图1。胶质瘤肿瘤表面图谱(TS-MAP)。(A)在完整患者、小鼠肿瘤、原发性GBM 2D和3d球形培养中揭开表面组和内吞细胞组的程序示意图(A1)。(A2)建立了可逆生物素化、高亲和HPLC和LC-MS/MS富集TS的工作流程，集成了使用GaAsP阵列共聚焦探测器(Airyscan)和FACS的高分辨率成像。(A3)一个内部的TS分类器(SURFME)用于过滤和分类真正的膜蛋白，通过匹配肿瘤切片的免疫荧光分析确定潜在的候选靶点，并在体外进行ADC治疗试验。(B)在2D和3D条件下培养的原代GBM细胞表面组和内吞细胞组的定量。如所示，在2D或3D中生长的U3065(上)和U3082(下)GBM细胞中，非生物素化(对照)、总表面生物素化(表面)、MesNa处理后的残留细胞表面信号(表面+ MesNa)和内吞表面蛋白(内化)的FACS分析的代表性直方图。(C) B中所述实验的定量分析，内吞蛋白表达为总表面组蛋白丰度的一部分。数据以三次独立实验的平均值±标准差表示，每次实验重复三次。MFI，中位荧光强度。 (D) High-resolution Airyscan imaging of surface and endocytosed biotinylated proteins (green) in GBM cells grown in 2D or 3D (shown in representative brightfield images), as indicated. (Scale bars, 5 μm [U3065 2D], 2 μm [U3065 2D Inset], 50 μm [U3065 and U3082 3D, Left], and 20 μm [U3065 and U3082 3D, Left].) (E) Airyscan imaging of endocytosed biotinylated proteins (green) and the early endosome marker EEA1 (magenta) in GBM cells grown in 2D or 3D, as indicated, after 1.5 h of endocytosis. (Scale bars, 5 μm, and 2 μm for Insets.) (F) Airyscan imaging for visualization of endocytosed surface proteins in the membrane of endolysosomal vesicles, as indicated by CD63-mCherry (magenta). Images were captured 45 min after initiation of endocytosis (see also Movies S1 and S2). (Scale bars, 2 μm.) (D–F) Shown are representative images from at least three independent experiments. White squares indicate zoomed areas shown in Insets. White arrows indicate colocalization. Credit: DOI: 10.1073/pnas.2114456119
