Animal Bifidobacterium lactis subspecies for enhancing anti-tumor immunity of lung cancer and application thereof

Abstract

The present invention provides an animal Bifidobacterium lactis subsp. lactis for enhancing anti-tumor immunity of lung cancer and an application thereof. The present invention separates and preserves animal Bifidobacterium lactis subsp. lactis SDZL01 for enhancing anti-tumor immunity of lung cancer, and names it as animal Bifidobacterium lactis subsp. lactis SDZL01 strain. Animal Bifidobacterium lactis subsp. lactis has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on November 18, 2024, and its preservation number is Bifidobacterium lactis subsp. lactis CGMCC No. 32692. Animal Bifidobacterium lactis subsp. lactis or its metabolite indole‑3‑acetic acid can be used to regulate the immune microenvironment of NSCLC or enhance immunotherapy. Animal Bifidobacterium lactis subsp. lactis can enhance anti-tumor immunity and can be used to prepare products that enhance immunotherapy of lung cancer. Animal Bifidobacterium lactis subsp. lactis can improve the response rate of cancer immunotherapy population and expand the population of tumor patients who benefit from cancer immunotherapy.

Description

Technical Field

[0001] The present invention relates to the technical field of bifidobacteria, and in particular to an animal Bifidobacterium lactis subspecies for enhancing anti-tumor immunity of lung cancer and an application thereof. Background Art

[0002] Lung cancer is currently the most common type of cancer and one of the leading causes of death among cancer patients worldwide. In 2022, there were 1.06 million new cases of lung cancer in my country and 730,000 lung cancer-related deaths. Non-small cell lung cancer (NSCLC), as the main subtype of lung cancer, accounts for about 85% of lung cancer, which seriously threatens the life and health of people in my country and even the world. my country is a high-incidence area of ​​NSCLC, but the effects of treatment methods such as surgery, chemotherapy and molecular targeted therapy are still unsatisfactory. In the past decade, emerging immunotherapy programs have run through all stages of NSCLC treatment and reshaped the NSCLC treatment landscape. In the 2023 CSCO NSCLC diagnosis and treatment guidelines, immunotherapy has become the standard treatment for patients with driver gene-negative advanced NSCLC, which has led to the approval of domestic immunotherapy drugs mainly based on sindilimab, tislelizumab and carrelizumab for the treatment of advanced NSCLC. In addition, in the most complex stage III NSCLC cases, adjuvant and neoadjuvant immunotherapy have also become powerful new treatment strategies that can significantly improve patient prognosis. Although immunotherapy has achieved phased results in the treatment of NSCLC, many patients inevitably develop immune resistance, and patients with primary resistance to immunotherapy usually have poor efficacy and rapid clinical progression, which is one of the difficulties in clinical treatment. Therefore, accurately screening potential beneficiaries of immunotherapy and improving the sensitivity of NSCLC patients to immunotherapy are the top priorities of current precision immunotherapy for NSCLC.

[0003] Bifidobacterium is the main probiotic in the intestinal flora and plays an important regulatory role in maintaining intestinal homeostasis. Bifidobacterium is closely related to the regulation of tumor microenvironment and regulates tumors in distant organs by producing anti-cancer metabolites. Many studies have shown that intestinal bifidobacteria can inhibit tumor progression. For example, Bifidobacterium bifidum can induce mitochondrial apoptosis of gastric cancer cells through the Akt-p53 pathway, thereby inhibiting the progression of gastric cancer. Bifidobacterium also plays an important role in regulating immunotherapy sensitivity. For example, Bifidobacterium longum RAPO enhances the efficacy of anti-PD-1 immunotherapy in triple-negative breast cancer mouse models by reducing the polarization of macrophages to M2 in the tumor immune microenvironment. Although bifidobacteria play an important role in inhibiting tumor progression, regulating the immune microenvironment, and improving the sensitivity of immunotherapy, the potential regulatory mechanism of intestinal bifidobacteria on the immune microenvironment of NSCLC has not yet been elucidated. Therefore, we propose an animal bifidobacterium that enhances lung cancer anti-tumor immunity and its application. Summary of the invention

