利用奈米鑽石連接SSEA-1抗體追蹤神經多形性膠質瘤細胞中SSEA-1蛋白的選擇性自噬路徑

Abstract

階段特異性胚胎抗原(SSEAs)是由醣蛋白或醣脂質所攜帶的醣化抗原表位，例如SSEA-1是胚胎或癌症幹細胞的標誌。奈米鑽石是一種以碳為基礎的生物相容性材料，被開發使用於細胞的標定和檢測。先前我們發現奈米鑽石會被泛素披覆並和自噬作用受器結合而進入選擇性自噬路徑，在此篇研究中，我們發現奈米鑽石連接SSEA-1抗體能和表達於神經膠質瘤細胞的SSEA-1蛋白結合並進入選擇性自噬路徑。我們探討來自神經膠質瘤患者的癌細胞株，包括會表現SSEA-1蛋白於S1R1細胞株，以及不表現的GBM8901細胞株。奈米鑽石連接SSEA-1抗體能和S1R1細胞的SSEA-1蛋白結合並提高細胞對奈米鑽石的攝取量，但GBM8901細胞則無此現象發生。以免疫沉澱法及多光子共軛焦螢光顯微鏡分析可觀察到奈米鑽石連接SSEA-1抗體複合體能與細胞SSEA-1蛋白結合。此外，我們發現奈米鑽石SSEA-1抗體複合體，能被細胞中的泛素披覆並被自噬作用受器SQSTM1和LC3辨識而運送到溶酶體。這些發現顯示奈米鑽-SSEA-1抗體複合體能夠藉由和SSEA-1蛋白結合而進入選擇性自噬路徑。我們利用奈米鑽石追蹤SSEA-1蛋白進入選擇性自噬作用，可能提供一個新的角度來探討癌症幹細胞與選擇性自噬作用之間的相關性。

Stage-specific embryonic antigens (SSEAs) are glycosylated epitope carried by glycoproteins or glycolipids. For example, SSEA-1 is a marker in embryonic or carcinoma stem cells. Nanodiamond (ND) is a biocompatible carbon-based nanomaterial, which is developed for cellular labeling and detection. Previously, we found that ubiquitin (Ub)-coated NDs bind to autophagy receptors for entry into selective autophagy pathway. In this study, we show ND conjugated SSEA-1 antibody (Ab) that could enter the selective autophagy pathway through bind to SSEA-1 proteins which expressed in glioblastoma multiforme stem (GBM) cells. We investigated the cells, that were from GBM patients, which expressed SSEA-1 protein expressed in the S1R1 cell line but not expressed in GBM8901 cell line. The ND-conjugated SSEA-1 Ab could bind to the SSEA-1 proteins and increased the uptake ability of NDs in the S1R1 cells but not in the GBM8901 cells. Furthermore, the ND-SSEA-1 Ab complexes bind with SSEA-1 proteins that can be observed by using immunoprecipitation and confocal microscopy. In addition, we found that the complexes of ND-SSEA-1 Ab could be coated on Ub and then recognized by autophagy receptors SQSTM1/p62 and LC3 to transport to the lysosomes. These findings reveal that ND-SSEA-1 Ab complexes could enter the selective autophagy pathway through binding to SSEA-1 proteins. We demonstrate that ND can be used for tracking of SSEA-1 proteins entering the selective autophagy pathway and may provide a new perspective to investigate relationship between cancer stem cell marker and selective autophagy pathway.