Abstract B001: Glioblastoma evolution and resistance is mediated by a shift in cell state and tumor microenvironmental interactions
Authors: Shankar Sunita , Ravikumar Visweswaran , Tripathy Arushi , Ji Sunjong , Loper Jackson , Hassan Zainab , Fehmi Ziad , Haydin Yacoub , Huang Dah-Luen , Bhadury Sagnik , Kulkarni Reva , Regier Jeffrey , Wahl Daniel , Rao Arvind , Rehemtulla Alnawaz , Al-Holou Wajd N. 
Published: March 2024

Glioblastoma (GBM) is a lethal disease characterized by inevitable recurrence. Recently, single cell sequencing studies of primary GBM have defined various tumor cell states, specifically the neural progenitor-like (NPC-like), oligodendrocyte progenitor (OPC-like), astrocyte-like(AC-like), and mesenchymal(MES-like) states. Yet, the role these cell states play in treatment resistance and recurrence is not well defined. Furthermore, the spatial tumor microenvironmental (TME) context of tumor cells clearly plays a role in treatment response. Here we utilize in vivo recurrence models, and single cell and spatial transcriptomic (ST) analyses of primary and recurrent GBM to determine the pathways and spatial TME interactions mediating recurrence. Experimental Design: To define the evolution of the tumor landscape from primary to recurrence, we performed single cell RNA sequencing from stereotactically resected primary GBM (178K cells from 26 patients) and at recurrence (170K cells from 12 patients). ST analyses were also completed, as well as in vitro and in vivo mechanistic studies. Results: We identified a notable shift in the proportion of cell states in response to treatment from primary to recurrence with a significant decrease in AC-like cells (72% vs 55%, p&lt;2e-16) and increase in MES-like cells (15% vs 34%, p&lt;2e-16). This suggests that AC-like cells may be more treatment sensitive compared to other cell states, especially MES-like cells. We then worked to define potential TME interactions of these cell states to define drivers of recurrence. ST analyses revealed NPC-like cells in the infiltrative edge, AC-like cells in the tumor core, and MES-like cells in the hypoxic zones. We performed pseudotime trajectory analysis which identified that NPC-like cells divided into 2 daughter lineages: 1) AC-like cells which form the bulk of primary tumors and 2) MES-like cells that make up a large proportion of the recurrence. We then found that THY1, a cell surface receptor, was highly expressed in NPC-like cells in the primary tumors. While at recurrence, there was a remarkable increase in THY1 expression coincident with a switch to a predominantly MES-like state. Furthermore, ST analyses revealed upregulation of stem-like pathways in THY1 regions, as well as co-localization of MES-like THY1 cells with monocytes in hypoxic zones(p&lt;0.001), suggesting an important role for TME interactions in mediating recurrence. To assess the biological implications of THY1, subsequent in vitro and in vivo studies revealed that THY1 knockout significantly decreased cell proliferation and tumor volume, and improved treatment response suggesting an important role for THY1 in driving treatment resistance and inevitable recurrence. Conclusions: Utilizing mechanistic and bioinformatic analyses, we show that a small population of NPC and MES-like THY1+ cells in primary GBM is likely to survive chemoradiation and mediate recurrence. Defining the interactions of these cell states in their TME and the mechanistic underpinnings of THY1 interactions may be critical to identifying new treatments.

Citation Format: Sunita Shankar, Visweswaran Ravikumar, Arushi Tripathy, Sunjong Ji, Jackson Loper, Zainab Hassan, Ziad Fehmi, Yacoub Haydin, Dah-Luen Huang, Sagnik Bhadury, Reva Kulkarni, Jeffrey Regier, Daniel Wahl, Arvind Rao, Alnawaz Rehemtulla, Wajd N. Al-Holou. Glioblastoma evolution and resistance is mediated by a shift in cell state and tumor microenvironmental interactions [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B001.