Tmic-56. deciphering the intricate glioblastoma tumor microenvironment and egfrviii transcript distribution through single-cell spatial profiling

Abstract INTRODUCTION Profiling the tumor microenvironment (TME) in glioblastoma (GBM) poses challenges due to its complex and heterogeneous nature. Analyzing diverse cell populations, deciphering dynamic cell-cell interactions, and overcoming spatial-temporal variations present significant challenges to characterizing the GBM TME. These challenges can, in part, be overcome using in situ sequencing (ISS) approaches that offer the advantage of directly analyzing gene expression within intact tissue, providing spatial and molecular context for understanding complex biological processes and tumor heterogeneity. Method/ RESULTS Here, we report a ‘first in world’ application of 10xGenomics Xenium ISS platform to profiling RNA expression profiles within GBM tissue, including primary and recurrent tumors, to establish single cell-level profiling of the TME. Our panel of canonical and custom GBM genes allowed us to establish profiles of high-resolution cell neighborhoods, intratumoral cell-types and cell-states, and the distribution of infiltrating tumor cells throughout the tissue. Exploiting the chemistry that underlies the Xenium platform, we also developed a novel ISS isoform expression assay that semi-quantitatively profiles a mutated variant of the epidermal growth factor receptor (EGFR). Data show that EGFRvIII, which has been shown to promote tumor growth, invasion, and resistance to therapies, is preferentially expressed within tumor-subsets within the GBM. Our EGFRvIII assay not only has the potential to act as a high sensitivity method of tumor diagnosis but its distribution will allow for correlation of this tumorigenic marker with activated pathways across the tissue. CONCLUSION ISS is a valuable tool for profiling the complex TME in GBM, enabling analysis of intratumoral cell types, cell states, and EGFRvIII distribution. Our study demonstrates its pioneering application, offering insights into the molecular landscape and potential for novel diagnostic signatures and improved treatment strategies for this aggressive cancer.