Application of sea cucumber polysaccharide in process of assisting anti-PD-1 in treating colon cancer

Abstract

The invention discloses application of sea cucumber polysaccharide in the process of assisting anti-PD-1 in treating colon cancer, and belongs to the technical field of medicine. The SCP pretreatment is combined with the anti-PD-1 immunotherapy, so that the curative effect of the anti-PD-1 immunotherapy is remarkably enhanced. Researches show that single use of the SCP has no significant inhibition effect on tumor growth, but the SCP can significantly enhance the anti-tumor effect of anti-PD-1 treatment. The SCP combined with anti-PD-1 treatment can increase the number of CD8 < + > T lymphocytes, especially IFN-gamma < + > cytotoxic CD8 < + > T cells, and reduce the proportion of regulatory CD4 < + > T cells at the same time. The SCP improves the immunotherapy effect by adjusting intestinal flora and increasing the relative abundance of beneficial bacteria such as bifidobacteria and fecal bacillus, and fecal flora transplantation experiments show that the tumor immunotherapy enhancing effect of the SCP is at least partially mediated by the intestinal flora.

Description

Technical Field

[0001] The invention relates to application of sea cucumber polysaccharide in assisting anti-PD-1 in treating colon cancer, and belongs to the technical field of medicine. Background Art

[0002] The immune system regulates immune responses, prevents overactivation, and maintains immune tolerance through immune checkpoint molecules such as PD-1, PD-L1, and CTLA-4. However, tumor cells evade host immune surveillance by overexpressing these immune checkpoint molecules and inhibiting the anti-tumor function of the immune system. In recent years, immune checkpoint inhibition therapy (ICB) has shown significant efficacy in the treatment of various malignancies such as melanoma, non-small cell lung cancer, and renal cell carcinoma as an innovative cancer treatment. Despite this, most colorectal cancer (CRC) patients will develop drug resistance when receiving aPD-1 treatment, resulting in limited therapeutic effects, and only a few patients with microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR) benefit from it. Therefore, overcoming the resistance to immune checkpoint inhibition therapy and improving the therapeutic effect have become research hotspots in the current field of tumor treatment. Summary of the invention

[0003] In order to solve the above problems, the present invention provides the use of sea cucumber polysaccharide in the preparation of a drug for treating cancer resistant to anticancer drugs.

[0004] In one embodiment of the present invention, the anticancer drug is anti-PD-1.

[0005] In one embodiment of the present invention, the cancer is colon cancer.

[0006] In one embodiment of the present invention, the sea cucumber polysaccharide is used to regulate intestinal microbiota.

[0007] In one embodiment of the present invention, the sea cucumber polysaccharide is used to increase the relative abundance of Bifidobacterium and Faecalibacterium.

[0008] In one embodiment of the present invention, sea cucumber polysaccharides are used to regulate intestinal flora and are used in combination with anticancer drugs to overcome drug resistance in colorectal cancer immunotherapy.

[0009] In one embodiment of the present invention, sea cucumber polysaccharides are used in conjunction with anticancer drugs, IFN-γ + Cytotoxic CD8 + Increased numbers of T cells, regulatory CD4 + The number of T cells decreases.

[0010] In one embodiment of the present invention, the drug is administered orally.

[0011] In one embodiment of the present invention, Bifidobacterium and Fecalibaculum are orally administered instead of sea cucumber polysaccharides.

[0012] In one embodiment of the present invention, sea cucumber polysaccharides are used as a dietary supplement and as an adjuvant therapy to improve the efficacy of immunotherapy.

[0013] In one embodiment of the present invention, the method for preparing sea cucumber polysaccharides specifically comprises:

[0014] The dried sea cucumber was powdered and defatted with CHCl3 / MeOH (4:1, v / v) for 24 hours. After filtration and drying, the precipitate was suspended in 1.5 L of 0.1 M sodium acetate buffer (pH 5.7) containing 3 g papain, 5 mM EDTA and 5 mM cysteine ​​and incubated at 60°C for 24 hours. The mixture was then centrifuged at 5000 rpm for 20 minutes. In order to precipitate the sulfated polysaccharide, 96 ml of 10% cetylpyridinium chloride aqueous solution was added to the supernatant. The precipitate was collected and dissolved in 500 ml of 3.0 m NaCl: ethanol (100:15, v / v) solution, and then further precipitated with three times the volume of ethanol at 4°C for 12 hours. The precipitate was then redissolved in distilled water, dialyzed with pure water for 48 hours, and finally freeze-dried to obtain sea cucumber polysaccharide (SCP).

