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EZH2/NOTCH-1/STAT3 Signaling in Triple-Negative Breast Cancer Spheroids  \nAlice Ilari 1, Emanuela Grassilli 2, Mario Mauri 1, Marina E. Cazzaniga 1, Serena Capici 3, Marialuisa Lavitrano 1 and Maria Grazia Cerrito 1, *  \nAcademic Editors: Elena Bonanno and Ana Cristina Gonçalves  \nReceived: 27 October 2025  \nRevised: 14 December 2025  \nAccepted: 16 December 2025  \nPublished: 22 December 2025  \nCopyright: © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license.  \n1 School of Medicine and Surgery, Milano-Bicocca University, Via Cadore 48, 20900 Monza, Italy; [a.ilari@campus.unimib.it](a.ilari@campus.unimib.it) (A.I.); [mario.mauri@unimib.it](mario.mauri@unimib.it) (M.M.); [marina.cazzaniga@unimib.it](marina.cazzaniga@unimib.it) (M.E.C.); marialuisa.lavitrano@unimib.it (M.L.)  \n2 Department of Life Sciences, Health and Health Professions, Link Campus University, Via del Casale di San Pio V 44, 00165 Roma, Italy; [e.grassilli@unilink.it](e.grassilli@unilink.it)  \n3 Phase 1 Research Centre, Fondazione IRCCS San Gerardo dei Tintori, Via Pergolesi 33, 20900 Monza, Italy; [serena.capici@irccs-sangerardo.it](serena.capici@irccs-sangerardo.it)  \n* Correspondence: mariagrazia.cerrito@unimib.it  \nAbstract  \nTriple 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—both at the mRNA and protein levels—by 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.  \nKeywords: Triple-Negative Breast Cancer (TNBC); metronomic therapy (mCHT); cancer stem cells (CSCs); stemness markers; EZH2; NOTCH-1; STAT3  \n1. Introduction  \nTriple-negative breast cancer (TNBC) is one of the most aggressive and therapeutically challenging breast cancer subtypes, characterized by the absence of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression [1,2] . This molecular profile results in a lack of targeted treatment options, leaving cytotoxic chemotherapy as the mainstay of treatment for most patients. Although many patients initially respond to maximum tolerated dose (MTD) chemotherapy, relapse is common and  \noften with an aggressive and treatment-resistant phenotype, resulting in shorter diseasefree survival and overall survival [3] . MTD—which in our experimental design corresponds to the STD treatment—mainly acts by inducing cytotoxicity in cancer cells, preferentially targeting the bulk of rapidly proliferating cells. This selective elimination process often fails to eradicate the quiescent CSC subpopulation, instead acti","cbCaigeweQmJFJJl","https://ap.wps.com/l/cbCaigeweQmJFJJl","pdf",5565647,22,"English","# Abstract\n# Keywords\n# 1. Introduction","[{\"question\":\"What is the main purpose of the study described in the abstract?\",\"answer\":\"To assess how metronomic chemotherapy with 5-fluorouracil and vinorelbine affects cancer stemness and specific signaling pathways in TNBC spheroids.\"},{\"question\":\"Which models were used to test metronomic treatment effects?\",\"answer\":\"Spheroids derived from two TNBC cell lines (BT-549 and MDA-MB-231) and a patient-derived primary cell line (MS-186).\"},{\"question\":\"How does metronomic chemotherapy differ from standard treatment in stemness marker effects?\",\"answer\":\"Compared with standard treatment, metronomic therapy achieves sustained suppression of stemness markers rather than partial and transient reduction.\"}]","Metronomic 5-Fluorouracil and Vinorelbine Reduce Cancer Stemness and Modulate EZH2/NOTCH-1/STAT3 Signaling in Triple-Negative Breast Cancer Spheroids | PDF",1790763746,55]