[0004] The purpose of the present invention is to solve the problems raised by the existing background technology. In order to achieve the above invention purpose, the present invention provides the following technical scheme: an animal Bifidobacterium lactis subspecies, including animal Bifidobacterium, the animal Bifidobacterium lactis subspecies is classified and named as: Bifidobacterium animalis subsp.lactis, The animal Bifidobacterium subsp. lactis can enhance anti-tumor immunity. The animal Bifidobacterium subsp. lactis has been deposited in the China General Microbiological Culture Collection Center (CGMCC) of the China Microbiological Culture Collection Committee on November 18, 2024. Its preservation number is CGMCC No. 32692. The address of the preservation unit is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The Bifidobacterium animalis subsp. lactis is used for enhancing the anti-tumor immunity of lung cancer.

[0005] An application of animal Bifidobacterium lactis subspecies in enhancing anti-tumor immunity of lung cancer, comprising animal Bifidobacterium lactis subspecies or its metabolite indole-3-acetic acid, for regulating the immune microenvironment of NSCLC or enhancing immunotherapy.

[0006] As a preferred technical solution of the present invention, the animal Bifidobacterium lactis subspecies and indole-3-acetic acid are used to regulate the polarization of macrophages M2 in the NSCLC microenvironment via the AHR / METTL3 / IL-6 axis.

[0007] As a preferred technical solution of the present invention, the animal Bifidobacterium lactis subspecies can regulate the polarization of macrophages in the NSCLC microenvironment and increase the proportion and function of CD8+T cells.

[0008] As a preferred technical solution of the present invention, the metabolite indole-3-acetic acid of Bifidobacterium animalis subsp. lactis inhibits the progression of NSCLC both in vivo and in vitro.

[0009] As a preferred technical solution of the present invention, the use of indole-3-acetic acid in inhibiting the progression of NSCLC both in vivo and in vitro.

[0010] As a preferred technical solution of the present invention, indole-3-acetic acid, a key metabolite of Bifidobacterium animalis subsp. lactis, may inhibit macrophage M2 polarization by reducing IL-6 expression in tumor cells, thereby regulating the application of NSCLC microenvironment.

[0011] As a preferred technical solution of the present invention, the animal Bifidobacterium lactis subspecies reduces macrophage M2 polarization and promotes M1 polarization; the animal Bifidobacterium lactis subspecies can promote the infiltration of CD8+T cells and increase the production of IFN-γ; the indole-3-acetic acid inhibits the proliferation of A549 cells; the indole-3-acetic acid changes the A549 cell cycle, including increasing the proportion of cells in the G1 phase and reducing the proportion of cells in the S phase, and induces apoptosis of A549 cells;

[0012] As a preferred technical solution of the present invention, a composition comprising Bifidobacterium animalis subsp. lactis or its metabolite indole-3-acetic acid is used to prepare a medicament for use in treating or preventing NSCLC.

[0013] As a preferred technical solution of the present invention, a composition comprising animal Bifidobacterium lactis subspecies or its metabolite indole-3-acetic acid and a PD-1 antibody drug.

[0014] Compared with the prior art, the present invention has the following beneficial effects: In the scheme of the present invention: the present invention isolates and preserves the animal Bifidobacterium lactis subspecies SDZL01 that enhances the anti-tumor immunity of lung cancer, and names it as the animal Bifidobacterium lactis subspecies Bifidobacterium animalis subsp. lactis SDZL01 strain. The strain can enhance anti-tumor immunity, indicating that the SDZL01 strain can improve the response rate of the cancer immunotherapy population and expand the population of tumor patients who benefit from cancer immunotherapy (immune checkpoint inhibitors); therefore, the animal Bifidobacterium lactis subspecies Bifidobacterium animalissubsp. lactis SDZL01 strain can be used to prepare products or medicines that enhance the immunotherapy of lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1The present invention provides a diagram of the effect of animal Bifidobacterium lactis subspecies on the microenvironment of NSCLC by regulating macrophage polarization and CD8+T cells. (A) Flow cytometry results show that animal Bifidobacterium lactis subspecies can significantly reduce the proportion of macrophages, reduce M2 polarization and promote M1 polarization; (B) Flow cytometry results show that animal Bifidobacterium lactis subspecies can promote the infiltration of CD8+T cells and increase the production of IFN-γ; (C) Multiple fluorescent immunohistochemistry technology confirms the regulatory effect of animal Bifidobacterium lactis subspecies on macrophage polarization and CD8+T cell infiltration in the tumor microenvironment. NSCLC, Non-small cell lung cancer.

