Recent developments on single-cell analytical methods have provided a clarifying view on cellular heterogeneity and its associated mechanistic and therapeutic implications. Nevertheless, single-cell studies on intracellular protein signaling activities can be confounded by the dynamic nature of signaling events. In addition, there is a pressing need of developing non-genetic analytical methods for clinical samples. These requisites call for a single-cell approach that can interrogate intracellular protein signaling dynamics while being compatible with other downstream analytical methods. A recently developed prototype method has enabled dynamic probing of intracellular protein signaling activities at single-cell resolution, without genetic modifications. The technology was based on intracellularly delivered epitope-targeting peptide probes, a microwell-based single-cell chip and high-speed imaging techniques. However, many obstacles exist that challenge the generalizability of this approach and limit its biomedical application. In this proposal, the aim is to significantly advance this proof-of- concept technology. Five specific goals are included: develop an automated screening protocol for improved screening efficiencies, implement a unique bicyclic peptide library for higher biding affinities, employ cell permeabilizers as a more generalizable delivery method, achieve simultaneous analysis of multiple signaling proteins, and integrate this technology with other single-cell detection methods. This integrated multiplex pipeline will provide an enabling technology that promises a more in-depth understanding of the interplay among protein signaling activities, protein expression levels and metabolism in biological processes.
Analyses of protein signaling activities at the single-cell level are essential to understanding many complex biological processes, such as cancer progression and immune responses. We plan to improve a prototype technology to monitor multiple signaling dynamics in large numbers of single cells. We further aim to integrate such measurements with the quantification of protein expression levels and metabolic activities, which promises a better analysis of the biological process.
