Subcellular transcriptome sequencing with single cell APEX-seq identifies regulators of cell-cell interactions.

Area of Science:

• Molecular Biology
• Genomics
• Cell Biology

Background:

• Single-cell RNA sequencing (scRNA-seq) offers insights into cell diversity but lacks subcellular resolution.
• RNA localization is critical for gene expression regulation, influencing splicing, translation, and protein function.
• Understanding transcript distribution is key to deciphering cellular heterogeneity and function.

Purpose of the Study:

• To introduce single-cell APEX-seq (scAPEX-seq), a novel method for mapping subcellular transcriptomes at single-cell resolution.
• To investigate the role of RNA localization in cell-cell interactions and uncover previously unresolved transcriptomic signatures.
• To characterize activated chimeric antigen receptor (CAR) T cell states and their functional impact on tumor cells.

Main Methods:

• Developed single-cell APEX-seq (scAPEX-seq) by integrating proximity labeling with droplet-based RNA sequencing.
• Optimized probe design and RNA recovery for enhanced sensitivity and efficiency.
• Applied scAPEX-seq to co-cultures of tumor cells with macrophages and CAR T cells.

Main Results:

• scAPEX-seq successfully mapped endoplasmic reticulum-associated transcripts in thousands of individual cells.
• Revealed interaction-dependent cell states and transcriptomic signatures in tumor-macrophage co-cultures, enriching for cell surface and secretory transcripts.
• Identified distinct activated CAR T cell states, including populations with upregulated NT5E or CTSW expression.
• Demonstrated that CTSW overexpression in CAR T cells promotes stem-like phenotypes, enhanced proliferation, and sustained tumor cell killing.

Conclusions:

• scAPEX-seq is a powerful and scalable method for profiling subcellular RNA populations at single-cell resolution.
• The technique enables the discovery of cell-cell interaction regulators missed by conventional scRNA-seq approaches.
• scAPEX-seq provides critical insights into the functional roles of RNA localization in cellular processes and disease.