Study Of Association Between Intestinal Microbiota And Tumor Microenvironment In DMMR/pMMR Colorectal Cancer

Part Ⅰ The Characteristics of intestinal microbiota in dMMR/pMMR colorectal cancer Objective: Colorectal cancer(CRC)is one of the most common malignant tumors.More and more studies have shown that the tumorigenesis and development of CRC are closely related to intestinal microbiota.The aim of this study integrated microbial ecology and tumor biology to compare and analyze the diversity and community compositions of intestinal microbiota in tumor tissues and paired adjacent non-tumor tissue from patients between deficient mismatch repair(dMMR)and proficient mismatch repair(pMMR)CRC,and to find specific dominant bacteria in intestinal microbiota of CRC patients with different mismatch repair status.Methods: In this study,we collected 230 cases of CRC tumor and paired adjacent non-tumor tissue samples.MMR status was determined byMMR proteins expressions by immunehistochemical staining in FFPE tissue,and defined as either dMMR(n=29)or pMMR(n=201).The bacterial 16 S r RNA genomic DNA from tumor tissues and paired adjacent non-tumor tissues were extracted by kit.The extracted DNA was amplified by employing primers targeting the V3-V4 hypervariable 16 S r RNA region,and then constructed a sequencing library.The original sequencing data was preprocessed for operational taxonomic unit(OUT)classification and species annotations.This study evaluated and analyzed the differences in gut microbial diversity and composition structure between dMMR and pMMR CRC tumor tissues and matched non-tumor tissue samples through bioinformatics analysis such as species taxonomic composition,Alpha diversity,Beta diversity,linear discriminant analysis effect size,species interaction correlation network analysis and functional gene prediction analysis of intestinal microbiota.Results:1.The Simpson,ACE and Chao1 indexes of dMMR tumor tissue group were significantly higher than those of pMMR tumor group(P<0.05).The α diversity of intestinal microbiota in dMMR CRC tumor tissues was significantly higher than that in non-tumor tissues(P<0.05).The Shannon and Simpson indexes of intestinal microbiota in pMMR CRC tumor tissues were significantly increased(P<0.001),while the richness index was significantly reduced(P<0.05).2.dMMR and pMMR CRC had significant differences in the community compositions of intestinal microbiota(P < 0.05).The community structure of the intestinal microbiota between dMMR and pMMR tumor tissues and paired adjacent non-tumor tissues was significantly different(P<0.05).3.At the phylum level,dMMR CRC patients were significantly enriched in Firmicutes(P<0.001),Actinobacteria(P=0.004),Fusobacteria(P<0.001)and Verrucobacteria(Verrucomicrobia,P<0.001);while the pMMR group was enriched in Proteobacteria(P<0.001).At the genus level,the abundance of Fusobacterium(LDA=2.20,P<0.001),Akkermansia(LDA=2.14,P<0.001),Bifidobacterium(LDA=3.45,P<0.001),Faecalibacterium(LDA=2.63,P=0.01),Streptococcus(LDA=3.98,P=0.01),Prevotella(LDA=2.381,P<0.01)in dMMR CRC patients was significantly increased.The pMMR CRC patients were significantly enriched in Serratia(LDA=2.14,P=0.03),Cupriavidus(LDA=4.37,P<0.001)and Sphingobium(LDA=4.98,P<0.001).4.Enterococcus and Ruminococcus were positively correlated,and Enterobacter and Serratia had a strong synergistic effect(P<0.05).5.The significantly enriched pathways in dMMR CRC patients included galactose metabolism pathway(P=0.02),N-glycan biosynthesis pathway(P<0.001),calcium signaling pathway(P=0.008)and other pathways.The pMMR CRC patients were enriched in the degradation pathways of valine,leucine and isoleucine(P<0.001),lysine degradation pathway(P=0.001),tryptophan metabolism and tyrosine metabolism pathways(P= 0.009),butyrate metabolism pathway(P=0.002)and lipopolysaccharide biosynthesis pathway(P<0.001).Conclusion:1.There were significant differences in the community diversity and composition of intestinal microbiota in CRC patients with different mismatch repair status.2.Fusobacterium and Akkermansia were dominant bacteria in the intestinal microbiota between dMMR and pMMR CRC patients.3.There were different functional pathways between dMMR and pMMR CRC patients,which might be the potential mechanism of intestinal microbiota in the tumorigenesis and development ofMMR-related CRC.Part Ⅱ The association between F.nucleatum and A.muciniphila abundance and prognosis of dMMR/pMMR colorectal cancerObjective: The aim of this partly study was to identify the specific bacteria between the dMMR and pMMR CRC patients at the species level using specific strain sequence primers.Next,we tested the abundance changes of Fusobacterium nucleatum(F.nucleatum)and Akkermansia muciniphila(A.muciniphila)in samples between dMMR and pMMR CRC patients to explore the potential of F.nucleatum and A.muciniphila as biomarkers for clinical predictor