The Preliminary Application Of Single Cell Whole Exome Sequencing On Clonal Evolution Of Metastatic Colorectal Cancer

Cancer has become a common health issue that affects all human beings.The death of cancer patients is mainly caused by tumor metastasis.Liver metastasis is common in colorectal carcinoma.With the application of next-generation sequencing technology in cancer research,the intertumoral and intra-tumoral genetic heterogeneity is detected in colorectal cancer.In metastatic colorectal cancer,the mechanism of tumor metastasis remains unknown.Single-cell sequencing can be used to reveal the genotype and phenotype of the tumor in single cell level,which is very suitable for studying tumor heterogeneity,cell lineage tracking and tumor microenvironment.With the application of single-cell sequencing technology in oncology and clinical research,the diagnosis and treatment of tumors will be greatly improved.In this study,we investigated the genetic characteristics and tumor clonal evolution within 102 single cells from a metastatic colorectal cancer patient.Firstly,we collected tumor tissues and adjacent tissues of primary tumor lesion and tumor tissues of liver metastases by surgical resection.We isolated high quality single cells by micromanipulator and performed single-cell whole genome amplification with multiple displacement amplification method.Somatic mutation in single cell and different tumor lesions were called by whole exome deep sequencing,and copy number variations of all tumor tissue were detected by low depth whole genome sequencing.We found that there are 60% common mutation sites and mutant genes,respectively,between primary and metastatic tumor,including TP53,BRAF,SMAD4 and other genes commonly found in colorectal cancer.It is inferred that the tumor metastasis happened in midstage of the tumor evolution.In addition,the mutations of tumor-driven genes in the evolution of metastatic colorectal cancer were studied by cell lineage analysis at single cell level.We revealed that the mutation pattern of APC gene may play an important role in the tumor development of the patient.We also found the driver gene mutation in the early stage of tumor formation and the mutation of SMAD4,a key driver gene,that might promote tumor metastasis in this patient.In the late stage of tumor evolution,CDH23 and DLG2 were mutated,which were related to cell division and adhesion.Overall,the tumor evolution process of metastatic colorectal cancer was deeply analyzed at single cell genome level in this study.We can infer the role of different cell subsets with their genetic characteristics among the tumor development.In the future,the breakthrough in throughput and cost of single-cell genome sequencing will arrive.Large-scale single-cell whole genome sequencing will uncover the mechanism of tumor metastasis.The development of single-cell sequencing and its clinical practice will have a profound impact on the clinical diagnosis and treatment in patients with metastatic cancer.