{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,11,6]],"date-time":"2026-11-06T12:35:20Z","timestamp":1793968520555,"version":"4.8.0"},"reference-count":45,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2025,12,22]],"date-time":"2025-12-22T00:00:00Z","timestamp":1766361600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Triple Negative Breast Cancers (TNBCs) are heterogeneous and aggressive tumors with a median overall survival of less than two years. Despite the availability of new drugs, the prognosis remains poor, implicating a more aggressive clinical course in the metastatic setting. This study investigated the effects of metronomic treatment (mCHT) with 5-fluorouracil (5-FU) plus vinorelbine (VNR) on spheroids derived from two different TNBC cell lines (BT-549 and MDA-MB-231) and a patient-derived primary cell line (MS-186). mCHT significantly reduced spheroid growth and altered spheroid architecture, with a pronounced effect in second-generation spheroids, enriched in self-renewing cancer stem cells (CSCs). Expression of CSC-related markers (CD44, CD133, NOTCH-1, and MYC) was more significantly altered\u2014both at the mRNA and protein levels\u2014by mCHT than by standard treatment (STD). In MS-186-derived spheroids, mCHT downregulated EZH2 and STAT3, key regulators of CSC maintenance, and reduced H3K27ac, suggesting a global epigenetic reprogramming. Unlike STD, which partially and transiently reduced stemness markers, mCHT achieved sustained suppression, indicating preferential targeting of therapy-resistant CSCs. These results indicate mCHT as a promising strategy for specifically aiming at the CSC-like compartment in TNBC, underscoring a therapeutic approach that reprograms key epigenetic networks and overcomes resistance to treatment.<\/jats:p>","DOI":"10.3390\/ijms27010123","type":"journal-article","created":{"date-parts":[[2025,12,23]],"date-time":"2025-12-23T11:30:18Z","timestamp":1766489418000},"page":"123","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Metronomic 5-Fluorouracil and Vinorelbine Reduce Cancer Stemness and Modulate EZH2\/NOTCH-1\/STAT3 Signaling in Triple-Negative Breast Cancer Spheroids"],"prefix":"10.3390","volume":"27","author":[{"given":"Alice","family":"Ilari","sequence":"first","affiliation":[{"name":"School of Medicine and Surgery, Milano-Bicocca University, Via Cadore 48, 20900 Monza, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5387-5925","authenticated-orcid":false,"given":"Emanuela","family":"Grassilli","sequence":"additional","affiliation":[{"name":"Department of Life Sciences, Health and Health Professions, Link Campus University, Via del Casale di San Pio V 44, 00165 Roma, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5876-6215","authenticated-orcid":false,"given":"Mario","family":"Mauri","sequence":"additional","affiliation":[{"name":"School of Medicine and Surgery, Milano-Bicocca University, Via Cadore 48, 20900 Monza, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1756-6935","authenticated-orcid":false,"given":"Marina E.","family":"Cazzaniga","sequence":"additional","affiliation":[{"name":"School of Medicine and Surgery, Milano-Bicocca University, Via Cadore 48, 20900 Monza, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Serena","family":"Capici","sequence":"additional","affiliation":[{"name":"Phase 1 Research Centre, Fondazione IRCCS San Gerardo dei Tintori, Via Pergolesi 33, 20900 Monza, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4852-1318","authenticated-orcid":false,"given":"Marialuisa","family":"Lavitrano","sequence":"additional","affiliation":[{"name":"School of Medicine and Surgery, Milano-Bicocca University, Via Cadore 48, 20900 Monza, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3347-5817","authenticated-orcid":false,"given":"Maria Grazia","family":"Cerrito","sequence":"additional","affiliation":[{"name":"School of Medicine and Surgery, Milano-Bicocca University, Via Cadore 48, 20900 Monza, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,22]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1038\/s41523-022-00468-0","article-title":"Triple negative breast cancer: Pitfalls and progress","volume":"8","author":"Zagami","year":"2022","journal-title":"npj Breast Cancer"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Cazzaniga, M.E., Capici, S., Cordani, N., Cogliati, V., Pepe, F.F., Riva, F., and Cerrito, M.G. 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