providing an experimental basis for enhancing intestinal cancer treatment.

Description

Technical Field

[0001] This invention relates to the field of cell biology, specifically to the application of microbial metabolites in tumor immune responses and methods and systems for screening microbial metabolites. Background Technology

[0002] Programmed death receptor 1 (PD-1) is an important immune checkpoint molecule, primarily expressed on activated T lymphocytes, B lymphocytes, and myeloid cells. The binding of PD-1 to its downstream protein, tyrosine phosphatase (SHP-2), which contains the Src homology 2 domain, can inhibit T cell activation and proliferation, thereby suppressing the immune response.

[0003] In the tumor microenvironment, tumor cells evade immune system surveillance by overexpressing programmed death receptor-ligand 1 (PD-L1), which binds to PD-1 on T cells, activating PD-1's recruitment of SHP-2. Therefore, the PD-1 / PD-L1 pathway has become an important target for immunotherapy. SHP-2, as a link in this pathway's signaling, also possesses the potential to target and regulate lymphocyte immunity.

[0004] Gut microbial metabolites play a crucial role in regulating the host immune response. Recent studies have shown that gut microbial metabolites can influence the efficacy of immunotherapy by modulating the PD-1 signaling pathway. Certain microbial metabolites, such as butyrate, can enhance T cell function by inhibiting histone deacetylases (HDACs) and suppressing PD-1 expression on T cells. Furthermore, the probiotic *Lactobacillus johnsonii* and its small molecule metabolite indolepropionic acid have been found to sustainably mobilize T cells to kill tumor cells.

[0005] Although some gut microbial metabolites targeting the PD-1 / PD-L1 pathway have been identified to enhance tumor therapy, these metabolites are few in number and have a weak effect on the immune response in colorectal cancer. Therefore, there is an urgent need for a new method to screen for more effective gut microbial metabolites to enhance the immune response in colorectal cancer. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, the purpose of this invention is to screen for new gut microbial metabolites that affect T cell function. By using a screening method based on PD-1 / SHP-2 pathway signal transduction, new gut microbial metabolites are screened out, and experimental evidence is provided for strategies to enhance colorectal cancer treatment with gut microbial metabolites.

[0007] The specific technical solution adopted in this application is as follows.

[0008] The application of microbial metabolites in tumor immune response, wherein the microbial metabolites are glycodeoxycholic acid (GDCA) and taurocholic acid (TCA).

[0009] Furthermore, the tumor is a colon cancer tumor.

[0010] Furthermore, glycodeoxycholic acid or taurocholic acid promote tumor growth by inhibiting T cell function.

[0011] Furthermore, inhibiting the production of glycodeoxycholic acid or taurocholic acid can enhance the immune response to colorectal cancer.

[0012] On the other hand, the present invention also provides a method for screening intestinal microbial metabolites based on PD-1 / SHP-2 pathway signal transduction, which screens out the microbial metabolites of the present invention: glycine deoxycholic acid and taurocholic acid, specifically including the following steps:

[0013] S1: Construction and validation of the PD-1 / SHP-2 screening system; among which,

[0014] S11, the C-terminus and N-terminus of firefly luciferase were fused to the ends of PD-1 and SHP-2, respectively, and constructed into overexpression plasmid vectors, named PD-1-C-luc and SHP-2-N-luc. PD-1-C-luc and SHP-2-N-luc were introduced into cells for overexpression. When the upstream protein of PD-1, programmed cell death-ligand 1, activated the recruitment of PD-1 to SHP-2, the divided luciferase protein halves approached each other, thereby restoring luciferase activity.

[0015] S12, PD-1-C-luc and SHP-2-N-luc plasmids were transfected into HEK293 cells, either separately or co-transfected, and the expression of the two plasmids was verified separately or simultaneously.

[0016] S13 and HEK293 cells were overexpressed with PD-1-C-luc and SHP-2-N-luc to establish a screening system. After culturing for 24 hours with or without PD-L1 and MB, luciferase activity was detected.

[0017] S2: Screening for gut microbial metabolites; among which...

[0018] A PD-1-C-luc / SHP-2-N-luc screening system was established in S21 and HEK293 cells. After co-culturing 510 intestinal microbial metabolites with HEK293 cells, luciferase activity was measured.

[0019] S22, The target metabolites that showed the effect of promoting or inhibiting the binding of PD-1 and SHP-2 in the preliminary screening were summarized and analyzed using the PD-1-C-luc / SHP-2-N-luc screening system.

