Democratised single cell proteomics uncovers tumor associated macrophage signature in CYLD cutaneous syndrome skin tumors

Single-cell proteomics (SCP) reveals cellular heterogeneity and biological insights inaccessible to bulk analysis. Existing limitations are cost, sample loss during processing, and accessibility to state-of-the-art instrumentation. We describe a label free SCP methodology in human tissue, combining fluorescence activated cell sorting (FACS), oil-immersion cell handling, mass-spectrometry, and neural-network derived spectral libraries which address these issues. We tested this methodology in a skin tumor syndrome, CYLD cutaneous syndrome (CCS), assessing tumor heterogeneity. Using a Bruker timsTOF HT platform we quantified > 4000 proteins, averaging [~]700 per cell, through a cost-effective pipeline without specialised liquid handling infrastructure. By utilising pre-existing bioinformatic tools from the scRNA-seq field we implemented a robust analysis methodology, discriminating between macrophages, dendritic cells and tumor keratinocytes, in an unbiased analysis of 419 CCS tumor cells. We validated the biological accuracy of cell annotations by cross referencing with each cells FACS markers. Furthermore, we identified a novel CCS tumor associated macrophage population which carried a tumor microenvironment remodelling signature. Our findings demonstrate an accessible SCP technology capable of yielding novel biological discoveries in clinical tissue. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/704354v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@6fe6a3org.highwire.dtl.DTLVardef@8f0ff9org.highwire.dtl.DTLVardef@61e037org.highwire.dtl.DTLVardef@1c76a43_HPS_FORMAT_FIGEXP M_FIG C_FIG IMPORTANTO_LIManuscripts submitted to Review Commons are peer reviewed in a journal-agnostic way. C_LIO_LIUpon transfer of the peer reviewed preprint to a journal, the referee reports will be available in full to the handling editor. C_LIO_LIThe identity of the referees will NOT be communicated to the authors unless the reviewers choose to sign their report. C_LIO_LIThe identity of the referee will be confidentially disclosed to any affiliate journals to which the manuscript is transferred. C_LI GUIDELINESO_LIFor reviewers: https://www.reviewcommons.org/reviewers C_LIO_LIFor authors: https://www.reviewcommons.org/authors C_LI CONTACTThe Review Commons office can be contacted directly at: office@reviewcommons.org