Identification and characterization of EGF receptor in individual exosomes by fluorescence-activated vesicle sorting
Identification and characterization of EGF receptor in individual exosomes by fluorescence-activated vesicle sorting
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DOI：
10.3402/jev.v5.29254
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发表时间：
2016-01-01
影响因子：
16
通讯作者：
Coffey, Robert J.
中科院分区：
文献类型：
--
作者：
Higginbotham, James N.;Zhang, Qin;Coffey, Robert J.
外泌体是一种40-130 nm的小细胞外囊泡，作为细胞间通讯、疾病生物标志物和潜在的药物递送载体，近年来已成为人们关注的焦点。目前尚不清楚一个细胞是否产生具有不同货物和可分离功能的不同种群的外泌体。为了解决这个问题，需要高分辨率的方法。使用商用流式细胞仪和直接标记的荧光抗体，我们展示了使用荧光激活囊泡分选（FAVS）分析和分选从DiFi细胞（人类结直肠癌细胞系）的条件培养基中通过顺序超离心分离的单个外泌体的可行性。用fas法检测单个DiFi外泌体上的EGFR和外泌体标志物CD9；此外，通过高分辨率随机光学重建显微镜，在流动分选的EGFR/CD9双阳性外泌体中大约100 nm的单个囊泡上鉴定了这两种标记物。我们提供的证据表明，使用单克隆抗体（mAb）可以识别“构象活性”EGFR (mAb 806)，可以在difi衍生的外泌体中评估EGFR的激活状态。使用人类抗原特异性抗体，FAVS能够检测从携带DiFi肿瘤异种移植的胸腺裸鼠血浆中分离的外泌体上的人类EGFR和CD9。采用多色fas同时鉴定3例正常人血浆源性外泌体上的CD9、EGFR和EGFR配体双调节蛋白（AREG）。这些研究证明了基于特定细胞表面标记的fas分析和分类单个外泌体的可行性。我们认为fas可能是监测结直肠癌和其他实体肿瘤患者循环外泌体中EGFR和AREG的有用工具。
Exosomes are small, 40-130 nm secreted extracellular vesicles that recently have become the subject of intense focus as agents of intercellular communication, disease biomarkers and potential vehicles for drug delivery. It is currently unknown whether a cell produces different populations of exosomes with distinct cargo and separable functions. To address this question, high-resolution methods are needed. Using a commercial flow cytometer and directly labelled fluorescent antibodies, we show the feasibility of using fluorescence-activated vesicle sorting (FAVS) to analyse and sort individual exosomes isolated by sequential ultracentrifugation from the conditioned medium of DiFi cells, a human colorectal cancer cell line. EGFR and the exosomal marker, CD9, were detected on individual DiFi exosomes by FAVS; moreover, both markers were identified by high-resolution stochastic optical reconstruction microscopy on individual, approximately 100 nm vesicles from flow-sorted EGFR/CD9 double-positive exosomes. We present evidence that the activation state of EGFR can be assessed in DiFi-derived exosomes using a monoclonal antibody (mAb) that recognizes "conformationally active" EGFR (mAb 806). Using human antigen-specific antibodies, FAVS was able to detect human EGFR and CD9 on exosomes isolated from the plasma of athymic nude mice bearing DiFi tumour xenografts. Multicolour FAVS was used to simultaneously identify CD9, EGFR and an EGFR ligand, amphiregulin (AREG), on human plasma-derived exosomes from 3 normal individuals. These studies demonstrate the feasibility of FAVS to both analyse and sort individual exosomes based on specific cell-surface markers. We propose that FAVS may be a useful tool to monitor EGFR and AREG in circulating exosomes from individuals with colorectal cancer and possibly other solid tumours.