Role Of Genomic Aberration In Tumorigenesis And Metastasis Revealed By Single-cell Sequencing

Background and Objective Malignant tumor is the major disease whichthreatens human life and health worldwide. Tumor recurrence and metastasis arethe main reasonswhichcause the high mortality. Tumor metastasis is a complex, multi-step process, in which cancer stem cells, circulatingtumor cells and micro-metastasismay playimportant roles. Genomic alterations occur in time of tumorigenesis and development. Therefore, exploring the genomic changes will reveal the molecular signature and mechanism of tumorigenesis and development. However, it is difficult to detect the genomic changes due to the small number of these samples. Traditional biological and clinical analysis is based on cell populations without considering the heterogeneity in tumor cells. The genomic characteristic and molecular mechanism in tumor recurrence and metastasis is unambiguous. Therefore, it is necessary to probe the genetic characteristics at single cell level. We plan to buildup a workflow throughout isolating single cancer stem cell(CSC), circulating tumor cell(CTC) and micro-metastasis lesions, single cell sequencing and bioinformatics analysis.We aim to explore the key driver events in whole genome during tumor metastasis process in single CSC, CTCand micro-metastasis lesions, and to reveal the genetic mechanism in tumorigenesis and metastasis to provide new insight for precision medicine.Methods 1) Fresh liver cancer tissues weredisaggregated into single cell suspensions, and stainedwith fluorescence cell surface marker antibodies. After sorting with FACs, single CSCs were picked up withmicropipette. 2) CTCswere enrichedby the Cell Search?system,and single CTCs were picked up with micropipette after reconfirming the fluorescence signals.Single nucleis were isolated from frozen tumor tissues and pickd up by micropipette. 3) After amplifying single cells with MALBAC, the whole genome libraries for sequencingwere prepared to investigate CNVs.Whole-exome sequencing was performed to investigate SNVs. 4) DNA was extracted from bulk cancer tissues, matched adjacent tissues and blood,to investigate single nucleotide variations(SNVs) after whole-exome sequencing. Mutation sites werevalidatedthrough target PCR and Sanger sequencing. 5) Tumor samples were obtainedfrom multi-lesions ofliver cancer bylaser capture microdissection(LCM) and then DNAwas extracted using commercial kit.Performed bioinformatic analysis to investigate CNVs and SNVs after sequencing,and validated mutation sites with Sanger sequencing.Results 1) CD44+CD90+stem-like cells accounted for 0.5% in liver cancer tissues. Large-scal CNVs in CD44+CD90+stem-like cells and CD44-CD90-cells in liver cancer tissues were reproducible, while heterogeneitystill existed based on focal regions. 2) Liver CSCs followed a consecutive progression model while their copy number variations accumulated during the progress. Liver cancer cells couldbe divided into different populations according to the CNV patterns in CSCs. 3) C: G>A:T transversionwas dominant in hepatocellular carcinoma. 4) Chromosome 1q amplification, chromosome 4q and 16 q deletion weredetected in all hepatocellular carcinoma patients. 5) CNVs in primary tumor cells wereheterogeneous in colon cancer while CTCs exhibited reproducible CNVs and were similar with lymphatic metastasis lesions. 6) CTCs exhibited reproducible copy number variations from the same patient in breast and gastric cancer, and distinct types of CNVs were detected in CTCs from prostate cancer patients. 7) SNVs analysis in CTCs from various cancer typesconfirmedthat mutations were enriched inextracellular matrix(ECM) related genes.Conclusions 1) CNVs in liver cancer stem cells and caner cells wereheterogeneous. Liver cancer stem cells followed a consecutive progression model, cancer cells wereoriginatedfrom liver cancer stem cells at different evolution stage. There werenormal stem cells in liver cancer tissues, the cell surface markerswere not specific to liver cancer stem cells. 2) Genomic variations in hepatocellular carcinomawerecomplex. Therefore,it wasnot comprehensive to analyze primary tumor or single lesion,the inter-patient heterogeneity should be fully considered for personalized therapy. 3) The evolution of CNVs in primary tumor cells followeda selective consecutive progression model whereas SNVsappeared to evolve sporadically. CTCs could be selected from a subclone of highly evolved primary tumor cells. CTCs exhibited reproducible CNVs in most of the patients, but had significant difference across cancer types.SNVs analysis revealedthat there were mutations in ECM related genes enriched in CTCs, we assumed that these genes might beinvolved in tumor metastasis.