# Reconstructing and comparing signal transduction networks from single-cell protein quantification data

## TL;DR

This paper introduces new methods to reconstruct and compare signal transduction networks using single-cell protein data, enabling insights into cellular differences without systematic perturbations.

## Contribution

The novel contribution is the development of scMRA and scCNR methods for reconstructing signaling networks from single-cell heterogeneity.

## Key findings

- scCNR reconstructs population-specific signaling networks with differing interaction strengths.
- The methods were validated on simulated data and applied to EGFR-inhibitor-treated keratinocytes.
- The approach recovers mechanistic signaling differences downstream of EGFR.

## Abstract

Signal transduction networks regulate many essential biological processes and are frequently aberrated in diseases such as cancer. A mechanistic understanding of such networks, and how they differ between cell populations, is essential to design effective treatment strategies. Typically, such networks are computationally reconstructed based on systematic perturbation experiments, followed by quantification of signaling protein activity. Recent technological advances now allow for the quantification of the activity of many (signaling) proteins simultaneously in single cells. This makes it feasible to reconstruct or quantify signaling networks without performing systematic perturbations.

Here, we introduce single-cell modular response analysis (scMRA) and single-cell comparative network reconstruction (scCNR) to derive signal transduction networks by exploiting the heterogeneity of single-cell (phospho-)protein measurements. The methods treat stochastic variation in total protein abundances as natural perturbation experiments, whose effects propagate through the network and hence facilitate the reconstruction and quantification of the underlying signaling network. scCNR reconstructs cell population-specific networks, where cells from different populations have the same underlying topology, but the interaction strengths can differ between populations. We extensively validated scMRA and scCNR on simulated data, and applied it to unpublished data of (phospho-)protein measurements of EGFR-inhibitor-treated keratinocytes to recover signaling differences downstream of EGFR. scCNR will help to unravel the mechanistic signaling differences between cell populations, and will subsequently guide the development of well-informed treatment strategies.

The code used for scCNR in this study has been deposited on Zenodo https://doi.org/10.5281/zenodo.17600937 and is also available as a Python module at https://github.com/ibivu/scmra. Additionally, data and code to reproduce all figures is available at https://github.com/tstohn/scmra_analysis.