[0016] Figure 2 The indole-3-acetic acid provided by the present invention is a key metabolite released by animal Bifidobacterium lactis subsp. (A) Next-generation sequencing showed that the genes of animal Bifidobacterium lactis were mainly related to its metabolic function; (B) Principal component analysis of non-targeted metabolomics showed that the metabolites of animal Bifidobacterium lactis were significantly different from those of the BHI control; (C) KEGG enrichment analysis of non-targeted metabolomics found that the metabolites of animal Bifidobacterium lactis were significantly associated with the tryptophan biosynthesis pathway; (D) Targeted metabolomics of Ba-CM and BHI controls showed that tryptophan and indole-related metabolites in animal Bifidobacterium lactis were significantly different from those of the BHI control, and the heat map showed that indole-3-acetic acid was the most significantly increased metabolite in animal Bifidobacterium lactis; (E) Experimental design of the germ-free mouse model; (F) Targeted tryptophan / indole pathway metabolomics analysis of feces, serum and lung tissues of germ-free mice showed that indole-3-acetic acid was the only metabolite that was significantly increased in the feces, serum and lung tissues of germ-free mice inoculated with Bifidobacterium lactis. Ba-CM, B. animalis subsp. lactisconditioned medium; IAA, Indole-3-acetic acid.

[0017] Figure 3The indole-3-acetic acid provided by the present invention inhibits the progression of NSCLC both in vivo and in vitro. (AB) After Lewis lung cancer cells were implanted subcutaneously in C57BL / 6 male mice, indole-3-acetic acid and PBS were gavaged daily; indole-3-acetic acid significantly inhibited the formation of NSCLC subcutaneous tumors; (C) MTT results showed that indole-3-acetic acid could inhibit the viability of A549 cells; (D) indole-3-acetic acid inhibited the cell cycle G1 / S phase conversion of A549 cell line; (E) indole-3-acetic acid promoted cell apoptosis of A549 cell line; (F) Western blot further verified that indole-3-acetic acid could regulate the protein levels of PCNA, Caspase 3, Cleaved Caspase 3, and Cyclin D1 in NSCLC cell line. NSCLC, Non-small cell lung cancer; IAA, Indole-3-acetic acid.

[0018] Figure 4 Indole-3-acetic acid, a key metabolite of Bifidobacterium animalis subsp. lactis provided by the present invention, may regulate the NSCLC microenvironment by reducing the expression of IL-6 in tumor cells. (A) Flow cytometry results showed that indole-3-acetic acid can reduce the proportion of macrophages in the tumor microenvironment, reduce M2 polarization and promote M1 polarization. In addition, indole-3-acetic acid can promote the infiltration of CD8+T cells and increase the production of IFN-γ; (B) PCR array showed that the expression of IL-6 was most significantly downregulated in A549 cells treated with Ba-CM; (C) qRT-PCR further verified that the expression of IL-6 was significantly downregulated in human NSCLC cell lines treated with Ba-CM. Ba-CM, B. animalis subsp. lactis conditioned medium; IAA, Indole-3-acetic acid; NSCLC, Non-small cell lung cancer. DETAILED DESCRIPTION

[0019] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0020] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely represents some embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. It should be noted that the embodiments of the present invention and the features and technical solutions in the embodiments can be combined with each other without conflict. It should be noted that similar numbers and letters represent similar items in the following drawings. Therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0021] Example 1: Please refer to Figure 1-Figure 4 , an animal Bifidobacterium lactis subspecies, the animal Bifidobacterium lactis subspecies is named as Bifidobacterium animalis subsp. lactis SDZL01 strain, the animal Bifidobacterium animalis subsp. lactis SDZL01 strain can enhance anti-tumor immunity, the animal Bifidobacterium lactis subspecies was deposited in the China General Microbiological Culture Collection Center (CGMCC) of the China General Microbiological Culture Collection Center (CGMCC) on November 18, 2024, and its preservation number is CGMCC No. 32692. The animal Bifidobacterium lactis subspecies is used to enhance the anti-tumor immunity of lung cancer.

