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

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, in vivo 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 in vivo, made these cells a suitable physiological 3D in vitro 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. 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. 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. 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. 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. 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. 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

ITIH5 mediates epigenetic reprogramming of breast cancer cells

Extracellular matrix (ECM) is known to maintain epithelial integrity. In carcinogenesis ECM degradation triggers metastasis by controlling migration and differentiation including cancer stem cell (CSC) characteristics. The ECM-modulator inter- α-trypsin inhibitor heavy chain family member five (ITIH5) was recently identified as tumor suppressor potentially involved in impairing breast cancer progression but molecular mechanisms underlying its function are still elusive

Cell Force-Driven Basement Membrane Disruption Fuels EGF- and Stiffness-Induced Invasive Cell Dissemination from Benign Breast Gland Acini

Local basement membrane (BM) disruption marks the initial step of breast cancer invasion. The activation mechanisms of force-driven BM-weakening remain elusive. We studied the mechanical response of MCF10A-derived human breast cell acini with BMs of tuneable maturation to physical and soluble tumour-like extracellular matrix (ECM) cues. Traction force microscopy (TFM) and elastic resonator interference stress microscopy (ERISM) were used to quantify pro-invasive BM stress and protrusive forces. Substrate stiffening and mechanically impaired BM scaffolds induced the invasive transition of benign acini synergistically. Robust BM scaffolds attenuated this invasive response. Additional oncogenic EGFR activation compromised the BMs’ barrier function, fuelling invasion speed and incidence. Mechanistically, EGFR-PI3-Kinase downstream signalling modulated both MMP- and force-driven BM-weakening processes. We show that breast acini form non-proteolytic and BM-piercing filopodia for continuous matrix mechanosensation, which significantly push and pull on the BM and ECM under pro-invasive conditions. Invasion-triggered acini further shear and compress their BM by contractility-based stresses that were significantly increased (3.7-fold) compared to non-invasive conditions. Overall, the highest amplitudes of protrusive and contractile forces accompanied the highest invasiveness. This work provides a mechanistic concept for tumour ECM-induced mechanically misbalanced breast glands fuelling force-driven BM disruption. Finally, this could facilitate early cell dissemination from pre-invasive lesions to metastasize eventually

The Acinar Cage: Basement Membranes Determine Molecule Exchange and Mechanical Stability of Human Breast Cell Acini

The biophysical properties of the basement membrane that surrounds human breast glands are poorly understood, but are thought to be decisive for normal organ function and malignancy. Here, we characterize the breast gland basement membrane with a focus on molecule permeation and mechanical stability, both crucial for organ function. We used well-established and nature-mimicking MCF10A acini as 3D cell model for human breast glands, with ether low- or highly-developed basement membrane scaffolds. Semi-quantitative dextran tracer (3 to 40 kDa) experiments allowed us to investigate the basement membrane scaffold as a molecule diffusion barrier in human breast acini in vitro. We demonstrated that molecule permeation correlated positively with macromolecule size and intriguingly also with basement membrane development state, revealing a pore size of at least 9 nm. Notably, an intact collagen IV mesh proved to be essential for this permeation function. Furthermore, we performed ultra-sensitive atomic force microscopy to quantify the response of native breast acini and of decellularized basement membrane shells against mechanical indentation. We found a clear correlation between increasing acinar force resistance and basement membrane formation stage. Most important native acini with highly-developed basement membranes as well as cell-free basement membrane shells could both withstand physiologically relevant loads (≤ 20 nN) without loss of structural integrity. In contrast, low-developed basement membranes were significantly softer and more fragile. In conclusion, our study emphasizes the key role of the basement membrane as conductor of acinar molecule influx and mechanical stability of human breast glands, which are fundamental for normal organ function

Capturing the differentiation process of MCF10A acini.

<p><b>A.</b> Equatorial cross sections through MCF10A acini demonstrate the differentiation process leading to apical and basal polarization. Basal polarization markers: laminin-5, collagen IV (BM formation). Apical polarization markers: GM-130 (Golgi apparatus). Proliferating-nuclear-antigen PCNA and active caspase-3 marked s-phase and apoptotic cells, respectively. DRAQ5 was used to counterstain cell nuclei, cytoskeletal F-actin was stained with actin-phalloidin. Note that stains were performed on different spheres for low (1–12 days)-, semi (13–24 days)-, highly (25–35 days)-matured states. Scale bars = 20 μm. <b>B.</b> Immunofluorescence data were used to infer the development of acinar structures depending on temporal EGF withdrawal. Temporal progression is highlighted with arrows. Acini were grouped according to their differential grades.</p

Intact collagen IV meshwork is essential for size-dependent molecule retardation.

<p><b>A.</b> Control stain of BM specific collagen IV protein after enzymatic collagenase IV treatment. Comparative detection of laminin-5 protein demonstrated the colocalization of residual collagen IV protein within the remaining BM structure. <b>B.</b> The time-lapse of dextran influx in collagenase IV treated MCF10A acini with highly-developed BM (semi-matured group) is independent of the dextran tracer size. Left panel (0 min): bright field images. Other panels: representative dextran signal appearance (contrast inverted images) at indicated locations was independent from dextran molecular weights. IC: Intercellular cleft signal. Scale bars = 20 μm.</p

Tight junction formation in MCF10A cells depends on culture conditions.

<p><b>A.</b> The tight junction (TJ) protein ZO-1 is visible as characteristic kissing points in 2D MCF10A monolayer cluster (white arrow head). <b>B.</b> Representative immunofluorescence stains for low- and highly-matured acini. Cortical F-actin signals (green) indicate acinar cell boundaries. ZO-1 protein (red) was only present as diffuse signal within the cytoplasm, irrespective of acinar-maturation states. The merged ZO-1 and F-actin stains demonstrate loss of colocalization and the absence of TJ-complexes. <b>C.</b> β-catenin signals indicate homogeneously distributed cell-cell contacts (adherence junctions) in both 2D MCF10A monolayer and in 3D MCF10A acini. Scale bars = 20 μm.</p

The basement membrane sustains the mechanical integrity of breast acini.

<p>The basement membrane sustains structural integrity without cellular networks. <b>A-A”.</b> Representative MCF10A acini embedded in EHS matrix were treated with OGP detergent. Acinar swelling and cell-BM separation are indicated by newly formed cell-free clefts during the first seven minutes (see inset). <b>A”‘.</b> Finally, acinar structures shrank to initial size. <b>B.</b> Only sparse contact points were visible between BM (red) and cell debris (green) after OGP-treatment. Nuclei (blue) were counterstained with DRAQ5. Scale bars = 20 μm. <b>C.</b> Schematics of the experimental AFM set up. <b>D.</b> AFM approach-retraction cycles as performed on the same 17-days old MCF10A acinus before (black) and after (red) OGP-treatment. <b>E.</b> Force values needed to reach a certain indentation depth into the acinus as directly read from the raw data in plot B. <b>F.</b> Plot illustrates the force distributions for increased indentation depths in native acini with low-developed BM (n = 40), native acini with highly-developed BM (n = 40) and OGP-treated acini with highly-developed BM (n = 31). Mean values with s.d. are shown. Differences between native and OGP-treated samples are highly significant (p < 0.001).</p