Abstract 3802: Integrating multiplex-staining and multiplex-DIA: Profiling the tumor microenvironment spatial proteome for precision cancer research
Authors: Zheng Xiang , Mund Andreas , Mann Matthias 
Published: March 2024

Abstract
Our innovative dual-dimensional multiplexing approach ushers in a new era of spatial proteomic exploration. Multiplex-staining deepens understanding of tumor-stroma interactions. Our robust dimethyl labeling-based multiplex-DIA workflow enhances throughput and proteome depth in the Deep Visual Proteomics (DVP) technology. Now, our audacious integration of multiplex-staining with multiplex-DIA holds the promise of pushing the boundaries of spatial proteome profiling. This innovation combines the power of mass spectrometry (MS) with clinical viability in 1) requiring just one FFPE section for spatial proteome analysis at the resolution of individual cell types; 2) ingeniously transmuting MS revelations into a clinically feasible staining strategy. A force in personalized oncology, our innovation unravels the intricate dynamics of the cancer ecosystem while ensuring direct clinical relevance.
We conducted multiplex immunofluorescent staining on human cancers using the MACSima platform, followed by cellular phenotyping and automated single-cell laser microdissection. Subsequently, we loaded dimethyl-labelled 3-plex tryptic peptides onto Evotips and performed LC-MS analysis using the Evosep One coupled to timsTOF SCP.
Pre- and post-22-plex staining proteome analysis confirmed no significant protein loss, validating microdissection compatibility and excellent recovery. The key findings include: 1) profiling of 21 distinct cell types/subtypes in human tonsil cancer tissues, resulting in the identification of 4941 protein groups from a cellular volume of 110,859 µm3, equivalent to approximately 55 cells; 2) observation of a loss of CD45RA/RO expression in tumor-infiltrating cytotoxic T cells, contributing to a reduction in MET and PDGF signaling activity within tumor cells; 3) the presence of abundant cytotoxic T cells, B cells, and plasma cells, coupled with a reduction in tumor cell proliferation, indicative of a hot tumor microenvironment. Proteome analysis revealed enhanced immune cell mobility and interactions with tumor cells. Moreover, our workflow facilitated the identification of potential diagnostic markers for mycosis fungoides, a marker-lacking skin lymphoma, where we achieved the identification of 2964 significant proteins out of a total of 6009. In addition, a rare lymphoma case study not only guided ABVD chemotherapy and suggested IL4 inhibition to address chemoresistance but also identified CDK, MCM, and PSMA/B/C/D/E inhibitors as promising candidates for targeted therapy. The integration of multiplex-staining and multiplex-DIA represents a significant stride in advancing personalized diagnostics and therapeutic strategies.
Citation Format: Xiang Zheng, Andreas Mund, Matthias Mann. Integrating multiplex-staining and multiplex-DIA: Profiling the tumor microenvironment spatial proteome for precision cancer research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3802.