[0022] The invention discloses an application of animal Bifidobacterium lactis subspecies in enhancing anti-tumor immunity of lung cancer, and an application of animal Bifidobacterium lactis subspecies or its metabolite indole-3-acetic acid in regulating the immune microenvironment of NSCLC or enhancing immunotherapy.

[0023] Application of Bifidobacterium animalis subsp. lactis and indole-3-acetic acid in regulating M2 polarization of macrophages in the NSCLC microenvironment via the AHR / METTL3 / IL-6 axis.

[0024] Bifidobacterium animalis subsp. lactis can regulate macrophage polarization in the NSCLC microenvironment and increase the proportion and function of CD8+T cells.

[0025] Indole-3-acetic acid, a metabolite of Bifidobacterium animalis subsp. lactis, inhibits the progression of NSCLC both in vitro and in vivo.

[0026] Indole-3-acetic acid has been shown to inhibit the progression of NSCLC both in vitro and in vivo.

[0027] Indole-3-acetic acid, a key metabolite of Bifidobacterium animalis subsp. lactis, may inhibit macrophage M2 polarization by reducing IL-6 expression in tumor cells, thereby regulating the application of NSCLC microenvironment.

[0028] Bifidobacterium animalis subsp. lactis reduces macrophage M2 polarization and promotes M1 polarization; Bifidobacterium animalis subsp. lactis promotes CD8+T cell infiltration and increases IFN-γ production; Indole-3-acetic acid inhibits A549 cell proliferation; Indole-3-acetic acid changes the A549 cell cycle, including increasing the proportion of cells in the G1 phase and decreasing the proportion of cells in the S phase, and induces A549 cell apoptosis;

[0029] The composition of animal Bifidobacterium lactis subspecies or its metabolite indole-3-acetic acid is used for preparing medicine, and the medicine is used for treating or preventing NSCLC.

[0030] A composition of animal Bifidobacterium lactis subspecies or its metabolite indole-3-acetic acid and a PD-1 antibody drug.

[0031] Experimental Example 1: The first part of the experiment: It was determined that Bifidobacterium animalis subsp. lactis and its metabolite indole-3-acetic acid regulate the immune microenvironment and inhibit the progression of NSCLC.

[0032] (1) Experimental verification of the reduction in the abundance of Bifidobacterium lactis in the feces and the indole-3-acetic acid content in the serum of NSCLC patients: Expand the collection of fecal and serum samples from NSCLC patients and healthy controls to detect the abundance of Bifidobacterium lactis in the feces of patients and the content of indole-3-acetic acid in the serum. Improve the verification of the correlation between Bifidobacterium lactis and indole-3-acetic acid and the clinical pathological characteristics and prognosis of NSCLC patients, such as staging, grade, and sensitivity to immunotherapy.

[0033] (2) Experimental verification of the inhibitory effects of animal Bifidobacterium lactis and indole-3-acetic acid on NSCLC: C57BL / 6 mice were selected as animal models, and mouse NSCLC cell lines CMT167 and Lewis were used to construct in situ and subcutaneous lung cancer tumor models. Mice were gavaged with animal Bifidobacterium lactis, indole-3-acetic acid and PBS, respectively. The abundance changes of animal Bifidobacterium lactis in mouse feces were detected by qRT-PCR technology; metabolomics was used to verify the changes in the content of indole-3-acetic acid in mouse feces, serum and tumor tissues. CyTOF mass spectrometry technology and multiplex fluorescence immunohistochemistry technology were further used to verify the effects of animal Bifidobacterium lactis and indole-3-acetic acid on the regulation of macrophage polarization and CD8+T cell infiltration and function in the immune microenvironment of NSCLC.

