{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T15:43:47Z","timestamp":1785426227805,"version":"3.56.0"},"posted":{"date-parts":[[2025,8,15]]},"group-title":"Cancer Biology","reference-count":40,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,8,15]],"date-time":"2025-08-15T00:00:00Z","timestamp":1755216000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"award":["R01CA218670"],"award-info":[{"award-number":["R01CA218670"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["P30CA051008"],"award-info":[{"award-number":["P30CA051008"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,7,24]]},"abstract":"<jats:p>Epithelial-mesenchymal transition (EMT) enables epithelial cancer cells to acquire mesenchymal-associated traits that can promote invasion and metastasis. Although distinct EMT-associated states have been linked to invasive and metastatic behavior, it remains unclear when these states arise during primary tumor progression, how they diversify, and whether metastatic competence is restricted to a particular EMT phenotype. Using single-cell RNA sequencing in a genetically engineered mouse model of triple-negative breast cancer (TNBC), together with functional studies of tumor organoids, we reconstructed the emergence of EMT-associated heterogeneity during tumor progression. We found that early malignant cells first lost mammary lineage identity, generating lineage-altered epithelial states with increased intrinsic plasticity. Rather than progressing through a single EMT program, these plastic states diversified through ERK1\/2-low and ERK1\/2-high EMT-associated programs. These programs generated distinct hybrid epithelial-mesenchymal states in early tumors and more uniform mesenchymal-like subpopulations at later stages, with canonical EMT features, diminished plasticity, and highly invasive behavior. Importantly, metastatic competence was not restricted to a single EMT-associated state: both heterogeneous hybrid cells and more uniform mesenchymal-like cells initiated metastases, with metastatic lesions retaining features of their initiating populations. Together, our results show that EMT-associated heterogeneity in TNBC emerges through early lineage-state disruption followed by parallel regulatory programs that generate distinct metastatic cell states rather than converging on a single highly metastatic phenotype.<\/jats:p>","DOI":"10.1101\/2025.08.15.670583","type":"posted-content","created":{"date-parts":[[2025,8,16]],"date-time":"2025-08-16T02:20:16Z","timestamp":1755310816000},"source":"Crossref","is-referenced-by-count":0,"title":["Multiple distinct metastatic cell states are induced by epithelial-mesenchymal plasticity"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-5799-9144","authenticated-orcid":false,"given":"Fahda","family":"Alsharief","sequence":"first","affiliation":[{"name":"Georgetown University"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3335-8426","authenticated-orcid":false,"given":"Robert K","family":"Suter","sequence":"additional","affiliation":[{"name":"Georgetown University"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3644-911X","authenticated-orcid":false,"given":"Apsra","family":"Nasir","sequence":"additional","affiliation":[{"name":"Georgetown University"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Idalia","family":"Cruz","sequence":"additional","affiliation":[{"name":"Georgetown University"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6961-0227","authenticated-orcid":false,"given":"Gray W","family":"Pearson","sequence":"additional","affiliation":[{"name":"Georgetown