Establishment Of Organoid Culture Technology For Circulating Tumor Stem Cells And Single Cell Isolation And Labeling Technology

Circulating tumor stem cells(CTSCs)play a crucial role in the tumor recurrence and metastasis,despite the rarity of them in the blood.Therefore,obtaining the information of circulating tumor stem cells is of great clinical significance for controlling tumor recurrence and metastasis in early clinical stage.However,the capture,enrichment and culture of circulating tumor cells(CTCs)is a technical difficulty.This study aims to establish a 3D organoid culture technology for CTSCs and develop a droplet microfluidic single-cell transcriptome detection platform.In this study,we firstly established a simulated tumor model to enrich tumor cells in the blood by the negative selection method,and then used serum-free conditioned medium to conduct 3D culture of the enriched cells.We observed that part of the cells isolated from blood proliferate into cell microspheres.Immunofluorescence results show that the microspheres obtained from 3D culture conditions not only express EpCAM,the marker of epithelial cells,but also express CD 133,the marker of tumor stem cells.Furthermore,we compared the expression levels of sternness-related genes of prostate cancer LNcaP cells and lung cancer H2347 cells in 3D culture and ordinary 2D culture by fluorescence quantitative PCR and western blot.The results show that the expression levels of stemnes-related genes,such as CD 133,CD44 and ABCG2,are significantly higher in the tumor cells obtained from 3D culture than those in 2D culture.In addition,in the serum-free of 3D culture system,the sphere forming rate of LNCaP cell line is(18.67±3.06)%.We also planted drug-resistant cancer stem cells in the 3D culture system,and found that these tumor cells showed stronger cell proliferation ability and formed tumor microspheres with larger diameter.In clinic practice,CTCs are enriched and isolated from lung cancer patients,and the tumor microspheres derived from CTSCs are successfully obtained through the 3D organoid culture technology.So far,the samples from 6 lung cancer patients are collected,3 of which successfully obtain lung cancer organoids and culture in vitro for more than 1 month,with a long-term culture success rate of 50%.Furthermore,the samples of lung cancer arganoids are fixed,embedded with paraffin,sectioned and stained with hematoxylin and eosin to analyze the morphological and structural characteristics.HE staining results indicate that lung cancer organoids manifest typical characteristics of cancer cells and tissue structureOn the other hand,in order to further study the molecular phenotypic feature of different tumor cells,we independently develop a high-throughput droplet microfluidic single-cell transcriptome detection platform based on the principle of "Drop-seq".By a microfluidic system,we successfully accomplish the co-encapsulation of the single cell and barcoded beads in oil-water emulsion droplets,namely the separation and labeling of single cells.When oil phase and water phase flow rate respectively is 200 and 30 p.L/min,and the cell and beads concentrations respectively is 250 and 400/μL,the optimum cell labeling rate is reached-(11.87±3.75)%.In order to evaluate the reliability of the established microfluidic single-cell transcriptome detection platform,we used a mixed human acute promblastic leukemia cell line HL-60 and mouse melanoma cell line B16-F10 as simulated samples for single-cell RNA-seq,and obtained the transcriptome data of 7535 cells in total.Among them,there are 70 cells mixed with human and mouse transcripts,accounting for only 0.9%of the total cells,indicating that the single-cell transcriptomic data obtained by the established microfluidic single-cell RNA-seq technology are of high accuracy.Subsequently,by the analysis of the single-cell transcriptomic data,we can not only distinguish the two cell types,but also screen out the specific expression markers of them.In conclusion,we establish an organoid culture technology for circulating tumor cells to enrich the circulating tumor stem cells.Lung cancer organoids are obtained by this method,and show the heterogeneity of circulating tumor cells and the feature of tumor issues.In addition,we also successfully develop a droplet microfluidic single-cell transcriptome detection platform,which can meet the requirements of oncology laboratory and show extensive potential for tumor research.