Influence of stress and microenvironmental factors in the progression from breast ductal carcinoma in situ to invasiveness

Abstract

One of the key events in the evolution of breast cancer is the transition from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC). The myoepithelial cell layer is disrupted in this transition allowing cancer epithelial cells to spread from the duct to the mammary stroma and thus, acquiring invasiveness. However, it is unknown which is the cause that triggers the rupture of the myoepithelial cell layer. Tumour microenvironment and in particular, the neuroendocrine factors released under stress situations, have an important role in the progression of breast cancer. Thus, the main aim of this work was to study how microenvironmental factors released under stress situations could influence the invasiveness of DCIS by affecting the integrity of the myoepithelial cell layer.

Due to the fact that there are not commercially available human myoepithelial cells, the isolation of human mammary epithelial and myoepithelial cells from reduction mammoplasties was first optimized, selecting the methodology that provided with the better properties to both epithelial and myoepithelial cells, in terms of proliferation capabilities, lineage-specific markers and functionality. Then, as senescence appearance and loss of antigens are common limitations in the primary cultures of mammary epithelial cells, the optimization of the media was performed, selecting the best media concerning proliferation capabilities, lineage-specific markers, senescence and functionality of epithelial and myoepithelial cells.

Then, as the necessary tools for the study of the effect of stress-related neuroendocrine factors had been achieved, 3D in vitro studies were performed with acinar structures formed by primary human epithelial and myoepithelial cells, which mimic the architecture found in the mammary gland. Different neuroendocrine factors (Substance P, Nerve Growth Factor, oxytocin, epinephrine, cortisol and corticosterone) were used in order to study how they affected the integrity of 3D acini. The results showed that glucocorticoids (cortisol and corticosterone) had the bigger impact in terms of acini formation and myoepithelial cell layer and BM disruption, traits that characterize the invasiveness of DCIS.

In order to shed more light into the role of glucocorticoids in the fate of DCIS, in vivo studies were performed, subjecting mice to immobilization stress that increased the levels of corticosterone in blood. Tumour cells that had the ability to form DCIS in mice were inoculated and the effect of stress was studied. Stress caused an earlier transition of DCIS to IDC, since it triggered the disorganization of the ductal architecture, loss of the BM and the apoptosis of myoepithelial cells.

Since the apoptosis of myoepithelial cells seemed to be an important characteristic in the invasiveness of DCIS, the fate of myoepithelial cells in patient samples with both DCIS alone or DCIS+IDC was studied, observing that the apoptosis of myoepithelial cells was a trait differentiating both sets of samples, since apoptotic myoepithelial cells were exclusively present in patient samples with DCIS+IDC.

Uno de los eventos más importantes en la evolución del cáncer de mama es la transición del carcinoma ductal in situ (DCIS) al carcinoma ductal invasivo (IDC). La capa mioepitelial se rompe en esta transición, permitiendo que las células epiteliales cancerosas se propaguen desde el ducto al estroma mamario, adquiriendo invasividad. Sin embargo, se desconocen cuáles son las causas que desencadenan la ruptura de la capa mioepitelial.

El microambiente tumoral y en particular, los factores neuroendocrinos secretados en situaciones de estrés tienen un papel importante en la progresión del cáncer de mama. Por ello, el objetivo principal de esta tesis es estudiar cómo los factores microambientales secretados en situaciones de estrés pueden influenciar en la invasión del DCIS afectando la integridad de la capa mioepitelial.

Dado que no existen células mioepiteliales humanas comerciales, el primer objetivo del trabajo ha sido optimizar la metodología de obtención de las células epiteliales y mioepiteliales mamarias, partiendo de mamoplastias de reducción. Además, debido a que la senescencia y la pérdida de antígenos específicos es un problema en los cultivos primarios de células mamarias, el segundo objetivo ha sido optimizar el medio de crecimiento de las células epiteliales y mioepiteliales.

Una vez obtenidas las herramientas necesarias, se realizaron cultivos 3D con las células mamarias primarias, obteniendo acinis formados por células epiteliales y mioepiteliales. El tratamiento con los factores neuroendocrinos demostró que los glucocorticoides (cortisol y corticosterona) causaban el mayor efecto en las estructuras ductales, ya que provocaban la apoptosis de las células mioepiteliales y la ruptura de la capa mioepitelial y la membrana basal.

Los estudios in vivo con la inoculación en ratones de células cancerosas capaces de formar DCIS, demostraron que el estrés aumenta los niveles de glucocorticoides (corticosterona) y provoca un adelanto en la transición del DCIS al IDC, causando la pérdida de la membrana basal y la apoptosis de las células mioepiteliales.

Además, dado que la apoptosis de las células mioepiteliales parece ser un rasgo característico en la invasión del DCIS, estudiamos dicha apoptosis en muestras de pacientes con DCIS y DCIS+IDC, mostrando que las células mioepiteliales apoptóticas son específicas en las pacientes con DCIS+IDC.