Abstract 41: The role of epithelial-to-mesenchymal transition on breast tumorigenesis cancer associated to mesenchymal stem cells
Authors: Rey Fernanda Marques , Pontes Carmen Lucia , Rosales Roberta Ribeiro , Russo José , Su Yanrong , Santucci-Pereirab Julia , Espreafico Enilza Maria , Tiezzi Daniel Guimarães 
Published: August 2018

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 Wharton's 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 Tukey's post-test were used for statistical analysis. Data were presented as mean ± standard deviation, p &lt;0.05. Results The co-culture of MCF-7 with MSCs showed change on localization of e-cadherin from the membrane to the cytoplasm. For n-cadherin, the co-localization were predominantly in membrane after MCF7 has been exposed to MSC. MCF-7 cells showed cytoplasm and nucleus co-localization on β-catenin biomarker. This phenomenon is confirmed by WB showing increase levels of e-cadherin in the cytoplasm fraction of MCF-7. Amoeboid to mesenchymal transition morphology was showed on 3D assay, this interaction phenomena establish the cell-cell communication to modify the profile on cell migration and determine the epithelial to mesenchymal morphology by MSC interaction with MCF-7. The co-culture of MCF-7 with 30% MSCs increases the number of mammospheres and the expression of CD44+/CD24-. On time-lapse assay, the MCF-7 cells show migration on the direction to MSC cells, moving toward these cells as a single-cell migration, with collective migration in response to MSC interaction spindle-shaped cells by detaching from the rest of the cell culture These cells exhibit a differential invasive movement pattern in response to MSC interaction. Conclusion MSC boost aggressiveness of the breast cancer cell MCF-7 indicating that interaction of the tumor microenvironment is determinant of the clonal expansion making this process a relevant part of the mechanism in metastatic dissemination.

Citation Format: Fernanda Marques Rey, Carmen Lucia Pontes, Roberta Ribeiro Rosales, José Russo, Yanrong Su, Julia Santucci-Pereirab, Enilza Maria Espreafico, Daniel Guimarães 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.