Functional and Mechanical <em>in vitro</em> Analyses of the Mammary Gland Basement Membrane as a Barrier During Cancer Invasion

Abstract

Among women, breast cancer is the most frequently diagnosed cancer and the leading cause of cancer deaths worldwide. Despite extensive research, the processes involved in invasion of malignant breast cancers are still not fully recognized. In general, the invasion in breast cancer is a highly coordinated process between cancer cells and their microenvironment. In the past decades, the basement membrane gained a crucial role as regulator of cell behavior. However, <em>in vivo</em> it is challenging to analyze the processes invasive cells use to break through the basement membrane. Therefore, three-dimensional cultivation of the non-transformed human mammary gland cell line MCF10A, which recapitulates the cellular organization found in mammary acini <em>in vivo</em>, made these cells a suitable physiological 3D <em>in vitro</em> breast gland model to study the role of the basement membrane during cell invasion. A unique feature of the MCF10A acini model is the tunable thickness of their basement membrane. <br /> It was hypothesized that basement membrane disruption and cell transmigration can be triggered by exogenous stress. Therefore, in this thesis the MCF10A acini model system was used to investigate to what extent the basement membrane integrity suppresses cell invasion. Thereby the interrelated factors like tumor-associated ECM-stiffening, growth factor stimulation, and actomyosin contractility were analyzed on their ability to induce cell invasion through the basement membrane in MCF10A acini. <br /> Using life cell imaging, invasion onset and overall incidence of cell-basement membrane transmigration were determined in dependency of both, normal breast- and tumor-like ECM stiffness. It could be demonstrated that a stiff matrix triggered cell invasion and increased invasion incidence compared to a soft matrix. Simultaneously, the basement membrane played a gatekeeper role by retaining the cells from invasion. Cell transmigration through the basement membrane could be further triggered by aberrant stimulation with epidermal growth factor (EGF) and showed that the mechanosensitivity of MCF10A acini could be switched-off by EGF, while the role of the basement membrane as a mechanical sustainer was strengthened. <br /> On the basis of these results, it was analyzed whether, and to what extent, the basement membrane disruption is accompanied by proteolytic degradation by matrix metalloproteinases (MMPs) during acinar invasion. For that, highly developed and thick basement membrane in MCF10A acini was first purposely weakened by type IV collagenase and showed that even on soft ECM, cells invaded the substrate in partial absence of the basement membrane, again indicating the crucial role of the basement membrane as a barrier. To demonstrate that during cell invasion in MCF10A acini the basement membrane was proteolytically weakened, MMP activity was inhibited. The results revealed a decrease of invasion incidence in MCF10A acini independent on substrate stiffness, and showed that MMPs are indeed involved in basement membrane degradation, but their activity was shown to be EGF dependent.<br /> Additionally, finger-like protrusions were observed in MCF10A acini reaching through the basement membrane. These actin-rich protrusions were hypothesized to be filopodia-like protrusions that are responsible for sensing of the extracellular microenvironment. These assumption indicating that MCF10A acini are mechanosensitive and able to respond to changes of ECM stiffness was analyzed by measuring forces generated by MCF10A acini during invasion. Thereby it was determined whether, and to what extent, basement membrane disruption is accompanied by altered cell force generation and quantitatively characterized the local invasion process in detail by traction force microscopy (TFM) and elastic resonator interference stress microscopy (ERISM). Interestingly, TFM analyses showed progressively increasing cell forces during cell-mediated basement membrane-breakdown and outgrowth. Additionally, the tumor-like ECM stiffness considerably contributed to generation of higher forces. By ERISM, local, vertical substrate deformations were detected during the early invasion phase in MCF10A acini, strengthening the contribution of the filopodia-like protrusion in being involved in mechanosensing.<br /> Based on these results, it was aimed to analyze which signaling pathway might be involved in induction of the invasive phenotype in MCF10A acini. It could be demonstrated that phosphoinositide 3 kinase (PI3K) is a crucial factor in upstream signaling pathway, as its inhibition led to a significant decrease of invasion incidence and retarded invasion onset.<br /> The results of this thesis demonstrate that the key mechanism of cancer cell invasion is a proteolytic-driven basement membrane transmigration mechanism which is activated by stiff matrix and aberrant EGF signaling. These findings highlight the crucial role of basement membrane-integrity as a mechanical barrier against breast cancer cell invasion.