{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T15:08:44Z","timestamp":1789052924561,"version":"build-2803163510"},"posted":{"date-parts":[[2026,2,9]]},"group-title":"Bioengineering","reference-count":43,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,2,9]],"date-time":"2026-02-09T00:00:00Z","timestamp":1770595200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"award":["2023-332386"],"award-info":[{"award-number":["2023-332386"]}],"id":[{"id":"https:\/\/ror.org\/001ader08","id-type":"ROR","asserted-by":"publisher"}]},{"award":["CGCATF-2023\/100006"],"award-info":[{"award-number":["CGCATF-2023\/100006"]}],"id":[{"id":"https:\/\/ror.org\/054225q67","id-type":"ROR","asserted-by":"publisher"}]},{"award":["OT2CA297242"],"award-info":[{"award-number":["OT2CA297242"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["5R01AI103867"],"award-info":[{"award-number":["5R01AI103867"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["P50CA092131"],"award-info":[{"award-number":["P50CA092131"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R35GM140888"],"award-info":[{"award-number":["R35GM140888"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["P30CA016042"],"award-info":[{"award-number":["P30CA016042"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["20163828"],"award-info":[{"award-number":["20163828"]}],"id":[{"id":"https:\/\/ror.org\/0184qbg02","id-type":"ROR","asserted-by":"publisher"}]},{"award":["20221408"],"award-info":[{"award-number":["20221408"]}],"id":[{"id":"https:\/\/ror.org\/0184qbg02","id-type":"ROR","asserted-by":"publisher"}]},{"award":["C-03920"],"award-info":[{"award-number":["C-03920"]}],"id":[{"id":"https:\/\/ror.org\/0184qbg02","id-type":"ROR","asserted-by":"publisher"}]},{"award":["W81XWH-20-1-0119"],"award-info":[{"award-number":["W81XWH-20-1-0119"]}],"id":[{"id":"https:\/\/ror.org\/0447fe631","id-type":"ROR","asserted-by":"publisher"}]},{"award":["2034835"],"award-info":[{"award-number":["2034835"]}],"id":[{"id":"https:\/\/ror.org\/021nxhr62","id-type":"ROR","asserted-by":"publisher"}]},{"award":["CH 2718\/1-1"],"award-info":[{"award-number":["CH 2718\/1-1"]}],"id":[{"id":"https:\/\/ror.org\/018mejw64","id-type":"ROR","asserted-by":"publisher"}]},{"award":["25YOUN17"],"award-info":[{"award-number":["25YOUN17"]}],"id":[{"id":"https:\/\/ror.org\/01npwtv09","id-type":"ROR","asserted-by":"publisher"}]},{"award":["CRI15364"],"award-info":[{"award-number":["CRI15364"]}],"id":[{"id":"https:\/\/ror.org\/02f3xk561","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,2,9]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>Cell\u2013cell interactions drive rapid and heterogeneous changes in gene expression, yet most transcriptomic methods either dissociate cells, losing pair identity and interaction timing, or infer communication indirectly from ligand\u2013receptor co-expression. Here we present Cell-Cell-seq, a scalable workflow for profiling defined cell pairs (\u201cdyads\u201d) with single-cell transcriptomic resolution. Cell-Cell-seq uses cavity-containing hydrogel microparticles (Nanovials) to confine two cells, synchronize contact onset, protect fragile conjugates during handling and sorting, and interface directly with droplet-based RNA sequencing. Using antigen-matched prostate tumor cells and engineered T cells as a model system, Cell-Cell-seq captured thousands of tumor\u2013T cell dyads and revealed broad functional and transcriptional heterogeneity across interactions. Dyads unmasked transient activation programs that were obscured in standard well-plate co-culture, consistent with asynchronous contact in bulk assays. To distinguish interaction-induced programs from the composite nature of dyad transcriptomes, we developed a pseudo-mixing framework that generates in silico pseudo-dyads to construct an empirical null distribution under \u201cno interaction,\u201d enabling statistically robust identification of emergent genes and partner-resolved attribution of responses. Dyad-resolved analysis further revealed coordinated cross-cell programs, including coupled chemokine expression consistent with bidirectional paracrine signaling and inverse coupling between tumor immunoregulatory programs and T cell activation. Finally, we introduce ccRepair to correct compositional dilution in mixed transcriptomes, improving interpretability while preserving genuine cross-cell coordination. Together, Cell-Cell-seq provides a generalizable platform for dissecting immune synapse biology and mapping interaction-dependent programs across heterogeneous cell populations, with applications in profiling tumor\u2013immune communication and functionally screening immunotherapies.