{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,4,1]],"date-time":"2026-04-01T11:46:30Z","timestamp":1775043990160,"version":"3.50.1"},"posted":{"date-parts":[[2026,3,29]]},"group-title":"Cancer Biology","reference-count":49,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,3,29]],"date-time":"2026-03-29T00:00:00Z","timestamp":1774742400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,3,29]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  We previously developed synthetic Notch (synNotch)-chimeric antigen receptor (CAR)-T cells to improve the safety and efficacy of CAR-T therapy for glioblastoma. In this system, an anti-EphA2\/IL13R\u03b12-dual-CAR is expressed only upon recognition of tumor- or brain-specific \u201cpriming\u201d antigens, EGFRvIII (termed E-SYNC cells) or brevican (B-SYNC), respectively, with E-SYNC currently under phase I clinical evaluation (\n                  <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"clintrialgov\" xlink:href=\"NCT06186401\">NCT06186401<\/jats:ext-link>\n                  ). However, tracking and profiling these engineered cells\n                  <jats:italic>in vivo<\/jats:italic>\n                  remain challenging, limiting our understanding of their activity and therapeutic potential. To address this gap, we developed a single-cell RNA-sequencing (scRNA-seq) workflow with custom spike-in probes for synNotch-CAR transcripts, enabling simultaneous detection of engineered cells and transcriptomic profiling.\n                  <jats:italic>In vitro<\/jats:italic>\n                  , integration of multiple probes using machine-learning-assisted classifiers detected 78.2% of E-SYNC cells and 60.0% of B-SYNC cells with 98.0% specificity. In a xenograft model, synNotch-positive cells were detected across the spleen, lung, and brain, with the highest frequency and most robust priming and activation observed in the brain. Single-cell transcriptomic analyses revealed tissue-specific differentiation programs, including cytotoxicity, proliferation, metabolic activity, and acquisition of tissue-resident memory phenotypes, shaped by both environmental cues and synNotch-mediated antigen recognition. In summary, this spike-in probe-enhanced scRNA-seq workflow enables robust detection and high-resolution characterization of synNotch-CAR-T cell dynamics and provides a broadly applicable platform for monitoring engineered immune cells in diverse clinical contexts.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>One Sentence Summary<\/jats:title>\n                  <jats:p>Our spike-in probe-enhanced single-cell RNA-sequencing method enables analysis of tissue-dependent activation and transcriptional states of synNotch-CAR-T cells, providing a robust and scalable platform for in vivo tracking and transcriptomic profiling of engineered cell therapies.<\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.03.26.713760","type":"posted-content","created":{"date-parts":[[2026,3,30]],"date-time":"2026-03-30T04:05:17Z","timestamp":1774843517000},"source":"Crossref","is-referenced-by-count":0,"title":["Spike-in probe-enhanced single-cell RNA-seq reveals post-infusion transcriptomic remodeling of \u201cprime-and-kill\u201d synNotch-CAR-T cells"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9191-8823","authenticated-orcid":false,"given":"Takahide","family":"Nejo","sequence":"first","affiliation":[{"name":"Department of Neurological Surgery, University of California, San Francisco, San Francisco, CA, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0944-3890","authenticated-orcid":false,"given":"Payal B.","family":"Watchmaker","sequence":"additional","affiliation":[{"name":"Department of Neurological Surgery, University of California, San Francisco, San Francisco, CA, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1167-0857","authenticated-orcid":false,"given":"Milos S.","family":"Simic","sequence":"additional","affiliation":[{"name":"UCSF Cell Design Institute and Department of Cellular & Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8141-7339","authenticated-orcid":false,"given":"Akane","family":"Yamamichi","sequence":"additional","affiliation":[{"name":"Department of Neurological Surgery, University of California, San Francisco, San Francisco, CA, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3760-2772","authenticated-orcid":false,"given":"Senthilnath","family":"Lakshmanachetty","sequence":"additional","affiliation":[{"name":"Department of Neurological Surgery, University of California, San Francisco, San Francisco, CA, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aishi","family":"Zhao","sequence":"additional","affiliation":[{"name":"Department of Neurological Surgery, University of California, San Francisco, San Francisco, CA, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jianwen","family":"Lu","sequence":"additional","affiliation":[{"name":"Department of Neurological Surgery, University of California, San Francisco, San Francisco, CA, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marco","family":"Gallus","sequence":"additional","affiliation":[{"name":"Department of Neurological Surgery, University of California, San Francisco, San Francisco, CA, USA"},{"name":"Department of Neurosurgery, University Hospital Muenster, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Heather L.","family":"Benway","sequence":"additional","affiliation":[{"name":"Department of Neurological Surgery, University of California, San Francisco, San Francisco, CA, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Robert","family":"Zhu","sequence":"additional","affiliation":[{"name":"UCSF Cell Design Institute and Department of Cellular & Molecular 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