Kirchhoff-Institute for Physics, Heidelberg University, Im Neuenheimer Feld 227, 69120 Heidelberg, Germany.
Department of Radiotherapy & Radiation Oncology, University Medical Center Hamburg - Eppendorf, Martinistr. 52, 20246 Hamburg, Germany.
Nanoscale. 2024 Aug 15;16(32):15240-15255. doi: 10.1039/d4nr01570c.
Super-resolution microscopy has been used to show the formation of receptor clusters and adapted lipid organization of cell membranes for many members of the ErbB receptor family. The clustering behaviour depends on the receptor size and shape, possibly ligand binding or expression activity. Using single molecule localization microscopy (SMLM), we also showed this typical clustering for the epidermal growth factor receptor variant III (EGFRvIII) in glioblastoma multiforme (GBM) cells. EGFRvIII is co-expressed with the wild type (EGFRwt) and both receptors are assumed to preferentially form hetero-dimers leading to transactivation and elevated oncogenic EGFR-signalling in GBM cells. Here, we analysed EGFRvIII and EGFRwt co-localization using our already described model system of the glioblastoma cell line DKMG, displaying endogenous EGFRvIII expression. Using EGFRvIII and EGFRwt specific antibodies, EGFR localization and their potential for dimerization in a given membrane cluster were analysed by dual colour SMLM supported by novel approaches of mathematic evaluations including Ripley statistics, persistent homology and similarity algorithms. Surprisingly, cluster analysis, Ripley point-to-point distance statistics for cluster geometry and persistent homology comparing cluster topology, revealed that both EGFRvIII and EGFRwt do primarily not form hetero-dimers but the results support the hypothesis that they tend to form homo-dimers. The ratio of homo-dimers obtained by this calculation was significantly higher (>5σ, standard deviation) than expected from randomly arranged points. In comparison, hetero-dimer formation was only slightly increased. We confirmed these data by immunoprecipitation, which show no co-precipitation of EGFRvIII and EGFRwt. Furthermore, we showed that the topology of the clusters was more similar among the same type than among the different types of receptors. Taken together, these data indicate that EGFRvIII does induce oncogenic signalling by homo-dimerisation and not preferentially by hetero-dimer formation with EGFRwt. These data offer a new perspective on EGFRvIII signalling which will lead to a better understanding of this tumour associated receptor variant in GBM.
超分辨率显微镜已被用于显示受体簇的形成和细胞膜的适应脂质组织，用于许多 ErbB 受体家族成员。聚类行为取决于受体的大小和形状，可能配体结合或表达活性。使用单分子定位显微镜 (SMLM)，我们还显示了这种典型的聚类现象，用于多形性胶质母细胞瘤 (GBM) 细胞中的表皮生长因子受体变体 III (EGFRvIII)。EGFRvIII 与野生型 (EGFRwt) 共同表达，并且假定这两种受体都优先形成异二聚体，导致 GBM 细胞中的转激活和升高的致癌性 EGFR 信号。在这里，我们使用已经描述的具有内源性 EGFRvIII 表达的神经胶质瘤细胞系 DKMG 的模型系统，分析 EGFRvIII 和 EGFRwt 的共定位。使用 EGFRvIII 和 EGFRwt 特异性抗体，通过双色 SMLM 进行 EGFR 定位及其在给定膜簇中的二聚化潜力分析，该分析得到了新的数学评估方法的支持，包括 Ripley 统计、持久同调以及相似性算法。令人惊讶的是，聚类分析、用于簇几何形状的 Ripley 点到点距离统计以及比较簇拓扑的持久同调显示，EGFRvIII 和 EGFRwt 主要不形成异二聚体，而是支持它们倾向于形成同二聚体的假说。通过该计算获得的同二聚体的比例明显高于（>5σ，标准偏差）随机排列的点预期的比例。相比之下，异二聚体的形成仅略有增加。我们通过免疫沉淀证实了这些数据，这些数据显示 EGFRvIII 和 EGFRwt 没有共沉淀。此外，我们表明，同一类型的簇之间的拓扑结构比不同类型的受体之间更相似。总之，这些数据表明 EGFRvIII 通过同二聚化而不是与 EGFRwt 优先形成异二聚化来诱导致癌信号。这些数据为 EGFRvIII 信号提供了新的视角，将导致对 GBM 中这种肿瘤相关受体变体的更好理解。