<\/jats:p>","DOI":"10.64898\/2026.02.05.704136","type":"posted-content","created":{"date-parts":[[2026,2,9]],"date-time":"2026-02-09T20:20:13Z","timestamp":1770668413000},"source":"Crossref","is-referenced-by-count":6,"title":["Systematic mapping of emergent transcriptional states in interacting single-cell dyads by Cell-Cell-seq"],"prefix":"10.64898","author":[{"given":"Sevana","family":"Baghdasarian","sequence":"first","affiliation":[{"name":"Department of Chemical and Biomolecular Engineering, University of California Los Angeles, Los Angeles, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qingyang","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Statistics and Data Science, University of California Los Angeles, Los Angeles, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Justin","family":"Langerman","sequence":"additional","affiliation":[{"name":"Department of Biological Chemistry, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhiyuan","family":"Mao","sequence":"additional","affiliation":[{"name":"Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA"},{"name":"Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, Los Angeles, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Heather","family":"Wright","sequence":"additional","affiliation":[{"name":"Department of Bioengineering, University of California Los Angeles, Los Angeles, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Caitlin","family":"Gee","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, Los Angeles, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Donghui","family":"Cheng","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, Los Angeles, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jami","family":"McLaughlin","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, Los Angeles, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John K.","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA"},{"name":"Division of Hematology\/Oncology, Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA"},{"name":"Biostatistics Program, Public Health Sciences Division, Fred Hutchinson Cancer Center, Seattle, WA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaojing","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9273-0278","authenticated-orcid":false,"given":"K. Christopher","family":"Garcia","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA"},{"name":"Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA"},{"name":"Parker Institute for Cancer Immunotherapy, Stanford University; Stanford, CA, USA"},{"name":"Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9288-5648","authenticated-orcid":false,"given":"Jingyi Jessica","family":"Li","sequence":"additional","affiliation":[{"name":"Department of Statistics and Data Science, University of California Los Angeles, Los Angeles, CA, USA"},{"name":"Interdepartmental Program of Bioinformatics, University of California Los Angeles, Los Angeles, CA, USA"},{"name":"Biostatistics Program, Public Health Sciences Division, Fred Hutchinson Cancer Center, Seattle, WA, USA"},{"name":"Department of Biostatistics, University of Washington, Seattle, WA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4461-4533","authenticated-orcid":false,"given":"Owen N.","family":"Witte","sequence":"additional","affiliation":[{"name":"Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA"},{"name":"Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, Los Angeles, CA, USA"},{"name":"Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA, USA"},{"name":"Parker Institute for Cancer Immunotherapy, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA"},{"name":"Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California Los Angeles, Los Angeles, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7796-3372","authenticated-orcid":false,"given":"Kathrin","family":"Plath","sequence":"additional","affiliation":[{"name":"Department of Biological Chemistry, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA"},{"name":"Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California Los Angeles, Los Angeles, CA, USA"},{"name":"Jonsson Comprehensive Cancer Center, University of California Los Angeles, Los Angeles, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3942-4284","authenticated-orcid":false,"given":"Dino","family":"Di Carlo","sequence":"additional","affiliation":[{"name":"Department of Bioengineering, University of California Los Angeles, Los Angeles, CA, USA"},{"name":"Jonsson Comprehensive Cancer Center, University of California Los Angeles, Los Angeles, CA, USA"},{"name":"California NanoSystems Institute (CNSI), University of California Los Angeles, Los Angeles, CA, USA"},{"name":"Partillion Bioscience, Pasadena, CA, USA"},{"name":"Department of Mechanical and Aerospace Engineering, University of California Los Angeles, Los Angeles, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026021200350886000_2026.02.05.704136v1.1","doi-asserted-by":"crossref","first-page":"381","DOI":"10.1038\/s41576-023-00685-8","article-title":"The diversification of methods for studying cell\u2013cell interactions and communication","volume":"25","year":"2024","journal-title":"Nat. Rev. Genet"},{"key":"2026021200350886000_2026.02.05.704136v1.2","doi-asserted-by":"publisher","DOI":"10.1038\/s41591-022-02104-7"},{"key":"2026021200350886000_2026.02.05.704136v1.3","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-022-01288-0"},{"key":"2026021200350886000_2026.02.05.704136v1.4","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-21246-9"},{"key":"2026021200350886000_2026.02.05.704136v1.5","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-020-01038-7"},{"key":"2026021200350886000_2026.02.05.704136v1.6","doi-asserted-by":"crossref","first-page":"2034","DOI":"10.1038\/s41596-024-01112-w","article-title":"Linking single-cell transcriptomes with secretion using SEC-seq","volume":"20","year":"2025","journal-title":"Nat. Protoc"},{"key":"2026021200350886000_2026.02.05.704136v1.7","doi-asserted-by":"publisher","DOI":"10.1021\/acsnano.1c11420"},{"key":"2026021200350886000_2026.02.05.704136v1.8","doi-asserted-by":"publisher","DOI":"10.1038\/s41565-023-01560-7"},{"key":"2026021200350886000_2026.02.05.704136v1.9","doi-asserted-by":"crossref","first-page":"e2203410119","DOI":"10.1073\/pnas.2203410119","article-title":"Physical and in silico immunopeptidomic profiling of a cancer antigen prostatic acid phosphatase reveals targets enabling TCR isolation","volume":"119","year":"2022","journal-title":"Proc. Natl. Acad. Sci"},{"key":"2026021200350886000_2026.02.05.704136v1.10","unstructured":"Chen, X. et al. Overcoming T cell tolerance to tumor self-antigens through catch-bond engineering. Sci. Press (2025)."},{"key":"2026021200350886000_2026.02.05.704136v1.11","doi-asserted-by":"crossref","first-page":"329","DOI":"10.1016\/j.cels.2019.03.003","article-title":"DoubletFinder: Doublet Detection in Single-Cell RNA Sequencing Data Using Artificial Nearest Neighbors","volume":"8","year":"2019","journal-title":"Cell Syst"},{"key":"2026021200350886000_2026.02.05.704136v1.12","doi-asserted-by":"crossref","unstructured":"Limoges, M.-A. , Cloutier, M. , Nandi, M. , Ilangumaran, S. & Ramanathan, S . The GIMAP Family Proteins: An Incomplete Puzzle. Front. Immunol. Volume 12-2021, (2021).","DOI":"10.3389\/fimmu.2021.679739"},{"key":"2026021200350886000_2026.02.05.704136v1.13","doi-asserted-by":"publisher","DOI":"10.1002\/eji.200425587"},{"key":"2026021200350886000_2026.02.05.704136v1.14","first-page":"545","article-title":"Role of the immediate early response 3 (IER3) gene in cellular stress response, inflammation and tumorigenesis","volume":"90","year":"2011","journal-title":"SFB 415 Specif. Pathophysiol. Signal Transduct. Pathw"},{"key":"2026021200350886000_2026.02.05.704136v1.15","doi-asserted-by":"publisher","DOI":"10.1016\/j.ccell.2024.04.011"},{"key":"2026021200350886000_2026.02.05.704136v1.16","doi-asserted-by":"publisher","DOI":"10.1084\/jem.185.3.491"},{"key":"2026021200350886000_2026.02.05.704136v1.17","doi-asserted-by":"crossref","first-page":"723","DOI":"10.1038\/s41592-023-01841-y","article-title":"Time-resolved assessment of single-cell protein secretion by sequencing","volume":"20","year":"2023","journal-title":"Nat. Methods"},{"key":"2026021200350886000_2026.02.05.704136v1.18","doi-asserted-by":"crossref","first-page":"1584","DOI":"10.1016\/j.ymthe.2025.02.024","article-title":"Targeting BCL11B in CAR-engineered lymphoid progenitors drives NK-like cell development with prolonged anti-leukemic activity","volume":"33","year":"2025","journal-title":"Mol. Ther"},{"key":"2026021200350886000_2026.02.05.704136v1.19","doi-asserted-by":"crossref","first-page":"2148850","DOI":"10.1080\/2162402X.2022.2148850","article-title":"BCL11B depletion induces the development of highly cytotoxic innate T cells out of IL-15 stimulated peripheral blood \u03b1\u03b2 CD8+ T cells","volume":"11","year":"2022","journal-title":"OncoImmunology"},{"key":"2026021200350886000_2026.02.05.704136v1.20","doi-asserted-by":"publisher","DOI":"10.1186\/s40425-017-0215-8"},{"key":"2026021200350886000_2026.02.05.704136v1.21","doi-asserted-by":"crossref","unstructured":"Binder, C. et al. CD2 Immunobiology. Front. Immunol. Volume 