and prognosis in CRC.Methods: The whole genomic DNA of dMMR and pMMR CRC tissue samples extracted by the kit were detected by quantitative Real-time PCR(q PCR)to assess the abundance of F.nucleatum and A.muciniphila in tumor tissues and non-tumor tissues of dMMR and pMMR CRC.Subsequently,the correlation between the abundance of F.nucleatum and A.muciniphila and the clinicopathological characteristics of dMMR and pMMR CRC patients was analyzed.Univariate analysis and COX multivariate proportional hazards regression analysis model were used to analyze the clinicopathological factors of CRC patient’s prognosis.Results:1.In dMMR CRC tumor tissues,the abundance of F.nucleatum was significantly higher than that in pMMR CRC group(P=0.045).And the abundance of F.nucleatum in dMMR and pMMR CRC tumor tissues was also significantly higher than that in non-tumor tissues(P<0.05).2.Compared with pMMR CRC tumor tissues,the abundance of A.muciniphila in dMMR CRC tumor tissues was significantly increased(P=0.001).And the abundance of A.muciniphila in dMMR and pMMR CRC tumor tissues was higher than that in non-tumor tissues(P<0.001).3.The abundance of F.nucleatum was significantly correlated with the tumor location and tumor size in pMMR CRC patients(P < 0.05).There no significant correlation was observed between F.nucleatum abundance and clinicopathological factors in dMMR CRC patients,such as tumor stage and tumor size(P>0.05).The abundance of A.muciniphila was not correlated with tumor location,tumor size and TNM stage in patients with dMMR and pMMR CRC(P>0.05).4.Significant differences were observed in neurological invasion,distant metastasis,TNM staging and F.nucleatum abundance related to progress free survival(PFS)of CRC.Neurological invasion and F.nucleatum abundance(HR=0.452,95%CI: 0.275-0.743,P=0.002)were independent factors of poor prognosis in CRC.The tumor differentiation,tumor location,tumor size,p T stage,distant metastasis and cancerous tubercle were related to overall survival(OS)of CRC.Among them,the tumor size,distant metastasis and cancerous tubercle were independent prognostic factors of OS in CRC patients(P<0.05).Conclusion:1.The abundance of F.nucleatum in dMMR CRC patients was significantly higher than that in pMMR patients,and F.nucleatum abundance in tumor tissues was also higher than that in non-tumor tissues.2.The abundance of A.muciniphila in dMMR CRC patients was significantly higher than that in pMMR CRC patients,and its abundance in non-tumor tissues was reduced.3.F.nucleatum could be considered as a poor prognostic factor of progress free survival in CRC patients.Part Ⅲ The effects of intestinal microbiota on immune microenvironment in dMMR/pMMR colorectal cancerObjective: The mechanism of interaction with intestinal microbiota and tumor immune system in CRC with different mismatch repair status is not clear.This part of study analyzed and compared the spatial distribution of immune cells in the tumor microenvironment between dMMR and pMMR CRC and the characteristics of checkpoint molecules.This study explored the correlation between the intestinal microbiota in CRC tissue samples with different microsatellite status and immune indicators in tumor immune microenvironment.Methods: Preoperative peripheral blood of dMMR and pMMR CRC patients was collected to analyze peripheral inflammatory indicators,such as NLR,PLR,LMR,AMC and SII.This study collected tumor tissues of dMMR and pMMR CRC,and the infiltration of tumor infiltrating lymphocytes(TILs)was detected by HE staining.Immunohistochemical staining analysis was used to detect the infiltration of CD8+ T cells and CD3+ T cells in the center of the tumor(CT)and invasive margin(IM)within the tumor microenvironment,and the expression level of immune checkpoint molecule Siglec-15.The spatial distribution characteristics of CD8+ T cells and CD3+ T cells infiltration and the expression difference of Siglec-15 between dMMR and pMMR CRC with different F.nucleatum and A.muciniphila abundance levels were analyzed.Then,we analyzed the correlation between the immunological indicators and clinicopathological characteristics of tumor microenvironment with different mismatch repair status.Univariate analysis and COX multivariate proportional hazards regression analysis model were used to analyze the prognostic factors of CRC patients.Spearman test was used to analyze the correlation between peripheral inflammatory indexes and immune indexes of tumor microenvironment and the intestinal microbiota.Results:1.TILs,CD3+ T and CD8+ T cells infiltration levels and immunescores of dMMR CRC patiens were significantly higher than those in pMMR group(P<0.001).The density of CD3+ and CD8+ T cells at invasive margin both in dMMR and pMMR CRC tumor tissues were higher than that in the center of the tumor(P<0.05).2.The CD3+ T cells and CD8+ T cells density within center of the tumor in the