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Volume 27
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10.3390/ijms27010123
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This is an early access version, the complete PDF, HTML, and XML versions will be available soon.
Article

Metronomic 5-Fluorouracil and Vinorelbine Reduce Cancer Stemness and Modulate EZH2/NOTCH-1/STAT3 Signaling in Triple-Negative Breast Cancer Spheroids

by
Alice Ilari
1,
Emanuela Grassilli
2,
Mario Mauri
1,
Marina E. Cazzaniga
1,
Serena Capici
3,
Marialuisa Lavitrano
1 and
Maria Grazia Cerrito
1,*
1
School of Medicine and Surgery, Milano-Bicocca University, Via Cadore 48, 20900 Monza, Italy
2
Department of Life Sciences, Health and Health Professions, Link Campus University, Via del Casale di San Pio V 44, 00165 Roma, Italy
3
Phase 1 Research Centre, Fondazione IRCCS San Gerardo dei Tintori, Via Pergolesi 33, 20900 Monza, Italy
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2026, 27(1), 123; https://doi.org/10.3390/ijms27010123 (registering DOI)
Submission received: 27 October 2025 / Revised: 14 December 2025 / Accepted: 16 December 2025 / Published: 22 December 2025
(This article belongs to the Special Issue Advances in Novel Therapeutic Strategies for Cancers)
Versions Notes

Abstract

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—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.
Keywords: Triple-Negative Breast Cancer (TNBC); metronomic therapy (mCHT); cancer stem cells (CSCs); stemness markers; EZH2; NOTCH-1; STAT3 Triple-Negative Breast Cancer (TNBC); metronomic therapy (mCHT); cancer stem cells (CSCs); stemness markers; EZH2; NOTCH-1; STAT3

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MDPI and ACS Style

Ilari, A.; Grassilli, E.; Mauri, M.; Cazzaniga, M.E.; Capici, S.; Lavitrano, M.; Cerrito, M.G. Metronomic 5-Fluorouracil and Vinorelbine Reduce Cancer Stemness and Modulate EZH2/NOTCH-1/STAT3 Signaling in Triple-Negative Breast Cancer Spheroids. Int. J. Mol. Sci. 2026, 27, 123. https://doi.org/10.3390/ijms27010123

AMA Style

Ilari A, Grassilli E, Mauri M, Cazzaniga ME, Capici S, Lavitrano M, Cerrito MG. Metronomic 5-Fluorouracil and Vinorelbine Reduce Cancer Stemness and Modulate EZH2/NOTCH-1/STAT3 Signaling in Triple-Negative Breast Cancer Spheroids. International Journal of Molecular Sciences. 2026; 27(1):123. https://doi.org/10.3390/ijms27010123

Chicago/Turabian Style

Ilari, Alice, Emanuela Grassilli, Mario Mauri, Marina E. Cazzaniga, Serena Capici, Marialuisa Lavitrano, and Maria Grazia Cerrito. 2026. "Metronomic 5-Fluorouracil and Vinorelbine Reduce Cancer Stemness and Modulate EZH2/NOTCH-1/STAT3 Signaling in Triple-Negative Breast Cancer Spheroids" International Journal of Molecular Sciences 27, no. 1: 123. https://doi.org/10.3390/ijms27010123

APA Style

Ilari, A., Grassilli, E., Mauri, M., Cazzaniga, M. E., Capici, S., Lavitrano, M., & Cerrito, M. G. (2026). Metronomic 5-Fluorouracil and Vinorelbine Reduce Cancer Stemness and Modulate EZH2/NOTCH-1/STAT3 Signaling in Triple-Negative Breast Cancer Spheroids. International Journal of Molecular Sciences, 27(1), 123. https://doi.org/10.3390/ijms27010123

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