Single-Cell Transcriptome Analyses Reveal Endothelial Cell Heterogeneity in Tumors and Changes following Antiangiogenic Treatment

Abstract

Angiogenesis involves dynamic interactions between specialized endothelial tip and stalk cells that are believed to be regulated in part by VEGF and Dll4-Notch signaling. However, our understanding of this process is hampered by limited knowledge of the heterogeneity of endothelial cells and the role of different signaling pathways in specifying endothelial phenotypes. Here, we characterized by single-cell transcriptomics the heterogeneity of mouse endothelial cells and other stromal cells during active angiogenesis in xenograft tumors as well as from adult normal heart, following pharmacologic inhibition of VEGF and Dll4-Notch signaling. We classified tumor endothelial cells into three subpopulations that appeared to correspond with tip-like, transition, and stalk-like cells. Previously identified markers for tip and stalk cells were confirmed and several novel ones discovered. Blockade of VEGF rapidly inhibited cell-cycle genes and strongly reduced the proportion of endothelial tip cells in tumors. In contrast, blockade of Dll4 promoted endothelial proliferation as well as tip cell markers; blockade of both pathways inhibited endothelial proliferation but preserved some tip cells. We also phenotypically classified other tumor stromal cells and found that tumor-associated fibroblasts responded to antiangiogenic drug treatments by upregulating hypoxia-associated genes and producing secreted factors involved in angiogenesis. Overall, our findings better define the heterogeneity of tumor endothelial and other stromal cells and reveal the roles of VEGF and Dll4-Notch in specifying tumor endothelial phenotype, highlighting the response of stromal cells to antiangiogenic therapies. These findings provide a framework for defining subpopulations of endothelial cells and tumor-associated fibroblasts and their rapid changes in gene expression following antiangiogenic treatment. .

Single cell genomic characterization reveals the cellular reprogramming of the gastric tumor microenvironment

“…ESM1 , ANGPT2 ), tip cell markers (e.g. COL4A1 , COL4A2 , DLL4 , MARCKSL1 ) and stalk cell markers ( ACKR1 , CD36 , SELP , VWF ) ( Supplementary Figure 11B, 11C) (34).…”

Extracellular Matrix in the Tumor Microenvironment and Its Impact on Cancer Therapy

“…As carcinoma-or tumor-associated fibroblasts (CAFs) are the main source of the ECM in tumors, it is necessary to have a closer look at the particularities of these cells (Bagordakis et al, 2016;Pankova et al, 2016;Pasanen et al, 2016). CAFs are found in all solid tumors (Puram et al, 2017;Zhao et al, 2018). They differ substantially from the quiescent, metabolically inactive fibroblasts found in normal connective tissue, as they are migratory, growth and immune response promoting, and synthetically active (reviewed in Kalluri, 2016).…”

Cells and gene expression programs in the adult human heart

“…Neighbouring clusters contained an arterial endothelial subpopulation (EC_art), as confirmed by specific enrichment of SEMA3G , EFNB2 , DLL4 , and a venous endothelial subpopulation (EC_ven) expressing a well-accepted venous marker NR2F2 38 and ACKR1 ( Figure 3A , Supplementary Figure C2 and Supplementary Table C2 ). ACKR1 was reported to also be expressed in endothelial stalk cells in cancer 39 , EC expanding in cirrhosis 40 and the endothelium of lymphnodes' post-capillary venules 41 .…”