[0020] S23, using the PD-1-C-luc / SHP-2-N-luc screening system to analyze bile acid metabolite pathway-related metabolites;

[0021] S24. Immunoprecipitation assay was used to analyze the effects of glycine deoxycholic acid and taurocholic acid screened on the binding of PD-1 and SHP-2 in HEK293 cells.

[0022] S25. Immunofluorescence assay was used to analyze the effects of glycodeoxycholic acid and taurocholic acid on the binding of PD-1 and SHP-2 in Jurkat T cells.

[0023] S3: Assay of T cell activation inhibition by glycodeoxycholic acid and taurocholic acid;

[0024] S4: Verification of the effects of glycodeoxycholic acid and taurocholic acid on inhibiting T cell function and promoting tumor growth.

[0025] Beneficial effects

[0026] This invention constructs a screening system by combining firefly luciferase with PD-1 / SHP-2. Various gut microbial metabolites are co-cultured independently with the screening system, and the influence of each metabolite on PD-1 pathway signaling is reflected by measuring the intensity of luciferase activity. This invention establishes a reliable, rapid, and effective screening and evaluation system for discovering gut microbial metabolites affecting the PD-1 signaling pathway and evaluating their efficacy, thereby screening for metabolites that play a role in the immune response to colorectal cancer and providing experimental evidence for enhancing anti-colorectal cancer therapy. Attached Figure Description

[0027] Figure 1 Construction and validation of the PD-1 / SHP-2 screening system. Among them:

[0028] A. Schematic diagram of the firefly luciferase screening system.

[0029] B. Transfect HEK293 cells with PD-1-C-luc and SHP-2-N-luc plasmids, either separately or together. After culturing the cells at 37°C and 5% CO2 for 24 hours, collect the cells and verify their expression by Western blotting.

[0030] C. HEK293 cells overexpressed with PD-1-C-luc and SHP-2-N-luc were cultured at 37℃ and 5% CO2 for 24 hours to establish a screening system. After culturing for another 24 hours with or without PD-L1 (5 μg / ml) and MB (1 μM), the culture medium was discarded, and the luciferase substrate D-Luciferin was added. The cells were incubated at 37℃ and 5% CO2 for 5 minutes, and then the luciferase activity was detected by a multi-functional microplate reader.

[0031] Data are expressed as mean ± SEM and analyzed by one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0032] Figure 2 Screening of gut microbial metabolites. Among them:

[0033] A. A PD-1-C-luc / SHP-2-N-luc screening system was established in HEK293 cells in 96-well plates. After co-culturing 510 intestinal microbial metabolites (10 uM) with the screening system for 24 hours, the luciferase activity was measured by the experimental method described above (adding luciferase substrate D-Luciferin, incubating at 37℃ and 5% CO2 for 5 minutes, and then detecting luciferase activity by a multi-functional microplate reader).

[0034] B. The target metabolites that showed the effect of promoting or inhibiting the binding of PD-1 and SHP-2 in the preliminary screening were summarized and analyzed using the PD-1-C-luc / SHP-2-N-luc screening system (n=3).

[0035] C. Under the same experimental conditions, the luciferase activity of bile acid metabolite pathway-related metabolites (10 uM) was measured using the PD-1-C-luc / SHP-2-N-luc screening system (n=3).

[0036] D. Immunoprecipitation assay to analyze the effects of GDCA and TCA on the binding of PD-1 and SHP-2: After overexpressing PD-1-C-luc / SHP-2-N-luc for 24 hours in HEK293 cells, GDCA (20 μM) and TCA (20 μM) were added and the cells were treated for another 24 hours. Cells were then collected and lysed. A portion of the cell lysate was collected as a control. Anti-PD-1 / IgG (1:500) was added to the remaining cell lysate, and the cells were incubated at 4°C on a shaker for 3 hours. Protein A agarose was then added and incubated overnight under the same conditions. The cell lysate and protein A agarose after overnight incubation were collected and used together with the cell lysate before incubation for Western blotting experiments.

