(H-281) Spatial Phospho-Proteomic Profiling of Cancer Tissues

Introduction:: Phosphorylation events in proteins play crucial roles in various cellular processes such as signal transduction, cell cycle regulation, and modulation of protein function which can ultimately contribute to our understanding of the efficacy and mechanisms of cancer therapeutics. Further, identification of alterations in phospho-signaling pathways or compensatory mechanisms may elucidate the molecular determinants of therapy resistance. Recently, intracellular proteins (including phosphorylation states) have been profiled in single cells in tandem with both chromatin accessibility and gene expression levels, demonstrating an approach to study the integration of cell signaling, gene regulation and expression in the same biological system. In this study, we utilize Deterministic Barcoding in-Tissue Sequencing (DBiT-seq) to map out the transcriptomic and phospho-proteomic landscape of different cancer types, including glioblastoma, aiming to elucidate complex intra-tumoral signaling networks, microenvironmental factors influencing phosphorylation events, and understand the functional significance of phosphoprotein localization in tumor biology.


Materials and Methods:: In this study, we curated a pilot panel of 9 human glioblastoma phospho-protein markers, which were conjugated in-house using Total-seq-B chemistry (BioLegend): pRPS6, NeuN, SOX2, Beta-actin, pERK1/2, GFAP, NPC, Beta 2-Microglobulin, and pSTAT3. We prepared 7 µm thick fresh-frozen and archived FFPE (Yale Pathology Tissue Services) glioblastoma tissue sections on poly-L-lysine coated slides. RNA integrity tests were performed on the fresh-frozen tissue sections, to ensure sample quality before experimental procedures began. To find the region of interest, Hematoxylin and Eosin (H&E) staining was performed on a section adjacent to that of which we performed DBiT-seq to sequence the whole transcriptome along with the 9 phospho-proteomic markers. Validation was performed using immunofluorescence (IF) staining of a few of the markers at a time, using EVOS (Thermo Fisher EVOS fl) at magnification of 10x with filters Cy5, RFP and GFP.


Results, Conclusions, and Discussions:: We adapted the DBiT-seq technology to capture both intracellular phospho-proteins and transcriptomic information from glioblastoma. Using this method, we were able to quantify our phospho-proteomic panel at spatial resolution, with just over 100 unique molecular identifiers (UMIs) per pixel, an expected number from phosphorylated proteins. Preliminary experimental UMAP depicted 4 distinct clusters, each enriched for one or two phospho-proteomic markers, and spatial patterning was detected for individual markers. GFAP and pERK1/2 were most highly expressed in glioblastoma across experiments while pRPS6 had relatively low expression. By building onto this panel, we aim to detect a more comprehensive understanding of phospho-proteomic networks in glioblastoma and expand out application to other cancers. Transcriptomic and ultimately epigenomic integration with our phospho-protein panel will elucidate mechanisms of inter and intra-cellular signaling networks, able to be adopted in diverse cancer biology contexts.

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