[0034] (3) Experimental verification of the regulation of macrophage polarization by Bifidobacterium animalis subsp. lactis and indole-3-acetic acid in vitro: Human NSCLC cells A549 and Calu-3 were treated with Bifidobacterium animalis subsp. lactis conditioned medium (Ba-CM), brain heart infusion (BHI), indole-3-acetic acid and PBS, respectively. The supernatants of the treated cells were collected and co-cultured with M0 macrophages. Flow cytometry was used to detect the polarization of macrophages.

[0035] Part 2 of the experiment: The mechanism by which Bifidobacterium lactis subsp. lactis and indole-3-acetic acid regulate M2 polarization of macrophages in the NSCLC microenvironment through the AHR / METTL3 / IL-6 axis in experimental animals.

[0036] (1) The regulatory mechanism of experimental animal Bifidobacterium lactis and indole-3-acetic acid on the expression of METTL3 and IL-6 and m6A modification: The human NSCLC cell lines A549 and Calu-3 were treated with Ba-CM and BHI, and indole-3-acetic acid and PBS, respectively. The cell pellets and cell supernatants were then collected to detect the expression of methyltransferases METTL3 and IL-6. The MeRIP-Seq / m6A-Seq technology was used to verify the regulatory effects of animal Bifidobacterium lactis and its metabolite indole-3-acetic acid on the m6A methylation modification of the NSCLC genome and the m6A methylation modification of the IL-6 gene. In addition, the differences in the expression of METTL3 and IL-6 in the tumor tissues of mice gavaged with Bifidobacterium lactis and indole-3-acetic acid and the control group were detected.

[0037] (2) The specific mechanism of IAA binding to AHR receptor was investigated by using surface plasmon resonance (SPR) technology to analyze the binding force between IAA and AHR receptor. The IAA pull-down experiment was used to explore whether IAA could directly bind to the recombinant AHR receptor protein.

[0038] (3) Whether the knockout of AHR receptor can reverse the regulatory effects of animal Bifidobacterium lactis and indole-3-acetic acid on the microenvironment: The AHR receptor of human NSCLC cell line was knocked out, and the knockout AHR receptor and control NSCLC cells were treated with Ba-CM and BHI, indole-3-acetic acid and PBS, respectively. The cell supernatant was collected and co-cultured with M0 macrophages. The M2 polarization of macrophages was detected by flow cytometry to determine whether the knockout of AHR receptor can reverse the regulatory effects of animal Bifidobacterium lactis and indole-3-acetic acid on macrophage polarization; the AHR receptor of mouse NSCLC cell line was knocked out, and in situ and subcutaneous tumor models were constructed. Mice were gavaged with animal Bifidobacterium lactis, indole-3-acetic acid and PBS, respectively, to determine whether the inhibitory effects of animal Bifidobacterium lactis and indole-3-acetic acid on NSCLC growth and the regulation of the immune microenvironment were eliminated after the knockout of AHR receptor at the animal model level.

[0039] (4) Experimental knockout of AHR receptors to reverse the inhibitory effects of animal Bifidobacterium lactis and indole-3-acetic acid on METTL3 and IL-6 expression and m6A modification: The AHR receptor of human NSCLC cell line was knocked out, and human NSCLC cells with AHR receptor knockout or control group were treated with Ba-CM and BHI, indole-3-acetic acid and PBS, respectively. Cell pellets were collected to detect the expression of METTL3 and IL-6, and m6A modification was detected using MeRIP-Seq / m6A-Seq technology. Cell supernatants were collected to verify the secretion of IL-6. The AHR receptor knockout mouse lung cancer orthotopic and subcutaneous tumor models were used, respectively, using animal Bifidobacterium lactis, indole-3-acetic acid and PBS for oral administration, to detect whether animal Bifidobacterium lactis and indole-3-acetic acid can downregulate METTL3 and IL-6 expression after AHR receptor knockout.

