| Objective:To investigate the effect of gut flora on the efficacy of PD-1inhibitor in the treatment of colorectal cancer through immunity and metabolic pathway.Methods:1. Clinical research73 patients of colorectal cancer with integrated clinical data were collected.The expression of PD-1 and Tim-3 was evaluated by immunohistochemical staining,the correlation of their expression and the prognosis of the patients was analyzed.2.Preclinical research64 male BALB/c mice were randomly divided into eight groups:group A,group a,group B,group b,group D,group d,group E,and group e which housed in specific pathogen-free(SPF)condition.The mice were fed with different antibiotics through drinking water to create the different gut flora.group A and group a were fed with sterile water,group B and group b were fed with vancomycin solution(concentration:0.25mg/m L),group D and group d were fed with colistin sulfate solution(concentration:2mg/m L),group E and group e were fed with combined antibiotic solution(concentration:ampicillin sodium 1mg/m L,streptomycin sulfate 5mg/m L,colistin sulfate 1mg/m L).The antibiotics were fed continuously until the end of the experiment.After 2weeks of feeding,the antibacterial efficacy of antibiotics was evaluated,and later,subcutaneous transplanted tumor models were established by using colon cancer cell line CT26.When the tumors volume grew to 50mm~3-100mm~3,group A,group B,group D,group E were treated with PD-1 inhibitor,and group a,group b,group d,group e were treated with isotype control(Ig G2a)through intraperitoneal injection,mice were injected 5 times at 3-day intervals,250?g/mouse each time.Continue to observe for 5 days after the last injection,at the end of experiment harvested fecal,tumor and serum samples.The tumor size was measured before each treatment and at the end of experiment to evaluate the efficacy of PD-1 inhibitor,at the same time fecal samples were harvested.Then fecal samples were sequenced by 16s r DNA and metagenome to screen the key gut flora which may affect the efficacy of PD-1 inhibitor and predict the functional pathway.3.Explore the mechanismSerum samples were tested by metabolomics to screen the differential metabolites and predict the key metabolic pathways.Flow cytometry,enzyme-linked immunosorbent assay(ELISA)and immunohistochemical(IHC)staining techniques were used to evaluate the change of peripheral immunity and tumor immunological microenvironment.And then the mechanism of how gut flora could affect the efficacy of PD-1 inhibitor was evaluated by correlation analysis.Results:1. The expression of PD-1 and Tim-3 was detected in colorectal cancer.The expression of PD-1 closely related to lymph node metastasis and TNM stage(P<0.05),also maintained negative correlation with prognosis(P<0.05).There was no significant correlation between the expression of Tim-3 and PD-1 or clinicopathological parameters which include prognosis.There was the worst prognosis in the patients with co-expression of PD-1 and Tim-3(P<0.05).2. Among the groups that treated with PD-1 inhibitor,the efficacy of group A and group B are significantly better than those of group D and group E(P<0.05).There was no significant difference in tumor size between groups a,b,d and e which treated with isotype control antibody.Only very few DNA was extracted from the feces in group E,and insufficient for sequencing.Sequencing of gut flora in groups A,B and D showed that the abundance of Bacteroides was higher in group D than that in group A and B(P<0.05),and the abundance of Prevotella?sp.?CAG:485,Parabacteroides?distasonis,Akkermansia?muciniphila,Bacteroides?uniformis was higher in group A and B than that in group D(P<0.05).Functional enrichment of differential genes of gut flora based on metagenomics shown that species of gut flora may affect the efficacy of PD-1 inhibitor in the treatment of colorectal cancer through several pathways which contain amino acid synthesis and metabolism pathway,phospholipid synthesis and metabolism pathway and some immune signal pathways.3. There were significant differences in serum metabolites in each group,but the enrichment of metabolic pathway was dominantly enriched in glycerol-phospholipid metabolism.And the concentration of Lyso PC(17:0)which was a metabolite of lysophosphatidylphospholipid was higher in group D and E than that in group A and B(P<0.05),and maintained negative correlation with the abundance of Prevotella?sp.?CAG:485(r=-0.68,P<0.05).Otherwise,the concentration of Lyso PC[20:4(8Z,11Z,14Z,17Z)]was higher in group A and B than that in group D and E(P<0.05),and maintained positive correlation with the abundance of Prevotella?sp.?CAG:485(r=0.51,P<0.05).In addition,the expression of IFN-?and IL-2 in tumor microenvironment was higher in group A and B than that in group D and E,especially group A(P<0.05).There was no difference between the groups of the proportion of CD4+T and CD8+T cells in peripheral blood.Conclusions:1.The expression of PD-1 in colorectal cancer was negatively correlated with prognosis.There was no significant correlation between PD-1and Tim-3expression in CRC.Combined analysis with these two markers will more valuable for evaluating the prognosis of CRC patients.2.Gut flora affected the efficacy of PD-1 inhibitor in the treatment of colorectal cancer.The increased abundance of Bacteroides may be key element for the poor response of PD-1 inhibitor in the treatment of colorectal cancer.On the contrast,the increased abundance of Prevotella?sp.?CAG:485,Parabacteroides?distasonis,Akkermansia?muciniphila,Bacteroides?unifor-mis may benefit from the treatment of PD-1 inhibitor.3. The concentration of serum lysophosphatidylcholine metabolites was related to the efficacy of PD-1 inhibitor in the treatment of colorectal cancer,and accompanied with changes of tumor immunological microenvironment.Therefore,gut flora may regulate the changes of tumor immunological microenvironment through affecting the metabolism of glycerophospholipids,thus influence the efficacy of PD-1 inhibitor on CRC. |
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