Phosphoprotein dynamics of interacting T cells and tumor cells by HySic.

Functional interactions between cytotoxic T cells and tumor cells are central to anti-cancer immunity. However, our understanding of the proteins involved is limited. Here, we present HySic (hybrid quantification of stable isotope labeling by amino acids in cell culture [SILAC]-labeled interacting cells) as a method to quantify protein and phosphorylation dynamics between and within physically interacting cells. Using co-cultured T cells and tumor cells, we directly measure the proteome and phosphoproteome of engaged cells without the need for physical separation. We identify proteins whose abundance or activation status changes upon T cell:tumor cell interaction and validate our method with established signal transduction pathways including interferon γ (IFNγ) and tumor necrosis factor (TNF). Furthermore, we identify the RHO/RAC/PAK1 signaling pathway to be activated upon cell engagement and show that pharmacologic inhibition of PAK1 sensitizes tumor cells to T cell killing. Thus, HySic is a simple method to study rapid protein signaling dynamics in physically interacting cells that is easily extended to other biological systems.

Informations de copyright

Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.

Déclaration de conflit d'intérêts

Declaration of interests S.I.-M. and D.S.P. are named inventors on patent P097110NL, which is unrelated to this work. D.S.P. is co-founder, shareholder, and advisor of Immagene, which is unrelated to this work. M.A. is currently lead scientific officer for Amigon, which has no relation to this work.