Tumoroid Construction Based On Microfluidic Chip Combined With Label-free Quantitative Proteomic Analysis

Recent years,microfluidic three-dimensional(3D)tumor culture technique has made great progress in tumor microenvironment simulation,cancer cascade mimic and drug screening.Meanwhile,as their functionality and complexity increase,it is more difficult for current chip models to selectively collect specific-layer samples from chip for further analysis.Moreover,a simplified and robust method for tumoroid formation with highly consistent size is relatively necessary.Here,we report a microfluidic chip,through a dual-flip strategy to implement straightforward tumoroid establishment.This platform guarantees stable batch-to-batch tumoroids formation and allows high resolution confocal imaging.Moreover,an initial cell density as low as 65 cells per chamber is efficient to deliver a tumoroid.With this chip,different-layer cells of interest could be collected from tumoroid for label-free quantitative(LFQ)proteomic analysis.For application demonstration,we constructed three different types tumoroid including hepatoblastoma(Hep G2),gastric carcinoma(MGC803-GFP)and lung carcinoma(A549).And we further verified this platform for A549 tumoroid proteomic analysis.Our data indicate that the out-layer cells of A549 tumoroid show extensively distinct proteomic expressions compared to two-dimensional cultured A549 cells.The up-regulated proteins are mainly related to tumorigenicity,proliferation and metastasis.And the differentially expressed proteins are mainly relevant to lipid metabolism pathway which is essential to tumor progression and proliferation.This platform enables cell morphological heterogeneity observation and proteomic expression heterogeneity analysis.And it also opens the way to numerous applications such as tracing the large-scale protein alterations induced by various environmental factors(such as drugs,mechanical stress,hypoxia,physical confinement,etc.)...