Abstract

Abstract Glioblastoma (GBM) is an aggressive brain cancer and is associated with very poor prognosis. Standard treatment involves surgical resection, post-operative radiation and TMZ chemotherapy. Further research into new therapeutic approaches which target chemo-resistant tumor propagating cells, are urgently required. The EphA3 receptor is frequently elevated in GBM, particularly in the mesenchymal subtype and expressed on tumor-initiating cells (Day et al. Cancer Cell 2013). Our data in GBM tissue shows that tumor cells expressing the high-affinity EphA3 ligand, ephrin A5; are distinct from EphA3 expressing cells. The ephrin A5-positive cells express the glial differentiation marker GFAP, are less proliferative and less stem cell-like. EphA3 is expressed on the vimentin-positive, highly proliferative cell population. Single cell RNA-sequencing revealed that EphA3 and ephrin A5 are highly expressed in recurrent GBM, and are markers of MES-like and AC-like cell states respectively. Ephrin A5 over-expression in-vivo led to extension of survival in orthotopic xenograft animal models. GBM aggressiveness and downregulation of stem cell markers. Spatial transcriptomics of xenograft tumors revealed a reduction in proliferation markers including Ki67, MCM7 and PCNA. We detected an increase in expression of AC-like cell-state with a concomitant reduction of the MES-like and NPC-like cell-states. Consistently, over-expression of ephrin-A5 in primary GBM lines led to a reduction in neurosphere formation, Ki67 staining and growth rates. Thus we propose that dual targeting of EphA3 and ephrin-A5 might better capture GBM tumour heterogeneity and lead to an extension GBM patient survival. Whilst better understanding the biology of ephrinA5 expression, we also aim to validate antibody-based approaches to perform dual targeting of EphA3 and ephrin A5, leading to an extension GBM patient survival.