Lulu Yan1,4, Jinyu Cheng2,4, Qing Nie3 and Xiaoqiang Sun1 1School of Mathematics, Sun Yat-sen University, Guangzhou 510275, China; 2Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China; 3Department of Mathematics and Department of Developmental and Cell Biology, NSF-Simons Center for Multiscale Cell Fate Research, University of California Irvine, Irvine, California 92697, USA

↵4 These authors contributed equally to this work.

Corresponding authors: sunxq6mail.sysu.edu.cn, qnieuci.edu Abstract

The emergence of spatial transcriptomics (ST) provides unprecedented opportunities to better decipher cell–cell communication (CCC). How to integrate spatial information and complex signaling mechanisms to infer functional CCC, however, remains a major challenge. Here, we present stMLnet, a method that takes into account spatial information and multilayer signaling regulation to identify signaling feedback loops within multilayer CCCs from ST data. To this end, stMLnet quantifies spatially dependent ligand–receptor signaling activity based on diffusion and mass action models, and maps it to intracellular targets. We benchmark stMLnet against seven representative methods and find that stMLnet performs better in both intercellular ligand–receptor inference and intracellular target gene prediction. We apply stMLnet to analyze data from diverse ST techniques like seqFISH+, Slide-seq v2, MERFISH, and Stereo-seq, verifying its robustness and scalability on ST data with varying spatial resolutions and gene coverages. Particularly, stMLnet reveals multilayer signaling feedback loops underlying the inflammatory response in ST data of COVID-19-infected lung tissue. Our study provides an effective tool for dissecting multilayer ligand/receptor-target regulation and multicellular signaling circuits from ST data, which can advance understanding of the mechanistic and functional roles of spatial CCC.

Received October 23, 2024. Accepted April 10, 2025.