Establishment Of Nasopharyngeal Carcinoma Circulating Tumor Single Cell Separation Method And CDNA Library

Background:Nasopharyngeal carcinoma(NPC)is the most commonly diagnosed head and neck cancer in Southeast Asia,with the high rates for cervical lymph node metastasis and distant metastasis.Most patients in the clinical diagnosis are already in the middle-late.With the development of radiotherapy,the therapeutic effect of early nasopharyngeal carcinoma has been improved,but distant metastasis is still the main reason of treatment failures.To explore the mechanism of distant metastasis and find out the biomarkers and therapeutic targets may be the possible breakthrough points to prevent and control NPC.Circulating tumor cells(CTCs)are tumor cells derived from the blood circulation,which are considered as the prerequisite fact for distant metastasis of tumor.Finding the CTC in the circulatory system and studying its biological characteristics may be helpful to reveal the mechanism of tumor distant metastasis.Smart-seq2 could analyze the transcriptomics of CTC from the level of single cell,which is more conducive to reveal the metastases mechanism of NPC.However,the using of the epithelial phenotype sorting circulating tumor cell technology is not yet fully defined tumor cells,limiting the clinical application of CTC,and brings to the single cell level analysis of the biological characteristics of CTCS bias.So,the key to analysis of NPC transcriptomics is establishing an unbiased NPC single CTC separation method and single CTC of NPC cDNA library.Objective:The main purpose of this study is to establish an effective method of capturing and identification CTCs from NPC,and build the nasopharyngeal tumor circulation single cell cDNA library which can be used in Smart-seq2.Methods:In this study,single-cell cDNA library of HONE1 was established in accordance with the Smart-seq2 steps,and made quality control for cDNA library by using real-time PCR.And then,we selected suitable method of removing red blood cells by comparing the influence of different methods on the single cell cDNA library,and compared expression of EpCAM and CK in primary nasopharyngeal tumor tissues and metastatic lymph nodes with immunohistochemical staining to found out suitable marker of CTCs of NPC.Subsequently,we built the CTC simulation model by mixing AGS-EBV-GFR cells with stable expression of green fluorescence and peripheral blood of healthy people,and explored efficiency and specificity of acquiring CTC by EpCAM-beads through detecting green fluorescence and DAPI staining.At last,CTCs were isolated from peripheral blood of NPC patients and single cell cDNA libraries were constructed,and made sure that single-cell cDNA library derived from CTCs of NPC by detecting EBV encode product LMP2A.The cDNA library was tested with Bioanalyzer system to check the quality whether meet the Smart-seq2 platform standards.Results:1.The target fragment was successfully amplified with real-time PCR primers hmPOLR2A,hmPOLR2A-2 and hmPOLR2A-1.The N-terminal and the C-terminal of RNA template had a closed initial concentration in the cDNA library.2.Erythrocytes were successfully removed by using red cell lysis buffer and lymphocyte separation medium.The success rate of building single cell cDNA library was 50%by using lymphocyte separation medium,while it was only 16.7%by red cell lysis buffer.3.EpCAM,CK expressed in both the primary tumors and the metastasis lesions.EpCAM expressed on the membrane surface,the expression of metastasis lesions was lower than that of the original site.4.In the CTC simulation model,EpCAM-beads for CTC efficiency could reach 56.7%,and all the cells were AGS cells,which observed green fluorescence 100%.5.There were 80%(4/5)single-cell cDNA library tested LMP2A in a NPC patient CTC.6.The fragment size of the single-cell cDNA library of NPC CTC was 600-2000bp,without or containing a small amount of below 500bp fragment,which met the requirements for building a Smart-seq2 platform.Conclusion:Greater unbiased separation of nasopharyngeal CTC single cells was achieved through isolating with lymphocyte separation medium,combining EpCAM-beads positive selection and detecting expression of EBV encoded product LMP2A.Based on these,a CTC cDNA library which meet the Smart-seq2 RNA sequencing platform could be built.