[0037] E. Immunofluorescence assay to analyze the effects of GDCA and TCA on the binding of PD-1 and SHP-2: Jurkat T cells were treated with GDCA (20 μM) and TCA (20 μM) at 37°C and 5% CO2 for 24 hours. Cells were then fixed with 4% paraformaldehyde at 4°C for 10 minutes. Anti-PD-1 and anti-SHP-2 were added to each well, and the cells were incubated overnight at 4°C. Free anti-PD-1 and anti-SHP-2 were washed away with PBS, and the corresponding fluorescent secondary antibodies were added. The cellular localization of PD-1 and SHP-2 was observed using a laser confocal microscope. (Green: Alexa) 488 conjugated secondary antibody, red: Alexa 594. Scale bar: 10μm.

[0038] Figure 3 Gut microbial metabolites inhibit T cell activation. Among them:

[0039] A. Statistical results of CD25 / CD69 fluorescence intensity (n=3): PBMCs were stimulated with anti-CD3 (5ug / ml) / anti-CD28 (3ug / ml) for 36 hours. After further treatment with different concentrations of GDCA (10, 20, 40uM) and TCA (10, 20, 40uM) for 24 hours, the cells were fixed in flow cytometry at 4℃ for 10 min. Then, the cells were incubated with fluorescently conjugated antibodies: anti-CD3 / CD45 / CD25 / CD69 at 4℃ for 30 min. The fluorescence intensity of CD25 / CD69 in CD3+CD45+ T cells in different treatment groups was detected by flow cytometry.

[0040] B. After stimulating human PBMCs with anti-CD3 (5ug / ml) / anti-CD28 (3ug / ml) for 36 hours, and then further treating them with GDCA (20uM) and TCA (20uM) for 24 hours, the cells were fixed with 4% paraformaldehyde at 4°C for 10 minutes. Fluorescently conjugated antibodies: anti-CD25-PE / anti-CD69 were added, and the cells were incubated overnight at 4°C in the dark. The expression changes of CD25 and CD69 molecules on the surface of human PBMCs under different drug treatments were observed using a laser confocal microscope (left figure). Fluorescence intensity statistics (right figure, n=3).

[0041] C. Human PBMCs were stimulated with anti-CD3 (5 μg / ml) / anti-CD28 (3 μg / ml) for 36 hours. PBMCs were first treated with fluorescein diacetate succinimide (CFSE) (dissolved in PBS containing 5% FBS, concentration 2 μM) and labeled for 3 minutes at 37°C in 5% CO2. Subsequently, they were treated with different concentrations of GDCA (10, 20, 40 μM) and TCA (10, 20, 40 μM) for 48 hours. After cell fixation, CD3 and CD45 molecules on the cell surface were labeled (method as above). Flow cytometry was used to detect changes in the green fluorescence intensity (CFSE) of CD3+CD45+ T cells in human PBMCs (left figure). Statistical results (right figure, n=3).

[0042] D. PBMCs were stimulated with anti-CD3 (5ug / ml) / anti-CD28 (3ug / ml) for 36 hours, followed by pretreatment with GDCA (20μM) and TCA (20μM) for 24 hours, and then co-cultured with human colon cancer cells SW480 (10:1) for 24 hours. SW480 cells were collected, fixed, and stained with PI; flow cytometry was used to analyze changes in cell fluorescence intensity. Human PBMCs were collected, fixed, and stained with CD3 / CD45 / CD8 / IFN-γ; flow cytometry was used to analyze changes in IFN-γ fluorescence intensity in CD3+CD45+CD8+ T cells (left figure) (specific procedures and experimental conditions during fixation and staining were the same as above). Statistical results (right figure, n=3).

[0043] E. PBMCs were stimulated with anti-CD3 (5ug / ml) / anti-CD28 (3ug / ml) for 36 hours, followed by pretreatment with GDCA (20μM) and TCA (20μM) for 24 hours, and then co-cultured with human colon cancer cells SW480 (10:1) for 24 hours. Human PBMCs were collected, fixed with 4% paraformaldehyde at 4°C for 10 minutes, and incubated overnight at 4°C in the dark with fluorescently conjugated antibodies: anti-CD8-FITC / anti-GZMB-PE. The expression of CD8 molecules on the surface of human PBMCs and intracellular protein granzyme B (GZMB) was detected using laser confocal microscopy (left figure). Statistical results (right figure, n=3).

[0044] Data are expressed as mean ± SEM and analyzed by one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0045] Figure 4 Gut microbial metabolites inhibit T cell function and promote tumor growth. Among them:

[0046] A. Animal model construction protocol: Male C57BL / 6 mice were subcutaneously inoculated with mouse colon cancer cells MC38 (5x105 / 100ul PBS / mouse). After tumor formation, the mice were randomly divided into groups (n=4 / group) and received PBS (100μl / mouse), GDCA (2.5mM, 100ul / mouse), or TCA (2.5mM, 100ul / mouse) every 3 days.