University"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025081901250684000_2025.08.15.670583v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/sj.bjc.6602787"},{"key":"2025081901250684000_2025.08.15.670583v1.2","doi-asserted-by":"publisher","DOI":"10.1158\/1078-0432.CCR-06-3045"},{"key":"2025081901250684000_2025.08.15.670583v1.3","doi-asserted-by":"publisher","DOI":"10.1007\/s10549-011-1935-y"},{"key":"2025081901250684000_2025.08.15.670583v1.4","doi-asserted-by":"crossref","first-page":"729","DOI":"10.1038\/s41598-021-04316-2","article-title":"Survival, treatment regimens and medical costs of women newly diagnosed with metastatic triple-negative breast cancer","volume":"12","year":"2022","journal-title":"Scientific Reports"},{"key":"2025081901250684000_2025.08.15.670583v1.5","doi-asserted-by":"publisher","DOI":"10.1172\/JCI77767"},{"key":"2025081901250684000_2025.08.15.670583v1.6","doi-asserted-by":"crossref","unstructured":"Echeverria GV , Ge Z , Seth S , Zhang X , Jeter-Jones S , Zhou X , et al. Resistance to neoadjuvant chemotherapy in triple-negative breast cancer mediated by a reversible drug-tolerant state. Sci Transl Med 2019;11","DOI":"10.1126\/scitranslmed.aav0936"},{"key":"2025081901250684000_2025.08.15.670583v1.7","doi-asserted-by":"crossref","unstructured":"Kvoka\u010dkov\u00e1 B , Rem\u0161\u00edk J , Jolly MK , Sou\u010dek K . Phenotypic Heterogeneity of Triple-Negative Breast Cancer Mediated by Epithelial-Mesenchymal Plasticity. Cancers (Basel) 2021;13","DOI":"10.3390\/cancers13092188"},{"key":"2025081901250684000_2025.08.15.670583v1.8","doi-asserted-by":"publisher","DOI":"10.1158\/0008-5472.CAN-14-3363"},{"key":"2025081901250684000_2025.08.15.670583v1.9","doi-asserted-by":"publisher","DOI":"10.1158\/0008-5472.CAN-20-0014"},{"key":"2025081901250684000_2025.08.15.670583v1.10","doi-asserted-by":"crossref","unstructured":"Grasset EM , Dunworth M , Sharma G , Loth M , Tandurella J , Cimino-Mathews A , et al. Triple-negative breast cancer metastasis involves complex epithelial-mesenchymal transition dynamics and requires vimentin. Sci Transl Med 2022;14:eabn7571","DOI":"10.1126\/scitranslmed.abn7571"},{"key":"2025081901250684000_2025.08.15.670583v1.11","unstructured":"Apsra N , Sharon C , Alec TM , Garrett TG , Raneen R , Molly EH , et al. The integration of Tgf\u03b2 and Egfr signaling programs confers the ability to lead heterogeneous collective invasion. bioRxiv 2023:2020.11.14.383232"},{"key":"2025081901250684000_2025.08.15.670583v1.12","doi-asserted-by":"publisher","DOI":"10.1172\/JCI65416"},{"key":"2025081901250684000_2025.08.15.670583v1.13","doi-asserted-by":"crossref","first-page":"1386","DOI":"10.3390\/biomedicines9101386","article-title":"Clinically Translatable Approaches of Inhibiting TGF-\u03b2 to Target Cancer Stem Cells in TNBC","volume":"9","year":"2021","journal-title":"Biomedicines"},{"key":"2025081901250684000_2025.08.15.670583v1.14","doi-asserted-by":"crossref","first-page":"2142","DOI":"10.1038\/s41467-020-16066-2","article-title":"Context specificity of the EMT transcriptional response","volume":"11","year":"2020","journal-title":"Nature Communications"},{"key":"2025081901250684000_2025.08.15.670583v1.15","doi-asserted-by":"publisher","DOI":"10.1016\/j.devcel.2018.05.027"},{"key":"2025081901250684000_2025.08.15.670583v1.16","doi-asserted-by":"crossref","unstructured":"Pearson GW . Control of Invasion by Epithelial-to-Mesenchymal Transition Programs during Metastasis. J Clin Med 