[0040] Part 3 of the experiment: The feasibility of enhancing the efficacy of immunotherapy with Bifidobacterium lactis subsp. lactis and indole-3-acetic acid in experimental animals.

[0041] (1) Whether Bifidobacterium lactis and indole-3-acetic acid can improve the sensitivity of NSCLC immunotherapy in vivo: Mouse orthotopic and subcutaneous tumor models were constructed using murine CMT167 and Lewis lung cancer cell lines. Mice were gavaged with animal Bifidobacterium lactis, indole-3-acetic acid and PBS, respectively, and treated with anti-PD-1 antibodies to explore whether animal Bifidobacterium lactis and indole-3-acetic acid can improve the sensitivity of NSCLC immunotherapy in vivo.

[0042] (2) Experimental study to investigate whether knocking out the AHR receptor can reverse the effects of animal Bifidobacterium lactis and indole-3-acetic acid on immunotherapy of NSCLC: AHR receptors in mouse NSCLC cells were knocked out, and in situ and subcutaneous lung cancer models were constructed. Mice were gavaged with animal Bifidobacterium lactis, indole-3-acetic acid, and PBS, respectively, and treated with anti-PD-1 antibodies to explore whether knocking out the AHR receptor can reverse the effects of animal Bifidobacterium lactis and indole-3-acetic acid on immunotherapy of NSCLC.

[0043] Experimental Example 2: An animal Bifidobacterium lactis subspecies. Experimental Figures on animal Bifidobacterium lactis subspecies: (1) Animal Bifidobacterium lactis subspecies regulates macrophage polarization and CD8+T cell function to affect the NSCLC microenvironment.

[0044] To clarify the regulatory effect of animal Bifidobacterium lactis subsp. lactis on the NSCLC microenvironment, we first analyzed Lewis subcutaneous tumor tissues of mice treated with animal Bifidobacterium lactis subsp. lactis and PBS control groups by flow cytometry. We found that animal Bifidobacterium lactis subsp. lactis could significantly reduce the proportion of total macrophages in the tumor microenvironment. Further analysis showed that animal Bifidobacterium lactis subsp. lactis could reduce the M2 polarization of macrophages and promote the M1 polarization ( Figure 1 A), in addition, Bifidobacterium animalis subsp. lactis promoted the infiltration of CD8+ T cells and increased the production of IFN-γ ( Figure 1 B). Next, we used multiple fluorescent immunohistochemistry to confirm the regulatory effects of animal Bifidobacterium lactis subsp. lactis on macrophage polarization and CD8+ T cells in the NSCLC microenvironment ( Figure 1 C). In summary, we determined that Bifidobacterium animalis subsp. lactis could regulate macrophage polarization in the NSCLC microenvironment and increase the proportion and function of CD8+T cells.

[0045] (2) Indole-3-acetic acid is a key metabolite released by Bifidobacterium animalis subsp. lactis.

[0046] In order to clarify what substances animal Bifidobacterium lactis subsp. lactis produces to regulate the progression of NSCLC, we first performed functional annotation analysis on the third-generation sequencing results of animal Bifidobacterium lactis subsp. lactis and found that the genes of animal Bifidobacterium lactis subsp. lactis were mainly related to its metabolic function ( Figure 2 A). Therefore, animal Bifidobacterium lactis subsp. lactis exerts its effects mainly by releasing metabolites. We further performed non-targeted metabolomics analysis on Ba-CM and BHI controls, and principal component analysis showed that the metabolites of animal Bifidobacterium lactis subsp. lactis were significantly different from those of BHI controls ( Figure 2 B), KEGG enrichment analysis found that the metabolites of Bifidobacterium animalis subsp. lactis were significantly associated with the tryptophan biosynthesis pathway ( Figure 2C). Therefore, we speculate that animal B. lactis subsp. lactis exerts its effects mainly by producing tryptophan-related metabolites. We then performed metabolomics analysis targeting the tryptophan pathway on Ba-CM and BHI controls. Principal component analysis revealed that tryptophan / indole-related metabolites in animal B. lactis subsp. lactis were significantly different from those in BHI controls ( Figure 2 D). To further clarify what tryptophan / indole-related metabolites produced by animal Bifidobacterium lactis subsp. lactis reach the lungs through the "gut-lung axis" to exert their effects, we constructed a germ-free mouse model and gavaged the germ-free mice with animal Bifidobacterium lactis subsp. lactis and PBS, respectively ( Figure 2 E). At the end of the experiment, we collected feces, serum, and lung tissues from germ-free mice for metabolomics analysis targeting the tryptophan / indole pathway. The results showed that indole-3-acetic acid was the only one that was both increased in Ba-CM ( Figure 2 D), and metabolites significantly increased in feces, serum and lung tissues of germ-free mice inoculated with animal Bifidobacterium lactis subsp. Figure 2 F). In summary, we preliminarily confirmed that indole-3-acetic acid is a key metabolite produced by animal Bifidobacterium.

