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

Abstract

PurposeGlioblastoma (GBM) is a lethal disease characterized by inevitable recurrence. Here we investigate the molecular pathways mediating resistance, with the goal of identifying therapeutic opportunities to target this tumor. Experimental DesignWe developed a longitudinal in vivo recurrence model utilizing patient-derived explants to produce paired specimens (pre- and post-recurrence) following temozolomide(TMZ) and radiation(IR). These specimens were evaluated for treatment response and to identify gene expression pathways driving treatment resistance. Findings were clinically validated using spatial transcriptomics of human GBMs. ResultsThese studies reveal in replicate cohorts, a gene expression profile characterized by upregulation of mesenchymal and stem-like genes at recurrence. Analyses of clinical databases revealed increased expression of this transcriptional profile to be significantly associated with worse median overall survival (248 days vs 430 days, p=0.0004), and upregulation of this profile at recurrence. Most notably, we identified upregulation of TGF{beta} signaling, and more than one-hundred-fold increase in THY1 levels at recurrence. Utilizing cell sorting, we observed that THY1-positive cells represented <10% of cells in the treatment-naive tumors and 75-96% in the recurrent tumors. We then isolated THY1-positive cells from treatment-naive patient samples and determined that they were inherently resistant to chemoradiation in orthotopic models. Additionally, using image-guided biopsies from treatment-naive human GBM, we conducted spatial transcriptomic analyses. This revealed rare THY1+ regions characterized by mesenchymal/stem-like gene expression, analogous to our recurrent mouse model samples, which co-localized with macrophages within the perivascular niche. Since TGF{beta} signaling contributes to a mesenchymal/stem-like phenotype, we inhibited TGF{beta}RI activity in vivo which resulted in decreased mesenchymal/stem-like protein levels, including THY1, and restored sensitivity to TMZ/IR in recurrent tumors. ConclusionsThese findings reveal that GBM recurrence may result from tumor repopulation by pre-existing, therapy-resistant, THY1-positive, mesenchymal/stem-like cells within the perivascular niche. Furthermore, our data demonstrate the promise of targeting upregulated pathways in resistant subclones as a novel mechanism to achieve therapeutic response, and specifically that THY1 expression may represent a biomarker of response to TGF{beta} inhibition.