[0047] B. From tumor implantation to sacrifice, the long and short diameters of the tumor in mice were measured every 3 days. After 5 measurements, statistical analysis was performed (V = 0.52 × long diameter × short diameter²). The tumor growth curve is shown as the average SEM. After the mice were sacrificed, the tumor tissue was taken for statistical analysis of volume (middle image) and weight (right image) (n = 4).

[0048] C. After euthanizing mice, spleens were harvested and thoroughly ground in PBS containing 5% FBS. The mixture was then filtered through a 70 μM cell sieve. The filtrate was collected, and the cell pellet was resuspended in erythrocyte lysis buffer and incubated at room temperature for 3 minutes. The cell pellet was collected again, washed twice with PBS containing 5% FBS, and then labeled with cell surface molecules CD3 / CD45 / CD8 / PD-1. The labeled cells were then fixed and permeabilized (using flow cytometry permeabilization buffer, incubated at 4°C for 10 minutes), followed by labeling with the intracellular protein GZMB. Flow cytometry was used to detect the fluorescence intensity of exhausted T cells (CD3+CD45+CD8+PD-1+) and cytotoxic T cells (CD3+CD45+CD8+GZMB+) in the spleens of mice from different treatment groups. Specific procedures and experimental conditions during fixation and staining were the same as described above.

[0049] Data are expressed as mean ± SEM and analyzed by one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Detailed Implementation

[0050] Example 1: Construction and Validation of the PD-1 / SHP-2 Screening System

[0051] The C-terminus and N-terminus of firefly luciferase were fused to the ends of PD-1 and SHP-2, respectively, and constructed into overexpression plasmid vectors named PD-1-C-luc and SHP-2-N-luc. PD-1-C-luc and SHP-2-N-luc were introduced into cells for overexpression. When the upstream protein of PD-1, programmed cell death ligand 1 (PD-L1), activated PD-1's recruitment of SHP-2, the dividing luciferase protein halves moved closer together, thereby restoring luciferase activity. Figure 1In a system co-expressing PD-1-C-luc and SHP-2-N-luc, the binding or inhibition of PD-1 and SHP-2 was reflected in the strength of luciferase activity. PD-1-C-luc and SHP-2-N-luc were successfully co-expressed in HEK293T cells. Figure 1 (B) Simultaneously, when PD-L1 activates PD-1's recruitment of SHP-2, we also detected a consequently restored luciferase activity (B). Figure 1 (C). It has been reported that methylene blue (MB) can block the recruitment of SHP-2 by PD-1.2 In our system, MB was verified to block this recruitment effect. Figure 1 (C)

[0052] Example 2 Screening of gut microbial metabolites

[0053] By detecting luciferase activity, substances similar to MB that can promote or inhibit the binding of PD-1 and SHP-2 can be screened. We obtained a compound library containing 512 human gut microbial metabolites, and co-incubated each metabolite with a PD-1-C-luc / SHP-2-N-luc screening system. From the compound library, we preliminarily screened target metabolites that can promote or inhibit the binding of PD-1 and SHP-2. Figure 2 A). Subsequently, to eliminate instability during the screening process, we conducted a secondary screening of the target metabolites initially selected. Figure 2 B) In both screenings, the hydrates of some bile acids exhibited stable effects. Therefore, we screened metabolites along the bile acid metabolic pathway again. Consistent with the results of the previous two screenings, the primary conjugated bile acid taurocholic acid (TCA) and the secondary conjugated bile acid glycodeoxycholic acid (GDCA) could relatively stably enhance enzyme activity. Figure 2 C).

[0054] Immunoprecipitation and immunofluorescence confocal microscopy further validated the role of GDCA and TCA in promoting the binding of PD-1 and SHP-2. Figure 2 D, E).

[0055] Example 3: Gut microbial metabolites inhibit T cell activation

[0056] The PD-1 / SHP-2 pathway is one of the core mechanisms regulating T cell function. It inhibits T cell activation, proliferation, and effector function through a tyrosine phosphorylation-dependent signaling cascade, playing a crucial role in tumor immune escape. Co-culturing GDCA and TCA with human peripheral blood mononuclear cells (PBMCs) and observing their effects using flow cytometry, revealed that both inhibit T cell activation. Figure 3A, B), proliferation ( Figure 3 C) and lethality ( Figure 3 D, E) indicate that the screening system described above can screen out gut microbial metabolites that regulate T cell function by affecting the binding of PD-1 and SHP-2.

