Validation of 3D Glandular Cultures To Investigate Endometrial Carcinogenesis
Nuria Eritja, Cristina Mirantes, David Llobet, Gemma Masip, Judit Pallares, Xavier Dolcet, Xavier Matias-Guiu. Hospital Universitari Arnau de Vilanova , University of Lleida, Irblleida, Lleida, Spain
Background: Development of three dimensional (3D) epithelial culture systems has emerged as a good approach to study the genes involved in the disruption of gland architecture, loss of epithelial polarity and increase of proliferation. We describe a novel 3D culture system of primary mouse endometrial epithelial cells which could be used to investigate the frequent alterations found in the endometrial carcinogenesis at the light of morphology, such as decreased E-Cadherin or PTEN expression or the effects of estrogenic stimulation.
Design: Epithelial cells from uterus were obtained after a mechanical and enzymatic digestion. After 24 hours of platting in plastic, cells were grown in a medium containing EGF plus Insulin and 3% of a reconstituted extracellular matrix (Matrigel). Matrigel allows cells to display a 3D organization comparable to that observed in vivo endometrium. We analyzed glandular polarity by immunofluorescense staining for different polarity markers. We inhibited E-Cadherin and PTEN expression by lentivirus-delivered shRNAs. We also exposed these cultures to a situation mimicking hyperestrogenism and we studied its effects on cell proliferation.
Results: The endometrial glands developed in our in vitro system conserved cytokeratin expression and were negative staining for vimentin, indicating its epithelial origin. Staining with phalloidin and GM130 indicated correct positioning of actin cytoskeleton and Golgi apparatus respectively. E-Cadherin and β-Catenin staining demonstrated stable cell-to-cell contacts. To validate our 3D culture for the study of disorders associated with tumor formation we infected epithelial cells with PTEN or E-Cadherin shRNAs. PTEN knock-down resulted in consistent increase of proliferation, measured by BrdU incorporation and Ciclin D1 expression. In cultures infected with E-Cadherin shRNA, cells were unable to form glandular structures and caused a complete loss of cell polarity accompanied by the acquisition of features associated with a malignant/migratory phenotype such as β-Catenin translocation, Golgi delocalization and polymerization of stress fibers. An excessive stimulation of estrogen caused an increase of proliferation and an enhanced gland size, but structures retained a correct architecture.
Conclusions: The results confirmed that glandular structures displayed the characteristic apicobasal polarity of glandular tissues, as well as the appropriate formation of cell-to-cell and cell-to-extracellular matrix contact, with patterns similar to those observed in intact endometrium.
Category: Gynecologic & Obstetrics
Wednesday, March 21, 2012 1:00 PM
Poster Session VI # 203, Wednesday Afternoon