Single-cell RNA sequencing has transformed our understanding of tissue complexity and heterogeneous cell states, yet provides little information about the subcellular organization of transcriptomes - despite the central role of RNA localization in splicing, translation, and function. Here we introduce single-cell APEX-seq (scAPEX-seq), a proximity labeling-based method for mapping subcellular transcriptomes at single-cell resolution. Improvements in probe design and RNA recovery enable APEX integration with droplet-based RNA-seq to capture endoplasmic reticulum–associated transcripts from thousands of individual cells. Applied to tumor–macrophage co-cultures, ER-targeted scAPEX-seq revealed interaction-dependent cell states and transcriptomic signatures by enriching for cell surface and secretory transcripts that are poorly resolved by conventional scRNA-seq. We further applied scAPEX-seq to short- and long-term co-cultures of HER2+ tumor cells with human chimeric antigen receptor (CAR) T cells, resolving distinct activated CAR T cell states, including populations characterized by upregulated NT5E or CTSW expression. We showed that overexpression of CTSW, a cathepsin protease, in CAR T cells promotes stem-like phenotypes, long-term proliferation, and sustained tumor cell killing. scAPEX-seq provides a powerful and scalable approach for profiling subcellular RNA populations, enabling the discovery of cell-cell interaction regulators missed by conventional approaches.
