The Simulated Optimization And Single-cell Analysis Based On The Herringbone Microfluidic Chip For Isolating Circulating Tumor Cells From Peripheral Blood

Circulating tumor cells(CTCs)shed from primary and metastasis tumors,then enter into blood circulation.They are the main resource of cancer metastasis.As a “liquid biopsy”,CTCs take advantages of repeatable detection,convienient sampling and minimal invasion,and have clinically important applications.CTCs as new tumor markers can prompt cancer metastasis and offer important information for early diagnosis and clinical stages.They are also served as key indicators for prognostic evaluation and monitor of treatment.In the meantime,the molecular characterization of CTCs enable targeted drug to further guide individual therapy.CTCs are extraordinarily rare and exhibit large functional heterogeneity.Therefore,it will be a great challenge to develop a high-sensitve,reliable and precision technology for capture and isolation of CTCs.Previous studies reported the single-sided herringbone chip for isolating CTCs from cancer blood,but they seldom carried out more deeper research focusing on the theoretical modeling and optimization of the herringbone chips.The paper developed an analytical model to optimize the geometric parameters and experimental conditions for enhacing CTCs capture efficiency by investigation of the effective contacts between cells and antibody-immobilized device surface.The study simulated the fluid flow within the single-sided herringbone chip to quantify how geometric parameters,flow rates and flow directions affect the cell-surface contact for cell capture.The results of simulated optimization are also successfully validatd by cell lines experiments.The optimized single-sided herringbone chip enables a 90±3% of capture efficiency of CTCs.In addition,the analytical model is also applied onto a novel double-sided herringbone chip.As a contrast to the geometrically optimized single-sided herringbone chip,the double-sided herringbone chip fails to exhibit higher effective cell-device contacts.However,the capture efficiency of CTCs directly from whole blood reached 94±4% using the optimized double-sided herringbone chip owing to the effects of leukocyte margination and plasma skimming.Although the capture efficiency of CTCs is high,it is confined to cell counts instead of further fuctional analysis.Given this,the paper describes one-step,microfluidics-based immunomagnetic isolation method of viable CTCs from the whole blood of non-small-cell lung cancer(NSCLC)patients.The identified CTCs are released from the chip and individually retrieved by a micromanipulator,followed by single-cell gene sequencing.Besides,The isolated CTCs are successfully cultured and grown ex vivo.In order to further identify the phenotype of CTCs,the paper utilizes a new integration of microwell array and beads-on-barcode antibody microarray(BOBarray).The released CTCs from the herringbone chip are transferred into microwell array.The number of microwells is far more than that of released cells,so there only could be a maximum of one cell per well.The proteins released from in-situ lysed single cell are captured using BOBarray.In the study,the protein markers quantify the multiplexed protein of lysed single-cell,including three organ-specific markers,two drug targets and the common leukocyte marker.Ultimately,the protein profile-based results could evaluate the origin of CTCs to guide further targeted therapy.In conlusion,this study has established the effective platform for CTCs capture and detection,which has advantages of high specificity,high sensitivity and reproducibility.It will provide valuable references for clinical application of CTCs.