Munoz, RaquelHileeto, DeniseCruz-Munoz, WilliamWood, Geoffrey A.Xu, PingMan, ShanViloria-Petit, AliciaKerbel, Robert S.2026-05-072026-05-072019-09-19https://doi.org/10.1371/journal.pone.0222580https://hdl.handle.net/10012/23253© 2019 Muñoz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Metronomic chemotherapy using the 5-FU prodrug uracil-tegafur (UFT) and cyclophosphamide (CTX) was previously shown to only modestly delay primary tumor growth, but nevertheless markedly suppressed the development of micro-metastasis in an orthotopic breast cancer xenograft model, using the metastatic variant of the MDA-MB-231 cell line, 231/LM2-4. Furthermore, a remarkable prolongation of survival, with no toxicity, was observed in a model of postsurgical advanced metastatic disease. A question that has remained unanswered is the seemingly selective anti-metastatic mechanisms of action responsible for this treatment. We assessed the in vivo effect of metronomic UFT, CTX or their combination, on vascular density, collagen deposition and c-Met (cell mediators or modulators of tumor cell invasion or dissemination) via histochemistry/immunohistochemistry of primary tumor sections. We also assessed the effect of continuous exposure to low and non-toxic doses of active drug metabolites 5-fluorouracil (5-FU), 4-hydroperoxycyclophosphamide (4-HC) or their combination, on 231/LM2-4 cell invasiveness in vitro. In the in vivo studies, a significant reduction in vascular density and p-Met[Y1003] levels was associated with UFT+CTX treatment. All treatments reduced intratumoral collagen deposition. In the in vitro studies, a significant reduction of collagen IV invasion by all treatments was observed. The 3D structures formed by 231/LM2-4 on Matrigel showed a predominantly Mass phenotype under treated conditions and Stellate phenotype in untreated cultures. Taken together, the results suggest the low-dose metronomic chemotherapy regimens tested can suppress several mediators of tumor invasiveness highlighting a new perspective for the anti-metastatic efficacy of metronomic chemotherapy.enAttribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/cancer treatmentbreast cancerinvasive tumorscancers and neoplasmscollagenscancer chemotherapymetastasisnecrosisArticle