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Phosphoprotein dynamics of interacting tumor and T cells by HySic

ABSTRACT: Functional interactions between cytotoxic T cells and tumor cells are central to anti-cancer immunity. Some of the proteins involved, particularly immune checkpoints expressed by T cells, serve as promising clinical targets in immunotherapy. However, our understanding of the complexity and dynamics of the interactions between tumor cells and T cells is only rudimentary. Here we present HySic (for Hybrid quantification of SILAC (Stable Isotope Labelling by Amino acids in Cell culture)-labeled interacting cells) as an innovative method to quantify protein and phosphorylation dynamics between and within physically interacting (heterotypic) cells. We show that co-cultured HLA/antigen/TCR-matched tumor and T cells engage in stable interactions, allowing for in-depth HySic analysis. This method does not require physical separation of the two cell types for subsequent MS proteome and phosphoproteome measurements using label-free quantification (LFQ). We demonstrate that HySic can be used to identify proteins whose abundance or activation status changes upon interactions between T cells and tumor cells. We validated HySic with established signal transduction pathways, including IFN. Using HySic we also identified the RHO/RAC/PAK1 signaling pathway to be activated upon T cell:tumor cell interaction. Pharmacologic inhibition of PAK1 sensitized tumor cells to T cell killing. Thus, HySic is a simple method to study short-term protein signaling dynamics in physically interacting cells, which can be easily extended to other biological systems.

INSTRUMENT(S): Orbitrap Exploris 480, Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Primary Cell, T Cell, Lung, Epithelial Cell

DISEASE(S): Non-small Cell Lung Carcinoma

SUBMITTER: Kelly Stecker

LAB HEAD: Kelly E.

PROVIDER: PXD046734 | Pride | 2023-12-20

REPOSITORIES: Pride

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			Action	DRS
	20200124_OR14_Ag2_Steck001_SI_Batch1_proteome_SA01.raw	Raw
	20200124_OR14_Ag2_Steck001_SI_Batch1_proteome_SA02.raw	Raw
	20200124_OR14_Ag2_Steck001_SI_Batch1_proteome_SA03.raw	Raw
	20200124_OR14_Ag2_Steck001_SI_Batch1_proteome_SA04.raw	Raw
	20200124_OR14_Ag2_Steck001_SI_Batch1_proteome_SA05.raw	Raw

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Phosphoprotein dynamics of interacting tumor and T cells by HySic

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