11-2020, (2020).","DOI":"10.3389\/fimmu.2020.01090"},{"key":"2026021200350886000_2026.02.05.704136v1.22","doi-asserted-by":"publisher","DOI":"10.1089\/ars.2011.4073"},{"key":"2026021200350886000_2026.02.05.704136v1.23","doi-asserted-by":"crossref","first-page":"111006","DOI":"10.1016\/j.celrep.2022.111006","article-title":"Tetraspanin CD53 controls T cell immunity through regulation of CD45RO stability, mobility, and function","volume":"39","year":"2022","journal-title":"Cell Rep"},{"key":"2026021200350886000_2026.02.05.704136v1.24","doi-asserted-by":"publisher","DOI":"10.1038\/s41596-020-0292-x"},{"key":"2026021200350886000_2026.02.05.704136v1.25","doi-asserted-by":"crossref","first-page":"629","DOI":"10.1038\/s41587-020-0442-2","article-title":"Dissecting cellular crosstalk by sequencing physically interacting cells","volume":"38","year":"2020","journal-title":"Nat. Biotechnol"},{"key":"2026021200350886000_2026.02.05.704136v1.26","doi-asserted-by":"crossref","first-page":"1887","DOI":"10.1038\/s41592-025-02744-w","article-title":"Ultra-high-scale cytometry-based cellular interaction mapping","volume":"22","year":"2025","journal-title":"Nat. Methods"},{"key":"2026021200350886000_2026.02.05.704136v1.27","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2007224117"},{"key":"2026021200350886000_2026.02.05.704136v1.28","doi-asserted-by":"publisher","DOI":"10.1084\/jem.185.4.731"},{"key":"2026021200350886000_2026.02.05.704136v1.29","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2021.08.020"},{"key":"2026021200350886000_2026.02.05.704136v1.30","doi-asserted-by":"crossref","unstructured":"Chu, H. Y. et al. Dickkopf-1: A Promising Target for Cancer Immunotherapy. Front. Immunol. Volume 12-2021, (2021).","DOI":"10.3389\/fimmu.2021.658097"},{"key":"2026021200350886000_2026.02.05.704136v1.31","doi-asserted-by":"crossref","first-page":"646","DOI":"10.3350\/cmh.2025.0102","article-title":"TM4SF1 \u2013 A new immune target for treatment of hepatocellular carcinoma: Editorial on \u201cTargeting TM4SF1 promotes tumor senescence enhancing CD8+ T cell cytotoxic function in hepatocellular carcinoma\u201d","volume":"31","year":"2025","journal-title":"CMH"},{"key":"2026021200350886000_2026.02.05.704136v1.32","doi-asserted-by":"crossref","unstructured":"Cheng, R. Y.-H. et al. SEC-seq: association of molecular signatures with antibody secretion in thousands of single human plasma cells. Nat. Commun. 14, (2023).","DOI":"10.1038\/s41467-023-39367-8"},{"key":"2026021200350886000_2026.02.05.704136v1.33","doi-asserted-by":"crossref","unstructured":"Koo, D. et al. Defining T cell receptor repertoires using nanovial-based binding and functional screening. Proc. Natl. Acad. Sci. 121, (2024).","DOI":"10.1073\/pnas.2320442121"},{"key":"2026021200350886000_2026.02.05.704136v1.34","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-49123-1"},{"key":"2026021200350886000_2026.02.05.704136v1.35","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms6940"},{"key":"2026021200350886000_2026.02.05.704136v1.36","doi-asserted-by":"publisher","DOI":"10.1039\/D1LC01156A"},{"key":"2026021200350886000_2026.02.05.704136v1.37","doi-asserted-by":"publisher","DOI":"10.1039\/c0lc00172d"},{"key":"2026021200350886000_2026.02.05.704136v1.38","doi-asserted-by":"publisher","DOI":"10.1039\/C3LC50561H"},{"key":"2026021200350886000_2026.02.05.704136v1.39","doi-asserted-by":"crossref","unstructured":"Mao, T. et al. Single-Target Pairing System (StarPair) for Large-Scale Interrogation of Cell\u2013Cell Interactions. Adv. Sci. n\/a, e13951 (2025).","DOI":"10.1002\/advs.202513951"},{"key":"2026021200350886000_2026.02.05.704136v1.40","doi-asserted-by":"publisher","DOI":"10.1101\/2024.08.15.608174"},{"key":"2026021200350886000_2026.02.05.704136v1.41","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.4380"},{"key":"2026021200350886000_2026.02.05.704136v1.42","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1038\/s43586-020-00010-1","article-title":"Chromatin accessibility profiling methods","volume":"1","year":"2021","journal-title":"Nat. Rev. Methods Primer"},{"key":"2026021200350886000_2026.02.05.704136v1.43","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-021-00869-9"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.02.05.704136","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,2,12]],"date-time":"2026-02-12T08:35:24Z","timestamp":1770885324000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.02.05.704136"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,2,9]]},"references-count":43,"URL":"https:\/\/doi.org\/10.64898\/2026.02.05.704136","relation":{},"subject":[],"published":{"date-parts":[[2026,2,9]]},"subtype":"preprint"}}