Abstract 4310: Ultra high-plex spatial proteogenomic investigation of giant cell glioblastoma multiforme immune infiltrates reveals distinct protein and RNA expression profiles
Authors: Bonnett Shilah , Rosenbloom Alyssa , Ong Giang , Conner Mark , Newhouse Daniel , New Felicia , Sato Hiromi , Pha Chi , Ilcisin Saskia , Lyssand John , Geiss Gary , Beechem Joseph M. 
Published: April 2023

The advancement of spatially resolved, multiplex proteomic and transcriptomic technologies has revolutionized and redefined the approaches to complex biological questions pertaining to tissue heterogeneity, tumor microenvironments, cellular interactions, cellular diversity, and therapeutic response. Most spatial technologies yield single analyte proteomic or transcriptomic datasets from separate formalin-fixed paraffin-embedded (FFPE) tissues sections. Multiple studies have demonstrated a poor correlation between RNA expression and protein abundance owing to transcriptional and translational regulation, target turnover, and post-translational protein modifications. Therefore, a workflow that accurately measures RNA and protein simultaneously within a single tissue section with distinct spatial context is critical to a more complete biological understanding of cellular interactions and activities. Such multimodal omic datasets of protein and DNA or RNA have been termed “spatial proteogenomics”. Here we present a novel spatial proteogenomic (SPG) assay on the GeoMx® Digital Spatial Profiler platform with NGS readout that enables ultra high-plex digital quantitation of proteins (147-plex) and RNA (whole transcriptome, &gt; 18,000-plex) from a single FFPE sample. We demonstrated high concordance, R &gt; 0.85, and minor change in sensitivity (&lt;11%) between the SPG assay and the single analyte GeoMx Whole Transcriptome Atlas and GeoMx NGS Protein assays. We used the SPG assay to interrogate 23 different glioblastoma multiforme samples across 4 pathologies. We observed clustering of both RNA and protein based on cancer pathology and anatomic location. The in-depth investigation of giant cell glioblastoma multiforme (gcGBM) revealed distinct protein and RNA expression profiles compared to that of glioblastoma multiforme (GBM). Spatial proteogenomics allowed simultaneous interrogation of critical protein post-translational modifications alongside whole transcriptomic profiles within the same distinct cellular neighborhoods. Within our dataset, we observed &gt;2-fold higher protein expression levels of phospho-GSK3β (Ser9) in gcGBM compared to GBM. Inactivation of GSK3β through phosphorylation has been shown to enhance proliferation of GBM cells. We also observed differential protein expression phosphorylated Tau variants. Phospho-Thr231 Tau was &gt;2-fold higher in GBM compared to gcGBM. Associated with neurodegenerative Alzheimer’s disease, changes in Tau expression and phosphorylation have also been observed in glioblastoma. Our study exemplifies the utility of the SPG assay in expanding our understanding of glioblastoma multiforme molecular pathology.

Citation Format: Shilah Bonnett, Alyssa Rosenbloom, Giang Ong, Mark Conner, Daniel Newhouse, Felicia New, Hiromi Sato, Chi Pha, Saskia Ilcisin, John Lyssand, Gary Geiss, Joseph M. Beechem. Ultra high-plex spatial proteogenomic investigation of giant cell glioblastoma multiforme immune infiltrates reveals distinct protein and RNA expression profiles. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4310.