Objectives: Eribulin mesylate (ERI) is a simplified synthetic macrocyclic ketone analog of the marine sponge natural product halichondrin B. We examined effects of ERI and Paclitaxel (PTX) on blood vessel cells by gene expression profiling (GEP) in mono- and co-culture assays of pericytes and endothelial cells. Activity of ERI and PTX against cell growth inhibition and pericyte-driven in vitro angiogenesis was also studied.
Methods: We first assessed IC50s of ERI and PTX in 4-day growth inhibition assay in isolated primary human brain vascular pericytes (HBVP) and human umbilical vein endothelial cells (HUVEC). Based on identified IC50s, cells were treated with 10X IC50s of ERI or PTX and GEP was analyzed at 24 h using either Affimetrix Human Genome U133 Plus 2.0 arrays or custom TaqMan Low Density Cards (TLDA) designed with 92 genes related to angiogenesis, metastasis/ Epithelial Mesenchymal Transition (EMT) and cell differentiation signal pathways. Inhibitory activity of drugs on the length of capillary-like networks was analyzed in co-culture of HUVEC with HBVP.
Results: ERI and PTX inhibited cell growth of HBVP at IC50s of 1.2 and 3.1nM and HUVEC was more sensitive than HBVP by 1.9 and 3.3 fold in cell growth assay, respectively. In HUVEC, most genes were down-regulated by both ERI and PTX treatments, while in HBVP about equal number of genes were up- or down-regulated with microarray analysis. Interestingly, 63% affected genes in HUVECs overlapped for both treatments. In HBVPs, altered gene signatures were drug-dependent and an overlap was limited by 16%. ERI specifically affected genes in HIF1 and caveolar-mediated signaling pathways while PTX regulated genes in HER2, PI3K/AKT and HGF signaling pathways among others. We confirmed obtained altered GEP using TLDA and identified 42 significant genes differentially regulated by ERI and PTX in HBVP. To examine effects on pericyte-driven in vitro angiogenesis, we compared length of capillary-like networks in co-culture of HUVEC with HBVP. ERI disrupted capillary-like networks starting at about 2 nM, while PTX showed limited inhibitory activity by less than 50% even at 100-1000 nM for 4 days treatments. In co-culture assay, TLDA data showed decreased expression levels of angiogenesis-related genes DLL4 (14% compare to control), PDGFRB (70%) and metastasis/EMT-related genes ZEB1 (53%), TGFB3 (54 %), VIM (62 %) after treatment with 10X IC50s of ERI (p<0.05).
Conclusions: Endothelial cells and pericytes responded differently to ERI and PTX treatments and effects of ERI on GEP of pericytes were distinct from PTX. ERI inhibited pericyte-driven in vitro angiogenesis at sub nM using co-culture assay of HUVEC with HBVP. Further analysis of the role of ERI on GEP of pericytes and pericyte-driven angiogenesis in anti-tumor activity will be warranted.
Citation Format: Sergei I. Agoulnik, Judith L. Oestreicher, Noel H. Taylor, Mai Uesugi, Hiroki Muto, Satoshi Kawano, Kentaro Takahashi, Kentaro Matsuura, Ken Aoshima, Junji Matsui, Yasuhiro Funahashi. Eribulin and Paclitaxel differentially affect gene expression profiling of blood vessel cells and in vitro angiogenesis in co-cultures of human endothelial cells with pericytes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3830. doi:10.1158/1538-7445.AM2013-3830