ABSTRACT
We employed scRNA sequencing to extensively characterize the cellular landscape of human liver from development to disease. Analysis of â¼212,000 cells representing human fetal, hepatocellular carcinoma (HCC), and mouse liver revealed remarkable fetal-like reprogramming of the tumor microenvironment. Specifically, the HCC ecosystem displayed features reminiscent of fetal development, including re-emergence of fetal-associated endothelial cells (PLVAP/VEGFR2) and fetal-like (FOLR2) tumor-associated macrophages. In a cross-species comparative analysis, we discovered remarkable similarity between mouse embryonic, fetal-liver, and tumor macrophages. Spatial transcriptomics further revealed a shared onco-fetal ecosystem between fetal liver and HCC. Furthermore, gene regulatory analysis, spatial transcriptomics, and in vitro functional assays implicated VEGF and NOTCH signaling in maintaining onco-fetal ecosystem. Taken together, we report a shared immunosuppressive onco-fetal ecosystem in fetal liver and HCC. Our results unravel a previously unexplored onco-fetal reprogramming of the tumor ecosystem, provide novel targets for therapeutic interventions in HCC, and open avenues for identifying similar paradigms in other cancers and disease.
Subject(s)
Carcinoma, Hepatocellular/pathology , Endothelial Cells/metabolism , Tumor Microenvironment/genetics , Adult , Animals , Carcinoma, Hepatocellular/genetics , Cell Line , Disease Models, Animal , Endothelial Cells/pathology , Female , Folate Receptor 2/metabolism , Gene Expression Profiling/methods , Humans , Liver/pathology , Liver Neoplasms/genetics , Macrophages/metabolism , Male , Membrane Proteins/metabolism , Mice , Receptors, Notch/genetics , Receptors, Notch/metabolism , Signal Transduction/genetics , Transcriptome/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
BACKGROUND: Despite recent rapid progress in method development and biological understanding of induced pluripotent stem (iPS) cells, there has been a relative shortage of tools that monitor the early reprogramming process into human iPS cells. METHODS: We screened the in-house built fluorescent library compounds that specifically bind human iPS cells. After tertiary screening, the selected probe was analyzed for its ability to detect reprogramming cells in the time-dependent manner using high-content imaging analysis. The probe was compared with conventional dyes in different reprogramming methods, cell types, and cell culture conditions. Cell sorting was performed with the fluorescent probe to analyze the early reprogramming cells for their pluripotent characteristics and genome-wide gene expression signatures by RNA-seq. Finally, the candidate reprogramming factor identified was investigated for its ability to modulate reprogramming efficiency. RESULTS: We identified a novel BODIPY-derived fluorescent probe, BDL-E5, which detects live human iPS cells at the early reprogramming stage. BDL-E5 can recognize authentic reprogramming cells around 7 days before iPS colonies are formed and stained positive with conventional pluripotent markers. Cell sorting of reprogrammed cells with BDL-E5 allowed generation of an increased number and higher quality of iPS cells. RNA sequencing analysis of BDL-E5-positive versus negative cells revealed early reprogramming patterns of gene expression, which notably included CREB1. Reprogramming efficiency was significantly increased by overexpression of CREB1 and decreased by knockdown of CREB1. CONCLUSION: Collectively, BDL-E5 offers a valuable tool for delineating the early reprogramming pathway and clinically applicable commercial production of human iPS cells.