Functional interactions between cytotoxic immune 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. This notwithstanding, our comprehension of the complexity and dynamics of the interactions between tumor cells and T cells is only rudimentary. To decipher intercellular communication at a molecular level and identify new drug targets for immunotherapy, better approaches are needed to measure signaling dynamics between engaged cells.

Here we present a method to quantify short-term protein and phosphorylation dynamics between and within physically interacting (heterotypic) cells. We barcode specific cell types using SILAC labeling prior to co-culture incubation. We next extract proteins from the entire mixed cell lysate and quantify cell-type-specific proteome and phosphorylation dynamics occurring during the initial hours of T cell:tumor cell engagement using a second quantification method, either LFQ or TMT. Adopting a SILAC-TMT approach has improved the throughput and sensitivity in our assay. By implementing real-time-search capability in our MS3-based TMT acquisition methods, we can specifically select for tumor-derived or T cell-derived peptides for quantification.

Using hybrid quantification we have been able to discover proteins contributing to functional T cell:tumor interactions and have identified drug targets that sensitize tumor cells to T cell killing. Thus, hybrid quantification is simple method to study short-term protein signaling dynamics in physically interacting cells, which can be easily extended to other biological systems.