This is especially relevant for cell-cell interactions (CCIs), mediated by surface/secreted proteins translated at the ER. We hypothesized APEX enrichment of ER transcripts would detect CCI-driven RNA localization changes AND boost sensitivity for low-abundance transcripts vs standard scRNA-seq.
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Overexpressing CTSW in CAR T cells → larger fraction of stem-like memory phenotype (CD62L+CD45RA+), increased proliferation, and dramatically better tumor killing over repeated rounds of antigen challenge!
Clinical relevance: Using the Cancer Immunology Data Engine (CIDE; 90 datasets, 8,575 tumors), high CTSW expression—largely in T and NK cells—correlates with improved immunotherapy response and better overall survival across multiple cancers including melanoma and lung cancer.
Today we report single-cell APEX-seq (scAPEX-seq)—a method for unbiased mapping of subcellular transcriptomes at single-cell resolution. It reveals cell states invisible to standard scRNA-seq and identifies regulators of CAR T function that improve solid tumor killing.
tinyurl.com/32pf6b8p
Why sequence compartment-specific RNAs instead of whole-cell transcriptomes?
Because RNA localization controls splicing, translation, and degradation. Changes in RNA localization (independent of abundance changes) are undetectable by conventional scRNA-seq.
First test: macrophage-tumor cocultures, scRNA-seq vs scAPEX-seq (APEX targeted to ER membrane). The difference was striking: scAPEX-seq showed greater cluster diversity and clearly separated cocultured from monocultured cells. ~10x more DEGs and far more ligand-receptor interactions detected.
To develop scAPEX-seq, we first redesigned bulk APEX-seq. Our updated method, "APEX-seq2", uses a phenol-azide probe + on-bead amplification to achieve 10x higher RNA recovery, 10x fewer input cells, half the processing time—all while preserving spatial specificity. Full protocol in our Methods.
Next: CAR T + tumor cells. Comparing 2hr vs 21-day cocultures, scAPEX-seq revealed a distinct "late effector" CAR T state missed by whole-transcriptome seq—with higher cytotoxic transcripts and reduced exhaustion markers. One gene enriched in this population: cathepsin W (CTSW).
Co-led by extraordinary trainees Dr. Bo Cai and Andrew Xue, with collaborators Rogelio Hernández-López and postdoc Qian Xue. Xiaojie Qiu and his postdoc Nianping Liu applied Dynamo to analyze CAR T state transitions. Thanks to NSF, CIRM, and Biohub for funding.
www.biorxiv.org/cgi/content/...
