The plates were left at 37C, 5% CO2, and 24C30 hours was the time allowed for invasion to occur. staining overlap.(TIF) pone.0222580.s005.tif (4.1M) GUID:?A6E337A9-DA3E-427D-9A0C-9E486F70C7D8 S6 Fig: Expression and cellular distribution of c-Met in treated 231/LM2-4 tumor xenografts. A strong nuclear and cytoplasmic expression of c-Met was observed in all treatment groups, with no noticeable difference.(TIF) pone.0222580.s006.tif (5.8M) GUID:?40E822E6-DA7B-4518-A67D-E924A0C718DF S7 Fig: Assessment of the invasive capacity of 231/LM2-4 cells treated with 5-FU or 4-HC by a 3D lrECM on-top assay using Matrigel as barrier. Representative examples of the different morphological phenotypes of the multicellular structures. (1M 5-FU): Mass BMS303141 structures (A): round morphology (1C4), collective cell migration as chains of few cells with smooth borders (11,16), buds (6), or as disorganized masses (22). Single-cell protrusions (26,27). Multicellular streaming with no apparent junction contacts (13,17). Dissemination of single tumor cells (pink 20,28) and group of cells (pink 19,29). Pseudo-Stellate Mass structures (B): multicellular collective protrusive migration with leading cells with invadopodia (3,10,14) or leading buds (11), and a loose assembly of individual round cells in multicellular structures (1,4,6,7). Dissemination of single tumor cells (pink 9) and group of cells (pink 17). Contact (pink 12) and fusion (pink 15) between different structures. Stellate structures (C): protrusive leading front with invadopodia (11) or leading buds (3,4,14). Multicellular invasive chains with 1C2 cells in diameter (2) or broad masses of cells (18). Collective cell dissemination (pink 15). An uncoordinated arrangement of the component cells in some multicellular chains (pink 13,17), contacts (pink 8,19), fusions (images 16,20,22) between different structures to form a large stellate structure. (0.01M 4-HC): Mass structures (A): round morphology (1C3), collective cell migration as chains of few cells with smooth borders (7), buds (5), or as disorganized masses (19). Single-cell protrusions (21,26). Multicellular streaming with no apparent junction contacts (10,11). Dissemination of single tumor cells (pink 16,25) and group of cells (pink 17). Pseudo-Stellate Mass structures (B): multicellular collective protrusive migration pattern BMS303141 containing leading cells with invadopodia (5,8) or leading buds (1), and a loose assembly of individual round cells in multicellular structures (4). Dissemination of single tumor cells (pink 2). Fusion between different structures (pink 18). Stellate structures (C): protrusive leading front with invadopodia (17) or leading buds (10). Multicellular invasive chains consisted of one or two cells in diameter (12) or broad masses of cells (8). Single cell dissemination (pink 16). An uncoordinated arrangement of the component cells in some multicellular chains (pink 21). Contacts (pink 14,23) or more commonly fusions (images 13,19,22) between different structures to form a large stellate structure.(TIF) pone.0222580.s007.tif (3.5M) GUID:?7E4BB253-5CE9-4AED-AD43-3711C4DEA97A S1 Table: Assessment of peritumoral and intratumoral collagen deposition in paraffin tumor sections. (DOCX) pone.0222580.s008.docx (13K) GUID:?1ACB05FE-0537-44B5-87A5-C0F503B3C913 S2 Table: Assessment of p-Met[Y1003] in paraffin tumor sections. (DOCX) pone.0222580.s009.docx (13K) GUID:?DFD0B685-8D55-4662-A6F7-31FB5DD91909 S1 Appendix: Assessment of the anti-metastatic effect associated with UFT+CTX therapy in the neoadjuvant setting in 231/LM2-4 breast cancer model. (DOCX) pone.0222580.s010.docx (23K) GUID:?5F622DB0-BBB5-4DF2-B1D4-68F112DD8131 Attachment: Submitted filename: 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 RAPT1 (5-FU), 4-hydroperoxycyclophosphamide (4-HC) or their combination, on 231/LM2-4 cell invasiveness 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 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. BMS303141 Introduction An investigational form of therapy known as low-dose metronomic chemotherapy has been studied both preclinically and clinically for almost two decades [1C5]. Metronomic chemotherapy refers to the BMS303141 close regular (continuous) administration of less than maximum tolerated doses (MTDs) with each administration of a conventional chemotherapy drug, generally over long periods, in the absence of any prolonged (e.g. 2C3 week) break periods [1C5]. The proposed main anti-tumor mechanisms mediated by metronomic chemotherapy include inhibition of angiogenesis [1,2,6], stimulation of adaptive T and possibly innate NK cell mediated immunity [7C11] and direct tumor cell killing . The relative impact of these different mechanisms can depend on and vary with the chemotherapy drug used, and likely other variables such as drug dose and schedule [9,10]. A prime example.