[0047] (3) Indole-3-acetic acid inhibits the progression of NSCLC both in vivo and in vitro.

[0048] In order to clarify whether Bifidobacterium animalis subsp. lactis exerts its inhibitory effect on NSCLC through indole-3-acetic acid, we used indole-3-acetic acid and PBS to gavage Lewis lung cancer subcutaneous tumor mouse model ( Figure 3 A), the results showed that indole-3-acetic acid can significantly inhibit the growth of NSCLC tumors ( Figure 3 AB). To further clarify the effect of indole-3-acetic acid on NSCLC, we treated A549 cell line with indole-3-acetic acid and PBS respectively. Cell proliferation experiments showed that indole-3-acetic acid could significantly inhibit the proliferation of A549 cells ( Figure 3 C). In addition, IAA can significantly change the A549 cell cycle, including increasing the proportion of cells in the G1 phase and decreasing the proportion of cells in the S phase ( Figure 3 D) and can induce apoptosis of A549 cells ( Figure 3 E). At the same time, we further used Western blot technology to verify that indole-3-acetic acid can significantly reduce the expression level of proliferation index PCNA and cell cycle regulatory protein CyclinD1, and increase the expression level of apoptosis-related protein Cleaved Caspase 3. These results suggest that indole-3-acetic acid, a metabolite of animal Bifidobacterium, inhibits the progression of NSCLC both in vivo and in vitro.

[0049] (4) Indole-3-acetic acid, a metabolite of Bifidobacterium animalis subsp. lactis, may inhibit the M2 polarization of macrophages in the NSCLC microenvironment by reducing IL-6 expression.

[0050] To clarify whether animal Bifidobacterium lactis subsp. lactis regulates the NSCLC microenvironment through indole-3-acetic acid, we performed flow cytometric analysis on Lewis lung cancer subcutaneous tumor tissues gavaged with indole-3-acetic acid and PBS. We found that indole-3-acetic acid could significantly reduce the proportion of macrophages in the tumor microenvironment. Further analysis showed that indole-3-acetic acid could reduce the M2 polarization of macrophages and promote the M1 polarization ( Figure 4 In addition, indole-3-acetic acid can promote the infiltration of CD8+ T cells and increase the production of IFN-γ ( Figure 4 A). To further elucidate the potential mechanism of Bifidobacterium animalis subsp. lactis and its metabolite indole-3-acetic acid in regulating the microenvironment of NSCLC, we performed cytokine and chemokine PCR array detection on A549 cells treated with Ba-CM and found that the expression of IL-6 was most significantly downregulated in A549 cells treated with Ba-CM ( Figure 4 B), we then used qRT-PCR to verify that IL-6 was significantly downregulated in human NSCLC cell lines A549 and Calu-3 treated with Ba-CM ( Figure 4 C). IL-6 plays an important regulatory role in the tumor microenvironment. IL-6 can promote macrophage polarization to M2 type, thereby promoting tumor development. Therefore, we preliminarily determined that indole-3-acetic acid, a key metabolite of animal Bifidobacterium, may inhibit macrophage M2 polarization by reducing IL-6 expression in tumor cells, thereby regulating the NSCLC microenvironment.