[0015] Beneficial effects:

[0016] (1) The technical solution of the present invention improves the efficacy of anti-PD-1 immunotherapy in colorectal cancer by regulating the intestinal microbiota using SCP. This method can not only increase the activity of immune cells, but also further promote the effect of immunotherapy by improving the composition of the intestinal microbiota and overcome the immune resistance of colorectal cancer.

[0017] (2) As a natural product, sea cucumber polysaccharides have good biocompatibility and safety and can be used as an adjuvant therapy in combination with existing immunotherapy.

[0018] (3) By regulating the intestinal microbiota, a new immunotherapy enhancement strategy is provided, which is expected to improve the limitations of current immunotherapy.

[0019] (4) This method is simple, easy to implement, and has high potential for clinical application.

[0020] (5) By using SCP to regulate the intestinal microbiota, the efficacy of anti-PD-1 immunotherapy in colorectal cancer was improved, reducing tumor volume by 42.78% and CD4+ There was no significant change in T cells overall, but CD8 + The percentage of T cells increased by 8.83%, and further T cell subtype analysis showed that the combined treatment promoted the effector function of T cells, INF-γ + CD8 + The proportion of T cells reached 25.40%. In contrast, Foxp3 + Regulatory CD4 + T cells, which often underlie the anti-tumor activity of immunotherapy, dropped dramatically to 4.53% after the combination treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Effect of SCP intervention on anti-PD-1 immunotherapy in CT-26 tumor-bearing mice, (A) Experimental schematic, (B) Tumor growth curve of each group, (C) Weight gain curve of each group;

[0022] Figure 2 Analysis of immune cells in tumors, (AD) CD4 + T cell ratio, CD8 + T cell ratio, IFN-γ + CD8 + T cell percentage, FOXP3 + CD4 + T cell percentage, (G) CD4 + 、CD8 + Representative immunohistochemical profiles of cells, scale bar = 100 μm;

[0023] Figure 3 Combined treatment can regulate intestinal flora and enhance intestinal immunity, (A) ACE and (B) Simpson index of intestinal flora in different treatment groups, (C) Lefse analysis of different treatment groups, (D) relative abundance of intestinal bacteria in mice in different treatment groups, relative mRNA expression of Bifidobacterium pseudocolonicum (E) and Faecalibaculum_rodentium (F) in fecal samples;

[0024] Figure 4 SCP partially enhances the anti-tumor effect of anti-PD-1mAb in CT-26 tumor-bearing mice through intestinal flora. (A) Schematic diagram of FMT experiment. Balb / C recipient mice were given antibiotics (ATB) by gavage before fecal microbiota transplantation, and then CT26 tumor cells were subcutaneously inoculated. (B) Tumor growth curves of each group. (C) Weight growth curves of each group. (DG) CD4 + T cell ratio, CD8 +T cell ratio, IFN-γ + CD8 + T cell percentage, FOXP3 + CD4 + T cell percentage;

[0025] Figure 5 Bifidobacterium pseudolongum and Faecalibaculum_rodentium enhance the anti-tumor effect of anti-PD-1mAb in Balb / c mice through intestinal microbiota. (A) Schematic diagram of the effect of Bifidobacterium plus anti-PD-1mAb on Balb / c mice. (B) Tumor growth curves of each group. (CF) CD4+T cell ratio, CD8+T cell ratio, IFN-γ + CD8 + T cell percentage, FOXP3 + CD4 + T cell percentage. DETAILED DESCRIPTION

[0026] Example 1 Effect of sea cucumber polysaccharides on the therapeutic effect of anti-PD-1 in CT26 tumor mice

[0027] Preparation method of sea cucumber polysaccharide:

[0028] The dried sea cucumber was powdered and defatted with CHCl3 / MeOH (4:1, v / v) for 24 hours. After filtration and drying, the precipitate was suspended in 1.5 L of 0.1 M sodium acetate buffer (pH 5.7) containing 3 g papain, 5 mM EDTA and 5 mM cysteine ​​and incubated at 60°C for 24 hours, and then the mixture was centrifuged at 5000 rpm for 20 minutes. In order to precipitate the sulfated polysaccharide, 96 ml of 10% cetylpyridinium chloride aqueous solution was added to the supernatant. The precipitate was collected and dissolved in 500 ml of 3.0 m NaCl: ethanol (100:15, v / v) solution, and then further precipitated with three volumes of ethanol at 4°C for 12 hours. The precipitate was then redissolved in distilled water, dialyzed with pure water for 48 hours, and finally freeze-dried to obtain SCP.

