{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,13]],"date-time":"2026-09-13T17:03:59Z","timestamp":1789319039833,"version":"build-2803163510"},"posted":{"date-parts":[[2026,9,10]]},"group-title":"Cell Biology","reference-count":42,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T00:00:00Z","timestamp":1788998400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"name":"National Cancer Institute","award":["R00CA259224"],"award-info":[{"award-number":["R00CA259224"]}]},{"name":"National Cancer Institute","award":["T32CA009594"],"award-info":[{"award-number":["T32CA009594"]}]},{"name":"National Cancer Institute","award":["R25CA221767"],"award-info":[{"award-number":["R25CA221767"]}]},{"name":"National Cancer Institute","award":["P30CA014599"],"award-info":[{"award-number":["P30CA014599"]}]},{"DOI":"10.13039\/100000861","name":"Burroughs Wellcome Fund","doi-asserted-by":"crossref","award":["1273341"],"award-info":[{"award-number":["1273341"]}],"id":[{"id":"10.13039\/100000861","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"crossref","award":["R35GM160032"],"award-info":[{"award-number":["R35GM160032"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"crossref","award":["K12GM146658"],"award-info":[{"award-number":["K12GM146658"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000005","name":"United States Department of Defense","doi-asserted-by":"crossref","award":["HT94252410401"],"award-info":[{"award-number":["HT94252410401"]}],"id":[{"id":"10.13039\/100000005","id-type":"DOI","asserted-by":"crossref"}]},{"id":[{"id":"https:\/\/ror.org\/052nngb38","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,10]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Cancer-associated fibroblasts (CAFs) exhibit extensive transcriptional and phenotypic heterogeneity and are highly plastic. Routine approaches to resolving CAF heterogeneity at the single-cell and protein levels are lacking, making it difficult to assess changes in CAF state in response to perturbations. Here, we present an optimized 28-marker spectral flow cytometry panel that uses unsupervised clustering to resolve CAF states across\n                  <jats:italic>in vivo<\/jats:italic>\n                  and\n                  <jats:italic>in vitro<\/jats:italic>\n                  models. Using this new fibroblast profiling panel (FPP), we show that LRRC15+ CAFs in murine pancreatic ductal adenocarcinoma are phenotypically distinct from canonical \u03b1SMA+ myofibroblasts, rather than representing a subset of this population. We find that marker expression is graded rather than discrete across clusters, indicating that CAF identity spans a continuum rather than fixed states. Metabolic profiling further shows that hypoxic CAFs were enriched for glucose uptake and an inflammatory phenotype. Using\n                  <jats:italic>in vitro<\/jats:italic>\n                  models, we further demonstrate how the FPP can resolve plasticity outcomes. In a 3D organoid co-culture model, direct contact between tumor cells and fibroblasts, but not organoid-derived factors alone, induces a stem-like CAF state also observed\n                  <jats:italic>in vivo<\/jats:italic>\n                  . In 2D, TGF\u03b2 and inflammatory\/hypoxic stimuli each enrich distinct, pre-existing CAF states rather than generating new ones. These findings establish our FPP as a practical method for resolving fibroblast heterogeneity at the protein level in cancer and beyond.\n                <\/jats:p>","DOI":"10.64898\/2026.09.07.749987","type":"posted-content","created":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T14:25:19Z","timestamp":1789136719000},"source":"Crossref","is-referenced-by-count":0,"title":["A Full Spectrum Fibroblast Profiling Panel Reveals Phenotypic and Metabolic Heterogeneity of Cancer-Associated Fibroblasts at the Single-Cell Level"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9473-6419","authenticated-orcid":false,"given":"Kevin Mu\u00f1oz","family":"Forti","sequence":"first","affiliation":[{"name":"Section of Hematology\/Oncology, Department of Medicine, Biological Sciences Division, The University of Chicago, Chicago, IL 60637, USA"},{"name":"University of Chicago Comprehensive Cancer Center, Chicago, IL 60637, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2312-0289","authenticated-orcid":false,"given":"Marta","family":"Storl-Desmond","sequence":"additional","affiliation":[{"name":"Section of Hematology\/Oncology, Department of Medicine, Biological Sciences Division, The University of Chicago, Chicago, IL 60637, USA"},{"name":"Committee on Cancer Biology, The University of Chicago, Chicago, IL 60637, USA"},{"name":"University of Chicago Comprehensive Cancer Center, Chicago, IL 60637, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-2910-0451","authenticated-orcid":false,"given":"Sayana E.","family":"Isaac","sequence":"additional","affiliation":[{"name":"Section of Hematology\/Oncology, Department of Medicine, Biological Sciences Division, The University of Chicago, Chicago, IL 60637, USA"},{"name":"Biological Sciences Collegiate Division, The University of Chicago, Chicago, IL 60637, 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