[0051] The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Although the present invention has been described in detail with reference to the above embodiments, the present invention is not limited to the above specific implementation methods. Therefore, any modification or equivalent replacement of the present invention; and all technical solutions and improvements thereof that do not depart from the spirit and scope of the invention are included in the scope of the claims of the present invention.

Claims

1. An animal Bifidobacterium lactis subsp., characterized in that It includes animal Bifidobacterium lactis subspecies, which is named as Bifidobacterium animalis subsp. lactis SDZL01 strain, and the Bifidobacterium animalis subsp. lactis SDZL01 strain can enhance anti-tumor immunity. The animal Bifidobacterium lactis subspecies has been deposited in the China General Microbiological Culture Collection Center (CGMCC) of the China General Microbiological Culture Collection Center (CGMCC) on November 18, 2024, and its preservation number is CGMCC No. 32692. The animal Bifidobacterium lactis subspecies is used to enhance the anti-tumor immunity of lung cancer.

2. An application of Bifidobacterium lactis subspecies in enhancing anti-tumor immunity of lung cancer, characterized in that: The invention comprises animal Bifidobacterium lactis subspecies or its metabolite indole-3-acetic acid, and is used for regulating the immune microenvironment of NSCLC or enhancing immunotherapy.

3. The use of animal Bifidobacterium lactis subspecies in enhancing anti-tumor immunity of lung cancer according to claim 2, characterized in that: The application of the animal Bifidobacterium lactis subspecies and indole-3-acetic acid in regulating macrophage M2 polarization in the NSCLC microenvironment through the AHR / METTL3 / IL-6 axis.

4. The use of animal Bifidobacterium lactis subspecies in enhancing anti-tumor immunity of lung cancer according to claim 3, characterized in that: The animal Bifidobacterium lactis subspecies can regulate the polarization of macrophages in the NSCLC microenvironment and increase the proportion and function of CD8+T cells.

5. The use of animal Bifidobacterium lactis subspecies in enhancing anti-tumor immunity of lung cancer according to claim 4, characterized in that: The metabolite indole-3-acetic acid of the animal Bifidobacterium lactis subspecies inhibits the progression of non-small cell lung cancer (NSCLC) both in vivo and in vitro.

6. The use of animal Bifidobacterium lactis subspecies in enhancing anti-tumor immunity of lung cancer according to claim 5, characterized in that: The indole-3-acetic acid is used to inhibit the progression of NSCLC both in vivo and in vitro.

7. The use of animal Bifidobacterium lactis subspecies in enhancing anti-tumor immunity of lung cancer according to claim 6, characterized in that: Indole-3-acetic acid, a key metabolite of Bifidobacterium animalis subsp. lactis, may inhibit macrophage M2 polarization by reducing IL-6 expression in tumor cells, thereby regulating the application of NSCLC microenvironment.

8. The use of animal Bifidobacterium lactis subspecies in enhancing anti-tumor immunity of lung cancer according to claim 7, characterized in that: The animal Bifidobacterium lactis subspecies reduces macrophage M2 polarization and promotes M1 polarization; the animal Bifidobacterium can promote the infiltration of CD8+T cells and increase the production of IFN-γ; the indole-3-acetic acid inhibits the proliferation of A549 cells; the indole-3-acetic acid changes the A549 cell cycle, including increasing the proportion of G1 phase cells and reducing the proportion of S phase cells, and inducing A549 cell apoptosis.

9. The use of animal Bifidobacterium lactis subspecies in enhancing anti-tumor immunity of lung cancer according to claim 8, characterized in that: The composition comprising animal Bifidobacterium lactis subsp. lactis or its metabolite indole-3-acetic acid is used for preparing a medicament for treating or preventing NSCLC.

10. The use of animal Bifidobacterium lactis subspecies in enhancing anti-tumor immunity of lung cancer according to claim 9, characterized in that: A composition comprising animal Bifidobacterium lactis subspecies or its metabolite indole-3-acetic acid and a PD-1 antibody drug.

Images