[0029] Experimental methods: This study used 6-8 week old male Balb / C mice and randomly divided them into four groups: control group, anti-PD-1 group, SCP group, and SCP+anti-PD-1 group.

[0030] Control group: Each mouse was supplemented with 200 μL of PBS every day for 10 days, and then about 1×106 CT26 cells were subcutaneously inoculated into the dorsal flank of the mouse, and PBS was continued to be given every day for 29 days;

[0031] Anti-PD-1 group: Each mouse was supplemented with 200 μL of PBS every day for 10 days, and then about 1×106 CT26 cells were subcutaneously inoculated into the dorsal flank of the mouse. Anti-PD-1 injections were started 8 days after tumor cell inoculation, once every three days, for a total of three injections, with a dose of 100 μg each time. At the same time, PBS was continued to be given every day for 29 days;

[0032] SCP group: 10 days of pretreatment with SCP, 200 mg / kg 体重 Then, approximately 1×106 CT26 cells were subcutaneously inoculated into the dorsal flank of mice, and SCP was continued to be administered daily for supplementation until day 29;

[0033] SCP+anti-PD-1 group: 10 days of pretreatment with SCP, 200 mg / kg 体重 Then, approximately 1×106 CT26 cells were subcutaneously inoculated into the dorsal flank of mice. Anti-PD-1 injections were started 8 days after tumor cell inoculation, once every three days for a total of three injections, with a dose of 100 μg each time. At the same time, SCP was continued to be administered daily for supplementation until day 29.

[0034] Tumor volume and body weight were measured. All mice were housed in a specific pathogen-free environment at a temperature of 22-25°C and a humidity of 40%-60%, and were provided with tap water and regular feed.

[0035] Experimental results: CT-26CRC cells (naturally resistant to anti-PD-1 treatment) were subcutaneously inoculated into mice with or without SCP pretreatment ( Figure 1 A). After cell inoculation, mice were injected with anti-PD-1 three times. SCP alone had no inhibitory effect on tumor growth compared with the control group (p<0.05). In addition, as expected, anti-PD-1 alone had minimal effect on tumor growth (p<0.05). In contrast, SCP combined with anti-PD-1 significantly reduced tumor volume by 42.78% compared with anti-PD-1 alone ( Figure 1 B). There was no significant difference in the body weight of mice in each group ( Figure 1 C), indicating that the combined treatment was well tolerated by mice. These data suggest that SCP can alleviate drug resistance in immunotherapy-refractory mouse tumor models.

[0036] Example 2 Effect of sea cucumber polysaccharides on tumor immune cells

[0037] Experimental method: Tumor cells were collected and placed in a culture dish containing 10 mL of PBS (containing 2% FBS), and then washed with PBS. The cells were then digested with 0.25% trypsin to obtain a single cell suspension. After digestion, medium containing fetal bovine serum was added to terminate the reaction, and the cell suspension was filtered with a 70 μm filter to remove any clumps. The cells were then resuspended in an appropriate volume of flow cytometry buffer and the concentration was adjusted to 1×10 6 cells / mL. 6 1 μg of fluorescently labeled antibody was added to each cell / μL, mixed gently, and incubated in the dark at 4°C for 30 minutes. After incubation, the cells were washed again with PBS to remove unbound antibodies. Finally, the cells were resuspended in flow cytometry buffer for analysis to evaluate characteristics such as cell surface markers and cell cycle status.

[0038] Experimental results: Compared with the anti-PD-1 group, CD4 + There was no significant change in T cells overall ( Figure 2 A) However, CD8 + The percentage of T cells increased by 8.83% ( Figure 2 B) Further T cell subtype analysis showed that the combination therapy promoted the effector function of T cells, INF-γ + CD8 + The proportion of T cells was higher (25.40%) ( Figure 2 C) In contrast, Foxp3 + Regulatory CD4 + T cells, which usually constitute the anti-tumor activity of immunotherapy, dropped sharply to 4.53% after the combination treatment ( Figure 2 D). Immunohistochemical analysis also confirmed an increase in the percentage of CD8+ cells in the tumor ( Figure 2 E). Taken together, these data suggest that SCPs can enhance effector T cell infiltration into tumors and promote responses to anti-PD-1 therapy in CT-26 tumors.

