A New Method For Single Cell RNA Analysis Of Circulating Tumor Microemboli

The major cause of cancer-associated mortality is tumor metastasis.Circulating tumor microemboli(CTM)is considered to be the "seed" of tumor metastasis in the process of tumor metastasis.The latest research has showed the high metastatic potential of CTM.The different cell types contained in the CTM provided an enabling environment.Therefore,further study of CTM could help reveal the mechanism of tumor metastasis.The single cell RNA sequencing could provide the transcriptome information located at the terminal of phenotype and the technical basis for the CTM analysis in single cell level.However,the following problems should be solved for singlecell RNA analysis.(1)The prevalence and amount of CTM are rare.It's necessary to constructing in vitro and in vivo CTM models to meeting the requirements of basic CTM research.(2)How to enrich complete CTM from blood and depolymerize the CTM into single cells without RNA degradation.Moreover,the separated single cells should be satisfied the requirements of single cell transcriptome analysis.(3)How to ensure efficient and uniform amplicon due to the rare initial amount of RNA in a single cell.Firstly,Alternative systems have been employed to "mimic" the CTMs due to blood samples contain significantly fewer CTMs than single CTCs.Cell clumps formed during the trypsinization of adherent cell lines or during their growth in non-adherent conditions have been used to investigate the metastatic potential of CTMs as the in vitro model systems.Cell clusters produced from both method with good cell viability and steady state.The number of single cells was one order of magnitude greater than the number of clusters during the trypsinization.The number of clusters and single cells was similar during their growth in non-adherent conditions.Hence,clusters produced from non-adherent conditions were better.In vivo CTM models were constructed by using mouse models to further simulating the physiological state of CTMs.We sampled the blood of tumor-bearing animals for presence of single or clustered CTCs using a terminal bleed.And we observed few CTMs per mouse.We evaluated three different cell cluster dissociation reagents and three different CTMs isolation methods.Tissue(tumor)dissociation/single cell isolation kit can't dissociate the cell cluster.Single cells were harvested using 0.25%trypsin-EDTA with serious RNA degradation in 2 minutes.Cell cluster could be dissociated by non-protein trypsin replacement in 2 minutes.And the single cells from clusters had the same RNA content as fresh single cells.We investigated the different CTMs isolation methods.The conditions of subtraction method are too harsh for CTMs isolation.CTMs were dissociated into single cells after isolation.The filter could keep the integrity of CTMs,while CTMs were anchored on the filter membrane by fluid pressure.The positive sorting method has a better recovery rate of CTMs and maintained the preferable integrity of the CTMs.The recovered CTMs could satisfy the requirement of single-cell RNA analysis.Beads-UMI-seq was built for the problem of single cell transcriptome technology.It was improved method of beads-seq.Six nucleotides of UMI was added to the primers.cDNAs could marked with different UMIs after reverse-transcribtion.we the amount of dATP and dTTP in the PCR process was optimized.As the results of optimized condition,products of cDNA library could meet the high-throughput sequencing requirements.The method not only retains the characteristics of efficiently amplify,but also adds molecular barcode to correct the bias of amplification.We have built a new method for single cell RNA analysis of CTMs based on in vitro and in vivo model-systems,pretreatment of CTMs and non-biased and efficient single-cell cDNA library on beads.The methods provide a technical support for CTM research.