Microscopy-based single-cell proteomic profiling reveals heterogeneity in DNA damage response dynamics.

Area of Science:

• Proteomics
• Cancer Biology
• Cellular Imaging

Background:

• Single-cell proteomics by mass spectrometry (SCoPE-MS) offers deep proteomic insights but cannot link proteomes to specific cell phenotypes.
• Understanding tumor heterogeneity requires methods that correlate proteomic data with cellular functions and behaviors.

Purpose of the Study:

• To develop a novel microscopy-based functional single-cell proteomic-profiling technology (FUNpro) for real-time screening, identification, and isolation of single cells based on phenotype.
• To apply FUNpro to investigate a rare subpopulation of U2OS osteosarcoma cells with an abnormal DNA damage response (DDR) after ionizing radiation (IR).

Main Methods:

• Development of FUNpro, a microscopy-based platform enabling functional single-cell proteomic profiling.
• Application of FUNpro to U2OS osteosarcoma cells treated with ionizing radiation to identify cells with prolonged DNA damage response.
• Proteomic analysis of the identified cell subpopulation.

Main Results:

• FUNpro successfully enabled real-time screening and isolation of single cells with dynamic or rare phenotypes.
• A specific subpopulation of U2OS cells exhibiting a prolonged DNA damage response post-ionizing radiation was identified and profiled.
• The protein PDS5A was identified as a key contributor to the abnormal DDR dynamics and enhanced cell survival after IR.

Conclusions:

• FUNpro technology effectively bridges the gap between single-cell proteomics and functional cell characteristics.
• The study identified PDS5A as a critical factor in the prolonged DNA damage response and survival of osteosarcoma cells, offering potential therapeutic targets.