[0039] Example 3 Effect of sea cucumber polysaccharide on intestinal flora of mice

[0040] Experimental methods: Mouse feces were collected and stored at -80°C for future extraction and analysis of DNA. Sequencing was performed by Magigene Technology (Guangzhou, China) using primers 341F and 806R to amplify the V3-V4 region of the bacterial 16S rRNA gene, and the raw data were generated by the Illumina Miseq PE250 platform. After truncation and removal of primer information, further analysis was performed using the QIIME2 platform. The DADA2 algorithm was used to denoise the sequences and generate representative sequences and ASV tables. Finally, the Bayesian classifier in the RDP database was used to classify the representative bacterial gene sequences with a confidence threshold of 80%.

[0041] Results: To characterize the effect of SCP on the intestinal flora of tumor-bearing mice, we used 16s RNA sequencing to detect the composition of intestinal flora in mice that received or did not receive SCP intervention. Compared with the control group, SCP treatment was associated with increased ACE ( Figure 3 A) and Simposon index decreased ( Figure 3 B) is related, indicating a higher microbial diversity. Microbial composition analysis showed that the microbial composition of the two groups of mice was different ( Figure 3 C).

[0042] Lefse analysis showed that the bacteria enriched in the SCP group included Lactobacillus, Faecalibaculum, Bifidobacteria, and Actinobacteria, while the bacteria enriched in the control group included probacobacteria, Akkermansia, and Clostridium ( Figure 3 DF).

[0043] Example 4 Fecal microbiota transplantation experiment verifies that intestinal microbiota is the pathway for sea cucumber polysaccharides to exert their effects

[0044] Experimental method: Fresh feces of the control group and SCP group were homogenized to ensure microbial activity. The homogenate was then resuspended in sterile saline (0.5 mL) in an anaerobic chamber. The solution was thoroughly mixed and centrifuged at 500g for 5 minutes (4°C). The supernatant was collected and centrifuged at 12000r / min for 5 minutes. Sterile saline was added to the precipitate in a ratio of 1:3, resuspended, centrifuged, and the precipitate was collected. All fresh bacterial solutions were prepared 30 minutes before fecal flora transplantation to prevent significant changes in the in vitro flora. One week after FMT, tumor cells were inoculated and mice were treated with SCP and / or anti-PD-1mAb according to the method of Example 1.

[0045] The recipient mice were pretreated by gavage with an antibiotic mixture containing 10 mg / mL of ampicillin, 10 mg / mL of neomycin, 10 mg / mL of metronidazole, and 5 mg / mL of vancomycin to consume the resident flora in the body. The donor materials were taken from the above-mentioned different groups of mice. According to the different sources of the donors, the recipient mice were divided into 4 groups: FMT control group, FMTanti-PD-1 group, FMT SCP group, and FMTSCP+anti-PD-1 group. Tumor cells were inoculated 7 days after FMT administration, and PD-1 monoclonal antibodies were injected 8 days after inoculation. Tumor volume was measured every 4 days. Tumor immune cells were detected according to the method described in Example 2.

[0046] Experimental results: The intestinal flora of different treatment groups were transferred into antibiotic-pretreated mice, which were then inoculated with tumor cells and treated with anti-PD-1 ( Figure 4 A). Microbiota from SCP-treated mice significantly enhanced the anti-tumor activity of anti-PD-1, whereas mice with microbiota from the control group did not benefit from immunotherapy ( Figure 4 BC). Without anti-PD-1 treatment, the microbiota itself had no positive effect on tumors. Tumor growth in the FMT SCP group tended to be slower than that in the FMT control group and the FMT control group + anti-PD-1 group. Immune cell analysis showed that CD8+T and IFN-γ + CD8+T cells increased significantly in tumors ( Figure 4 EF), whereas in mice transplanted with the SCP-treated mouse microbiota, regulatory Foxp3 + CD4+T cell decrease ( Figure 4 G). Taken together, these results suggest that the enhancing effects of SCP are mediated, at least in part, by the gut microbiota.

