Dynamic Network Biomarker Study Based On B Cell Of Colorectal Adjacent Tissues

Colorectal cancer(CRC)is one of the top five common gastrointestinal malignancies in terms of morbidity and mortality worldwide.Despite significant advances in surgery and multimodal treatment,survival rates at the progressive stage remain low.A growing evidence suggests that the para-cancerous tissue of the tumor,an intermediate state with mature B cells in the immune microenvironment for antigen presentation and further differentiation into plasma cells,in turn plays a key role in the progressive stage of colorectal cancer.If we can quantify the mature B cells in the para-cancerous tissues and intervene at an early stage to prevent CRC from advanced further,we can effectively improve the survival rate of patients.Dynamic Network Biomarker(DNB)is a method to model the temporal gene expression network based on Hidden Markov Model(HMM),which can systematically quantify the tumor metastasis process,detect the critical transition period,and explore the group of DNB molecules that dominate the network.Thus,exploring biomarkers of pre-metastatic mature B cells in lymph nodes in CRC is essential to stop irreversible progression.To identify such early warning biomarkers of mature B cells,this study performed single-cell Smart-seq2 fulllength sequencing of immune cells(CD45+)isolated from eight paired multi-regional CRC samples and analyzed the obtained mature B cells in para-cancerous tissues of CRC patients from stage I to stage IV using a dynamic network biomarker(DNB)method to identify core gene clusters and screened for potentially functionally important core genes,and finally constructed an animal model of core gene B-cell-specific knockout(CKO)using a gene editing method based on CRISPR/Cas9.The results of the study are as follows:1.Single cell transcriptome analysis of 725 B cells from CRC whole immune atlas identified six B cell subtypes,one of which was identified as CD74+ CD20+ BCL11A+CCR7+ B cells,exhibiting characteristics of a mature B cell subtype.2.DNB analysis of single-cell transcriptome profiles of mature B cells at different stages in adjacent tissues of CRC using the DNB method revealed that the peak of DNB appeared in phase II,the critical period of lymph node metastasis,when the standard deviation(Std)of this group of DNB molecules showed a significant upward trend in phase II,and the intragroup correlation coefficient(Inpcc)also showed a dramatic upward change in phase II,and the inter-group The correlation coefficient(Outpcc)reached the trough of four periods in phase II,indicating that phase II is a critical period before CRC lymph node metastasis.3.Further analysis revealed that DNB molecules underwent flipping changes in network expression levels,i.e.,some gene groups were lowly expressed in the early stage and highly expressed in the advanced stage;on the contrary,some genes that were highly expressed in the early stage were lowly expressed in the advanced stage.Moreover,this study found that DNB genes drive differential gene expression.For example,in the Janus kinase(JAK)-signal transducer and activator of transcription(STAT)pathway,reversal of downstream differential gene expression levels was triggered by DNB,which would affect the ability of mature B cells to differentiate into plasma cells;the study also found similar findings in immune functions such as antigen presentation.4.This study also found that DNB was comprehensively ranked by criteria based on core genes,differential genes,paraneoplastic specificity and high expression in phase II.DHX9 was the core DNB gene for phase II-specific paraneoplastic tissues based on DNB ranking.DHX9 and its neighboring network genes are involved in the dynamic expression of B-cell related pathways.In stage I-III,this group of molecules is reversed at the network level.DHX9 and DHX family have an important prognostic value in CRC as verified by external databases.High DHX9 expression levels imply a better prognosis.These results suggest that DHX9 has further research value as a candidate gene.5.To further investigate the effect of DHX9 on its progression in B cells in CRC,this study proposed to construct DHX9 B cell-specific knockout(CKO)mice,and DHX9 Flox F1 generation mice(DHX9 Flox / +)have been obtained.In conclusion,this study used DNB to provide early warning of mature B-cell subtypes in colorectal cancer paraneoplastic tissues and predicted that stage II could be used as a critical period before CRC lymph node metastasis;DNB genes were found to drive differential gene expression,and DHX9 in mature B cells in CRC paraneoplastic tissues could also be used as a biomarker of prognostic value in CRC progression,providing CRC new clues to potential immunotherapy targets for early intervention.