Abstract LB013: Subclonal evolution and expansion of spatially distinct THY1-positive cells is associated with recurrence in glioblastoma
Authors: Al-Holou Wajd N. , Wang Hanxiao , Ravikumar Visweswaran , Oneka Morgan , Verhaak Roel G. , Kim Hoon , Pratt Drew , Camelo-Piragua Sandra , Speers Corey , Wahl Daniel , Shankar Sunita , Hollon Todd , Sagher Oren , Heth Jason , Muraszko Karin M. , Lawrence Theodore S. , deCarvalho Ana C. , Mikkelsen Tom , Rao Arvind , Rehemtulla Alnawaz 
Published: June 2022

Abstract
Purpose: Glioblastoma (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 Design: We 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.
Results: These 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 TGFβ signaling, and more than one-hundred-fold increase in THY1 levels at recurrence. Utilizing cell sorting, we observed that THY1-positive cells represented &lt;10% of cells in the treatment-naïve tumors and 75-96% in the recurrent tumors. We then isolated THY1-positive cells from treatment-naïve patient samples and determined that they were inherently resistant to chemoradiation in orthotopic models. Additionally, using image-guided biopsies from treatment-naïve 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 TGFβ signaling contributes to a mesenchymal/stem-like phenotype, we inhibited TGFβRI activity in vivo which resulted in decreased mesenchymal/stem-like protein levels, including THY1, and restored sensitivity to TMZ/IR in recurrent tumors.
Conclusions: These 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 TGFβ inhibition.
Citation Format: Wajd N. Al-Holou, Hanxiao Wang, Visweswaran Ravikumar, Morgan Oneka, Roel G. Verhaak, Hoon Kim, Drew Pratt, Sandra Camelo-Piragua, Corey Speers, Daniel Wahl, Sunita Shankar, Todd Hollon, Oren Sagher, Jason Heth, Karin M. Muraszko, Theodore S. Lawrence, Ana C. deCarvalho, Tom Mikkelsen, Arvind Rao, Alnawaz Rehemtulla. Subclonal evolution and expansion of spatially distinct THY1-positive cells is associated with recurrence in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB013.