In Vivo Time-Resolved Single-Cell RNA-Seq Reveals Chemotherapy-Induced Transcriptional Dynamics in Tumor Infiltrating Lymphocytes.

Single-cell RNA sequencing (scRNA-seq) using metabolic RNA labeling enables detailed analysis of dynamic gene expression within single cells. However, most studies are limited to in vitro settings, restricting the exploration of in vivo transcriptomic dynamics. To address this, we developed scDyna-seq, a time-resolved scRNA-seq method for in vivo applications using 4-thiouridine (4sU) labeling. scDyna-seq efficiently captures nascent RNA, allowing for precise tracking of gene expression in both in vitro and in vivo contexts, including crossing the blood-brain and blood-fetal barriers. It is also compatible with other single-cell multiomics approaches. In a mouse bladder cancer model, scDyna-seq revealed that cisplatin (cis-diaminodichloroplatinum, CDDP) induced significant dynamic changes in tumor-infiltrating lymphocytes, particularly in genes related to costimulation, effector functions, and exhaustion, which were not detected by conventional methods. When coupled with scTCR-seq, scDyna-seq showed increased TCR clonal expansion linked to CDDP-induced immunogenic death and neoantigen production. In conclusion, scDyna-seq offers safe, precise in vivo RNA labeling as well as single-cell analysis, expanding our understanding of cellular dynamics and facilitating research in health and disease.