Formation of EGFRwt/EGFRvIII homo- and hetero-dimers in glioblastoma cells as detected by single molecule localization microscopy.

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.