Rapid Isolation Of Circulating Tumor Cells And Their Ex Vivo Culture

Circulating tumor cells(CTCs)are free tumor cells that shed from tumor sites and enter into blood circulation.CTCs represent a reliable source of tumor cells for the molecular characteristics of the original tumor.CTCs are generally believed to be the main source of cancer metastasis.1mL of peripheral blood contains 5.0~6.0×10~9 red blood cells,while the circulating tumor cells may be only 0~100.And a large amount of data show that circulating tumor cells have significant heterogeneity.It has been found that a large fraction of CTCs are apoptotic due to the loss of matrix-derived survival signals,immune attack or circulatory shear stress.Only a small CTC subset is capable of surviving and initiating distant metastases.Thus identifying molecular characteristics enables the further understanding of the tumor occurrence and development.Despite the continuous development of CTCs detection technology in recent years,its main application is still limited to counting.The extraordinary rarity of CTCs makes the subsequent molecular and functional analysis technically challenging.Ex vivo expansion of CTCs is believed to break this bottleneck and therefore provides sufficient biological material for animal models drug resistance and drug susceptibility and other pre-clinical experiments.In this study,we describe microfludics-based immunomagnetic isolation method to directly isolate CTCs from the whole blood of lung adenocarcinoma patients.It has not only high capture rate but also high viability.The experiment was divided into two groups.We performed single-cell gene detection by immunofluorescent staining,counting,micro-operation to get CTCs.On the other hand,CTCs were enriched in a small volume of liquid to achieve higher cell culture density.Then in vitro culture conditions were optimized to achieve amplification of CTCs.Ultimately glucose uptake analysis and whole exon sequencing were performed.