[0057] Example 4: Gut microbial metabolites inhibit T cell function and promote tumor growth

[0058] A mouse subcutaneous tumor model was constructed using mouse colon cancer cells MC38. After tumor formation, the tumor-bearing mice were injected peritumorally with PBS, GDCA, and TCA. Figure 4 (A). Changes in tumor volume and weight in mice showed that GDCA and TCA significantly promoted tumor growth. Figure 4 (B) After euthanizing the mice, the spleens were harvested for T cell function testing. It was found that GDCA and TCA increased the proportion of exhausted T cells in the spleens of tumor-bearing mice, while simultaneously inhibiting T cell cytotoxic activity. Figure 4 (C)

[0059] The above evidence suggests that GDCA or TCA promotes tumor growth by inhibiting T cell function, providing strong support for the feasibility and effectiveness of the screening method used in this study. Furthermore, enhancing the immune response to colorectal cancer by inhibiting the production of microbial metabolites such as GDCA and TCA may become a novel strategy for improving the efficacy of colorectal cancer treatment.

Claims

1. The application of microbial metabolites in tumor immune response, characterized in that, The microbial metabolites are glycine deoxycholic acid and taurine.

2. The application of the microbial metabolites according to claim 1 in tumor immune response, characterized in that, The tumor is a colon cancer.

3. The application of the microbial metabolites according to claim 1 or 2 in tumor immune response, characterized in that, Glycine deoxycholic acid or taurocholic acid promotes tumor growth by inhibiting T cell function.

4. The application of the microbial metabolites according to claim 1 or 2 in tumor immune response, characterized in that, Inhibiting the production of glycodeoxycholic acid or taurocholic acid can enhance the immune response to colorectal cancer.

5. A method for screening gut microbial metabolites based on PD-1 / SHP-2 pathway signal transduction, characterized in that, The screening process for glycodeoxycholic acid and taurocholic acid as described in claim 1 specifically includes the following steps: S1: Construction and validation of the PD-1 / SHP-2 screening system; among which, S11, the C-terminus and N-terminus of firefly luciferase were fused to the ends of PD-1 and SHP-2, respectively, and constructed into overexpression plasmid vectors, named PD-1-C-luc and SHP-2-N-luc. PD-1-C-luc and SHP-2-N-luc were introduced into cells for overexpression. When the upstream protein of PD-1, programmed cell death-ligand 1, activated the recruitment of PD-1 to SHP-2, the divided luciferase protein halves approached each other, thereby restoring luciferase activity. S12, PD-1-C-luc and SHP-2-N-luc plasmids were transfected into HEK293 cells, either separately or co-transfected, and the expression of the two plasmids was verified separately or simultaneously. S13 and HEK293 cells were overexpressed with PD-1-C-luc and SHP-2-N-luc to establish a screening system. After culturing for 24 hours with or without PD-L1 and MB, luciferase activity was detected. S2: Screening for gut microbial metabolites; among which... A PD-1-C-luc / SHP-2-N-luc screening system was established in S21 and HEK293 cells. After co-culturing 510 intestinal microbial metabolites with HEK293 cells, luciferase activity was measured. S22, The target metabolites that showed the effect of promoting or inhibiting the binding of PD-1 and SHP-2 in the preliminary screening were summarized and analyzed using the PD-1-C-luc / SHP-2-N-luc screening system. S23, using the PD-1-C-luc / SHP-2-N-luc screening system to analyze bile acid metabolite pathway-related metabolites; S24, Immunoprecipitation assay to analyze the effect of glycine deoxycholic acid and taurocholic acid screened by steps S21-S23 on the binding of PD-1 and SHP-2 in HEK293 cells. S25. Immunofluorescence assay was used to analyze the effects of glycodeoxycholic acid and taurocholic acid on the binding of PD-1 and SHP-2 in Jurkat T cells. S3: Assay of T cell activation inhibition by glycodeoxycholic acid and taurocholic acid; S4: Verification of the effects of glycodeoxycholic acid and taurocholic acid on inhibiting T cell function and promoting tumor growth.

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