Isolation,Detection And Characterization Of Circulating Tumor Cells In Peripheral Blood

Circulating tumor cells(CTCs)are the source of malignant tumor metastasis.The isolation and detection of CTCs in peripheral blood plays an important role in early diagnosis,efficacy monitoring,drug resistance monitoring,prognosis evaluation and individualized treatment of tumors.In this paper,based on hydrodynamics,isolating by size of epithelial tumor cells and immunofluorescence staining technology,a set of CTCs disc separation device was built,and a set of CTCs isolation and detection method was established.The separation membrane with the most suitable pore diameter was selected according to the recovery rate of tumor cells and the residual rate of leukocytes.Using lung cancer cell A549 as a cell model,the methodology was evaluated by constructing "A549+5 m L PBS detection system" and "A549+5 mL healthy person venous blood detection system".This method was compared with the CD-PRIME equipment of Clinomics company in Korea,and the blood samples of clinical patients were separated by this method.The results showed that the most suitable separation membrane was the ?8 ?m pore diameter membrane.The overall stability and repeatability of this method were good(CV<5%).In 5 mL PBS detection system,the recovery rate of tumor cells was 73.59% ±2.14%,and a minimum of 2 tumor cells could be detected.In 5 m L healthy venous blood detection system,the recovery rate of tumor cells was 70.05% ±5.89%,and a minimum of 3 tumor cells could be detected,that is,the minimum detection limit for the separation of CTCs by this method is 3 CTCs/5 mL peripheral blood.The recovery rate of tumor cells by the separation method established in this paper(72.05% ±2.62%)was slightly lower than that of CD-PRIME(74.58% ±0.72%),which basically reached the level of market commercialization.This method was successfully applied to the isolation and detection of CTCs in blood samples of patients with clinical lung cancer and the CTCs were successfully isolated from blood samples of 4 patients.On the basis of verifying the feasibility of this method,the A549 cells that mixed in 1 m L healthy venous blood were successfully isolated,eluted and cultured.The transcriptional level of CK18 gene in A549 cells was detected by qRT-PCR technique,and the mutation of EGFR gene was detected by first-generation sequencing and ddPCR technique.The results showed that the transcriptional level of CK18 gene in lung cancer A549 cells was higher than that in normal lung cells,and trace mutations of EGFR-T790 M and EGFR-L858 R were detected in A549 cells by ddPCR technique,but no mutations were detected by first-generation sequencing..The circulating tumor cell isolation and detection method established in this paper could successfully isolate CTCs,from peripheral blood.The tumor cells isolated by this method had biological activity,could be cultured in vitro,and could be characterized at gene level or transcriptional level.The results of this paper provided a laboratory basis for the isolation,identification and characterization of circulating tumor cells in peripheral blood.