RNA-Seq of human U87MG-GFP cells from sub-cutan xenografts as a function of distance from blood vessels

We devised a novel methodology for sorting stroma-free tumor cells according to their relative distance from BVs by injecting a vascular perfusion marker (Hoechst 33342). Briefly, post 4 weeks of sub cutaneous implantation of U87MG-GFP cell line into dorsal flank of NOD-SCID mice, the mice were injected i.v. with Hoechst dye. Mice sacrificed, tumors retrieved, single cell dissociated and FACS sorted to isolate cells located at progressive distance from blood vessels based on perfusion dye intake. Three fractions namely - Hh (close to BVs), Hm (intermediate) and Hl (farther from BVs) were FACS sorted. RNA was extracted from the sorted cells and RNA-Sequencing performed.

本研究开发了一种全新的实验方法，可根据无基质肿瘤细胞与血管（blood vessels，简称BVs）的相对距离对其进行分选，具体通过注射血管灌注标记物霍赫斯特33342（Hoechst 33342）实现。简言之，将U87MG-GFP细胞系皮下植入NOD-SCID（非肥胖糖尿病/重症联合免疫缺陷）小鼠的背部侧翼，待4周后通过静脉（intravenous，简称i.v.）注射霍赫斯特染料。随后处死小鼠并获取肿瘤组织，对其进行单细胞解离，再基于灌注染料的摄取水平，通过荧光激活细胞分选（Fluorescence-Activated Cell Sorting，简称FACS）分离与血管呈不同梯度距离的肿瘤细胞，最终得到三类细胞组分：紧邻血管的Hh、处于中间距离的Hm，以及远离血管的Hl。从分选得到的各组分细胞中提取总RNA，并完成RNA测序（RNA-Sequencing）实验。