2019;8","DOI":"10.3390\/jcm8050646"},{"key":"2025081901250684000_2025.08.15.670583v1.17","doi-asserted-by":"publisher","DOI":"10.1038\/S41580-020-0237-9"},{"key":"2025081901250684000_2025.08.15.670583v1.18","doi-asserted-by":"publisher","DOI":"10.1038\/s41576-023-00601-0"},{"key":"2025081901250684000_2025.08.15.670583v1.19","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-018-0040-3"},{"key":"2025081901250684000_2025.08.15.670583v1.20","doi-asserted-by":"publisher","DOI":"10.1038\/s41556-022-00877-0"},{"key":"2025081901250684000_2025.08.15.670583v1.21","doi-asserted-by":"publisher","DOI":"10.1038\/s41556-022-01045-0"},{"key":"2025081901250684000_2025.08.15.670583v1.22","doi-asserted-by":"publisher","DOI":"10.1038\/sj.onc.1203280"},{"key":"2025081901250684000_2025.08.15.670583v1.23","doi-asserted-by":"publisher","DOI":"10.1186\/gb-2007-8-5-r76"},{"key":"2025081901250684000_2025.08.15.670583v1.24","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1038\/s41580-018-0080-4","article-title":"New insights into the mechanisms of epithelial-mesenchymal transition and implications for cancer","volume":"20","year":"2019","journal-title":"Nat Rev Mol Cell Biol"},{"key":"2025081901250684000_2025.08.15.670583v1.25","doi-asserted-by":"publisher","DOI":"10.1158\/0008-5472.CAN-20-0014"},{"key":"2025081901250684000_2025.08.15.670583v1.26","doi-asserted-by":"crossref","unstructured":"Khateb M , Perovanovic J , Ko KD , Jiang K , Feng X , Acevedo-Luna N , et al. Transcriptomics, regulatory syntax, and enhancer identification in mesoderm-induced ESCs at single-cell resolution. Cell Reports 2022;40","DOI":"10.1101\/2022.03.29.486247"},{"key":"2025081901250684000_2025.08.15.670583v1.27","doi-asserted-by":"crossref","first-page":"1333","DOI":"10.1038\/s41592-021-01282-5","article-title":"Single-cell chromatin state analysis with Signac","volume":"18","year":"2021","journal-title":"Nature Methods"},{"key":"2025081901250684000_2025.08.15.670583v1.28","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-019-0969-x"},{"key":"2025081901250684000_2025.08.15.670583v1.29","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.4463"},{"key":"2025081901250684000_2025.08.15.670583v1.30","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.91.23.11236"},{"key":"2025081901250684000_2025.08.15.670583v1.31","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.cellbio.20.010403.095114"},{"key":"2025081901250684000_2025.08.15.670583v1.32","doi-asserted-by":"publisher","DOI":"10.1038\/nm.2000"},{"key":"2025081901250684000_2025.08.15.670583v1.33","doi-asserted-by":"crossref","unstructured":"Nasir A , Camacho S , McIntosh AT , Graham GT , Rahhal R , Huysman ME , et al. The integration of Tgf\u03b2 and Egfr signaling programs confers the ability to lead heterogeneous collective invasion. eLife Sciences Publications, Ltd; 2023.","DOI":"10.7554\/eLife.87991"},{"key":"2025081901250684000_2025.08.15.670583v1.34","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-018-07538-7"},{"key":"2025081901250684000_2025.08.15.670583v1.35","doi-asserted-by":"publisher","DOI":"10.1016\/j.ccell.2021.05.005"},{"key":"2025081901250684000_2025.08.15.670583v1.36","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-pathmechdis-051222-122423"},{"key":"2025081901250684000_2025.08.15.670583v1.37","doi-asserted-by":"publisher","DOI":"10.1084\/jem.20181827"},{"key":"2025081901250684000_2025.08.15.670583v1.38","doi-asserted-by":"publisher","DOI":"10.1038\/nature05760"},{"key":"2025081901250684000_2025.08.15.670583v1.39","first-page":"25","article-title":"Astrocytic laminin-211 drives disseminated breast tumor cell dormancy in brain","volume":"3","year":"2022","journal-title":"Nat Cancer"},{"key":"2025081901250684000_2025.08.15.670583v1.40","doi-asserted-by":"publisher","DOI":"10.1016\/j.ccell.2024.08.015"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.08.15.670583","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,25]],"date-time":"2026-07-25T03:30:11Z","timestamp":1784950211000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.08.15.670583"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,8,15]]},"references-count":40,"URL":"https:\/\/doi.org\/10.1101\/2025.08.15.670583","relation":{},"subject":[],"published":{"date-parts":[[2025,8,15]]},"subtype":"preprint"}}