Subclonal evolution and expansion of THY1-positive cells is associated with recurrence in glioblastoma

Glioblastoma(GBM) is a lethal disease characterized by treatment resistance and recurrence. To investigate the mechanisms that drive treatment resistance in GBM, we developed a longitudinal in vivo recurrence model utilizing patient-derived GBM explants to produce paired specimens pre- and post-recurrence following temozolomide(TMZ) and radiation(IR) therapy. These studies revealed in replicate cohorts, a common gene expression profile upon recurrence, characterized by an upregulation of transcripts associated with a mesenchymal and stem cell phenotype, including TGF{beta}1, TGF{beta}2, SOX2, ZEB2, GLI2 and THY1(CD90), with greater than one-hundred-fold increase in THY1 levels. Analyses of clinical databases revealed the association of this transcriptional profile with worse overall survival and elevation in recurrent tumors. We then isolated THY1-positive cells from treatment-naive patient samples which demonstrated inherent resistance to chemoradiation when implanted intracranially. Additionally, using image-guided biopsies from treatment-naive human GBM we conducted spatial whole transcriptomic analyses. This revealed rare THY1+ regions characterized by elevation of the mesenchymal and stem-like gene expression profile, previously identified in our in vivo recurrent samples, which co-localized with a macrophage gene signature within the perivascular niche. Since TGF{beta} signaling contributes to a mesenchymal/stem-like phenotype and therapeutic resistance, and to investigate its effect on THY1, we inhibited TGF{beta}RI kinase activity in vivo which resulted in decreased expression of genes characteristic of a mesenchymal/stem-like phenotype, including THY1. Notably, TGF{beta}RI inhibition restored sensitivity to TMZ/IR in recurrent tumors in vivo. These studies reveal that post-TMZ/IR recurrence may result from tumor repopulation by pre-existing, therapy-resistant, THY1-positive mesenchymal/stem-like cells within the perivascular niche. One Sentence SummaryTHY1 positive tumor cells drive resistance in glioblastoma