Study On The Relationship Between Gut Microbiota Diversity And Anti-tumor Immunity Of Breast Cancer Patients

Objectives:Breast cancer(BC)is the most common malignant tumor and the primary cause of cancer-associated mortality in women worldwide.Although numerous treatments,including chemotherapy,radiotherapy,endocrine therapy and targeted therapy,are currently available for BC,the response of patients greatly varies.The reason may be due to differences in anti-tumor immunity.Why are there so many differences?Previous studies revealed that the composition of the gut microbiota was a major environmental factor that varied among individuals,which might affect systemic immunity.The gut microbiota is demonstrated to initiate the differentiation of T cells,the expansion of specific molecular subsets and the activation state of innate antigen-presenting cells(APCs),which may eventually affect priming of the systemic immune response.In addition,the gut microbiota may improve toutcomes of cancer treatments by impairing inflammatory tone in response to different therapeutic protocols.There are many manifestations of anti-tumor immunity in the body,for breast cancer,the function of tumor infiltrating lymphocytes(TILs)is an important index.In BC,extensive tumor infiltration by cytotoxic CD8~+T cells is markedly associated with patient survival and response to therapies.Furthermore,the baseline expression of TILs can predict the pathological complete response(p CR)result following neoadjuvant chemotherapy in patients with BC,which is an important prognostic indicator.In addition,cytokines in serum,such as IFN-?and IL-2,are also important indicators.The aim of this study is to investigate whether the diversity of gut microbiota are related to the different expression of TILs in breast cancer patients.Using animal models to study whether the intervention of gut microbiota can affect its own anti-tumor immunity and find the key strains to provide a preclinical basis for future trials in the human body.Methods:1. Effect of gut microbiota diversity on the expression of TILs in breast cancer.A total of 80 patients with BC were divided into three groups based on the expression of TILs,as follows:High expression of TILs(TIL-H),medium expression of TILs(TIL-M)and low expression of TILs(TIL-L).DNA of the gut microbiota was determined by Illumina sequencing and taxonomy of 16S ribosomal RNA genes.A Chi-square test and Unifrac analysis of?-diversity were applied to assess the association between clinical characteristics and diversity of the gut microbiota.2. Effect of human intervention on the growth of tumor growing in mice.Balb/c mice were divided into three groups,antibiotic perfusion group(SA),bifidobacterium perfusion group(SB)and blank control group(SC),respectively.Three groups of mice were pretreated with antibiotics,Bifidobacterium and sterile water for three weeks,the frequency was once every other day.Three weeks later,fresh stool samples from three groups of mice were collected for meta16S DNA sequencing.Subsequently,the mice were inoculated with 4T1 breast cancer cells to observe the tumorigenesis time,tumor growth rate and tumor size,and draw the growth curve.3.Effects of gut microbiota differences on tumor immunity in mice.The mice were sacrificed,the serum and tumor of the mice were retained,and the tumor was divided into two parts,one part and the serum were analyzed by ELISA,detection of IFN-?an4d IL-2,the other part HE staining and immunohistochemical staining,detection of TILs and CD8~+T cells.Results:1.The?-diversity distribution was statistically significant(weighted Uni Frac,P<0.01;unweighted Uni Frac,P<0.01)when comparing the TIL-L and TIL-H groups and when comparing the three groups(TIL-H vs.TIL-M vs.TIL-L).2. The number of OUTs was 397,583 and 573 in SA group,SC group and SB group,respectively.In the ? diversity analysis of weighted(P=1.5015e-10)and unweighted(P=5.5914e-05),there were significant differences in gut microbiota among the three groups.Compared with SC group and SB group,Bacteroides was dominant in SA group.Tumor growth curve was statistically significant in SA group compared with SB group(P=0.010).3. There were differences in serum and tumor IFN-?and IL-2 in the three groups.SB group had the highest concentration and SA group had the lowest concentration.Conclusions:Collectively,the diversity of the gut microbiota was associated with the expression of TILs in patients with BC.Human intervention in the gut microbiota of mice could change the tumor growth status of tumor-bearing mice and change the concentration of IFN-?and IL-2in serum and tumor.