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The objective of this study is to identify and target the influence of tumor microenvironment with characteristics cancer stem cell (CSC) and clonal expansion on breast cancer population. For this purpose we are using the human breast epithelial cells (MCF-7) co-cultured with 1%, 10% or 30% mesenchymal stem cells (MSC) derived from Wharton9s jelly. Flow cytometry: cell surface markers CD44+CD24−/low expression was determined by flow cytometry on MCF-7 cells after exposition for 5 days with MSC. Mammospheres formation was determined by using MCF-7 and MSC seeded in ultra-low-adhesion culture plates for ten days, dissociated and purified by FACS using the CD44+CD24−/low markers. Further cell characterization was performed using the LSM 780 multiphoton and Fiji software, for e-cadherin, β -catenin and n-cadherin expression. Time Lapse migration assay was done using eight well chamber and photographed at 10X magnifications at 20 min intervals for 72 hours. The sorted co-culture and MCF-7 cells were used for cytoplasmic extraction and Western blot analysis of the e-cadherin expression level. MSC and MCF-7 seeded in a collagen matrix, fixed and photographed at 10X and 20X magnifications using Fiji software were used for the 3D reconstruction. The ANOVA with Tukey9s post-test were used for statistical analysis. Data were presented as mean ± standard deviation, p Citation Format: Fernanda Marques Rey, Carmen Lucia Pontes, Roberta Ribeiro Rosales, Jose Russo, Yanrong Su, Julia Santucci-Pereirab, Enilza Maria Espreafico, Daniel Guimaraes Tiezzi. The role of epithelial-to-mesenchymal transition on breast tumorigenesis cancer associated to mesenchymal stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 41.