F.nucleatum-low/negative group was significantly higher than that in the F.nucleatum-high group of dMMR CRC(P<0.05).The CD8+ T cells infiltration density at invasive margin in the F.nucleatum-low/negative group was significantly higher than that in center of the tumor(P=0.042).The density of CD3+ T cells and CD8+ T cells at invasive margin in the F.nucleatum-low/negative and F.nucleatum-high groups were significantly higher than that in center of the tumor in pMMR CRC patients(P<0.001).Compared with A.muciniphila-high group,the CD3+ T cells density within center of the tumor in the A.muciniphila-low/negative group of dMMR CRC patients was was significantly decreased(P=0.03).The density of CD8+ T cells and CD3+ T cells at invasive margin in A.muciniphila-high group was significantly higher than that in center of the tumor in the same tissue(P=0.024).The CD3+ T cells density of the A.muciniphila-low/negative group was significantly lower than that of the A.muciniphila-high group(P=0.045).The density of CD3+ T cells and CD8+ T cells at invasive margin of A.muciniphila-low/negative and A.muciniphila-high groups were higher than that of center of the tumor of pMMR CRC patients(P<0.001).3.The density of CD8+ T cells within center of the tumor in dMMR CRC was related to tumor size(P<0.001)and pathological type(P=0.001).There was a correlation between CD8+/CD3+ T cell ratio within center of the tumor in dMMR CRC and tumor size(P=0.045),and CD8+/CD3+ T cell ratio in the whole tumor microenvironment had a significant correlation with tumor size(P=0.039).The density of CD3+ T cells within center of the tumor in pMMR CRC was significantly correlated with tumor size(P=0.009),p T stage(P=0.002),TNM stage(P=0.002),distant metastasis(P=0.034)and neurological invasion(P=0.023).There was a significant correlation between the density of CD3+ T cells at invasive margin and TNM stage(P=0.009),liver metastasis(P=0.024),and distant metastasis(P=0.002).The density of CD8+ T cells within center of the tumor was related to p T stage(P=0.010),TNM stage(P=0.029)and neurological invasion(P=0.047).Significant correlations were observed between CD8+ T cells density at invasive margin and TNM staging(P=0.001),liver metastasis(P=0.024),distant metastasis(P=0.005),cancerous tubercle(P=0.020),and neurological invasion(P=0.033).CD8+/CD3+ T cell ratio within center of the tumor in the microenvironment was related to tumor size(P=0.021).The immunescore was significantly correlated with p T stage(P=0.006),TNM stage(P=0.007),distant metastasis(P=0.012),and cancerous tubercle(P=0.012).4.The tumor cells of tumor microenvironment significantly expressed Siglec-15 in pMMR CRC patients(P<0.001).The abundance of F.nucleatum and A.muciniphila were not related to the expression levels of Siglec-15 between dMMR and pMMR CRC patients(P>0.05).There was a significant correlation between Siglec-15 expression of dMMR CRC and pathological type(P=0.006),p N stage(P=0.001)and TNM stage(P=0.045).The expression level of Siglec-15 in dMMR CRC tissue was related to tumor size(P=0.041)and CD8+ T cells density at invasive margin(P=0.048).5.The CD3+ T cells within center of the tumor(P=0.002),CD8+ T cells at invasive margin(P=0.016)and CD8+/CD3+ T cell ratio within center of the tumor(P=0.006)in tumor microenvironment were related to PFS of CRC patients.Among them,CD3+ T cells within center of the tumor(HR=2.647,95%CI: 1.516-4.620,P=0.001)was an independent prognostic factors of CRC patients.6.The F.nucleatum abundance of CRC was positively correlated with peripheral blood NLR and SII(P<0.05),while it was negatively correlated with CD3+ T cells within center of the tumor(P=0.024).The abundance of A.muciniphila,TILs,CD3+ T cells and CD8+ T cells within center of the tumor and invasive margin and immunescores were negatively correlated with F.nucleatum in pMMR CRC(P<0.05).The level of Siglec-15 expression was negatively correlated with CD3+ T cells(P=0.025)and CD8+ T cells at invasive margin(P=0.010).There was a negative correlation between the abundance of F.nucleatum and CD8+/CD3+ T cell ratio at invasive margin in dMMR CRC(P=0.021).CD3+T cells,CD8+ T cells within center of the tumor and invasive margin and immunoscore of CRC tissues were positively correlated with the abundance of A.muciniphila(P<0.05).Conclusion:1.The density of CD3+ and CD8+ T cells within center of the tumor and invasive margin were different due to the abundance of F.nucleatum and A.muciniphila in tumor microenvironment between dMMR and pMMR CRC.2.Siglec-15 was highly expressed in pMMR CRC patients,suggesting that it might become a new checkpoint molecule for alternate immunotherapy;3.There was an interaction between F.nucleatum and A.muciniphila,mismatch repair status and immune cells in CRC.It was speculated that F.nucleatum had different interaction modes in the tumor microenvironment with different mismatch repair status of CRC.