[0047] Example 5 Effect of sea cucumber polysaccharide-enriched intestinal bacteria on the anti-PD-1 therapeutic effect in CT26 tumor mice

[0048] Experimental method: Faecalibaculum rodentium and Bifidobacterium pseudolongum were cultured in a fortified Clostridium medium, and the mixture was placed under anaerobic conditions at 37 ° C for 48 hours. After incubation, Faecalibaculum rodentium and Bifidobacterium pseudocolonicum were cultured by centrifugation at 13800g for 10 minutes at 4 ° C. The bacteria were then resuspended in sterile 0.1M PBS. Balb / C mice were gavaged with two bacteria (Faecalibaculumrodentium and Bifidobacterium pseudolongum) rich in the intestinal flora of sea cucumber polysaccharides-treated mice. Balb / C mice were divided into 6 groups: ABX group (mixed antibiotic intervention), ABX+anti-PD-1 group, BP group (Bifidobacterium pseudolongum intervention), BP+anti-PD-1 group, FR group (Faecalibaculum rodentium intervention), FR+anti-PD-1 group. The inoculated mouse modeling method and tumor immune cell detection method refer to Examples 1 and 2.

[0049] Results: To investigate whether specific commensal bacteria contribute to the effects of SCP anti-PD-1 therapy, we selected two bacterial groups that were abundant in the SCP treatment group, Bifidobacteria pseudolongum and Fecalibaculum rodentium, and tested their effects on the immunotherapy effect of CT-26 tumor mice ( Figure 5 A). Supplementation with B. pseudocolonicum or B. fecalcitrium significantly improved the response of tumor-bearing mice to anti-PD-1 treatment. Compared with anti-PD-1 treatment alone, the addition of B. pseudocolonicum or B. fecalcitrium with anti-PD-1 treatment reduced tumor volume by 70.18% and 64.69%, respectively ( Figure 5 B) Similar to SCP treatment, treatment with Bifidobacterium pseudocolonicum or Faecalibacterium faecium can increase intratumoral CD8 T cells + cell( Figure 5 D) and cytotoxic INF-γ + CD8+T cells ( Figure 5 E) levels and reduce the inhibitory regulatory FoxP3 + CD4 + The proportion of T cells ( Figure 5 F). In summary, B. pseudocolonicum or B. faecalis could reproduce the enhancing effects of SCP and FMT, indicating that these two bacteria may be key bacteria in enhancing the efficacy of anti-PD-1.

[0050] The present invention found that we demonstrated that sea cucumber polysaccharides can sensitize mouse CRC tumors to anti-PD-1 treatment by regulating tumor-infiltrating lymphocytes and remodeling the intestinal microbiota. In addition, this promoting effect is mediated by the microbiota and two intestinal commensal bacteria, Bifidobacterium pseudogliae and Faecalibacterium. These findings suggest that adding SCP to the diet or taking drugs with sea cucumber polysaccharides as the main raw material may be a potential way to combat anti-PD-1 resistance in colorectal cancer.

[0051] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of sea cucumber polysaccharides in the preparation of drugs for treating cancers resistant to anticancer drugs.

2. The use according to claim 1, characterized in that: The anticancer drug is anti-PD-1.

3. The use according to claim 1, characterized in that: The cancer is colon cancer.

4. The use according to claim 1, characterized in that: The sea cucumber polysaccharide is used for regulating intestinal microflora.

5. The use according to claim 1, characterized in that: The sea cucumber polysaccharide is used to increase the relative abundance of bifidobacteria and faecalis.

6. The use according to claim 1, characterized in that: By regulating intestinal flora through sea cucumber polysaccharides and using them together with anti-cancer drugs, the drug resistance in colorectal cancer immunotherapy can be overcome.

7. The use according to claim 1, characterized in that: Sea cucumber polysaccharides are used in conjunction with anticancer drugs, IFN-γ + Cytotoxic CD8 + Increased numbers of T cells, regulatory CD4 + The number of T cells decreases.

8. The use according to claim 1, characterized in that: The drug is administered orally.

9. The use according to claim 1, characterized in that: Oral administration of Bifidobacterium and Fecalibaculum was used instead of sea cucumber polysaccharides.

10. The use according to claim 1, characterized in that: The preparation method of sea cucumber polysaccharide includes: making dry sea cucumber into powder and defatting it with CHCl3 / MeOH, filtering and drying, dispersing the precipitate in 0.1M sodium acetate buffer with a pH of 5.7, and incubating it at 60°C for 24 hours, centrifuging to retain the supernatant, adding 10% cetylpyridinium chloride aqueous solution to the supernatant, collecting the precipitate and dissolving it in 3.0M NaCl / ethanol solution, and then further precipitating it with three times the volume of ethanol at 4°C for 12 hours, and then redissolving the precipitate in distilled water, dialyzing it with pure water for 48 hours, and finally freeze-drying it to obtain sea cucumber polysaccharide.

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