During the progression from ductal carcinoma in situ (DCIS) to invasive breast cancer (IBC), cells must overcome the physically restraining basement membrane (BM), which compartmentalizes the epithelium from the stroma. Since the extracellular matrix (ECM) of the epithelial and stromal compartments are biochemically and physically distinct from one another, the progression demands a certain degree of cellular plasticity for a primary tumor to become invasive. The epithelial-to-mesenchymal transition (EMT) depicts such a cell program, equipping cancer cells with features allowing for dissemination from the epithelial entity and stromal invasion at the single-cell level. Here, the reciprocal interference between an altering tumor microenvironment and the EMT phenotype was investigated in vitro. BM-typical collagen IV and stroma-typical collagen I coatings were applied as provisional 2D matrices. Pro-inflammatory growth factors were introduced to improve tissue mimicry. Whereas the growth on coated surfaces only slightly affected the EMT phenotype, the combinatorial action of collagen with growth factor TGF-β1 induced prominent phenotypic changes. However, EMT induction was independent of collagen type, and cellular accessibility for EMT-like changes was strongly cell-line dependent. Summarizing the entire body of data, an EMT-phenotyping model was used to determine cellular EMT status and estimate EMT-like changes. The miR200c-mediated reversion of mesenchymal MDA-MB-231 cells is reflected by our EMT-phenotype model, thus emphasizing its potential to predict the therapeutic efficacy of EMT-targeting drugs in the future.

Glioblastoma multiforme is a devastating disease that has attracted enormous attention due to poor prognosis and high recurrence. Small interfering RNA (siRNA) in principle offers a promising therapeutic approach by the downregulation of disease-related genes via RNA interference. For efficient siRNA delivery to target sites, cationic polymers are often used in preclinical studies for the protection of siRNA and complex formation based on electrostatic interactions. In an effort to develop biocompatible and efficient nanocarriers with a translational outlook for optimal gene silencing at reduced toxicity, we synthesized two sets of nylon-3 copolymers with variable cationic content (DM or NM monomer) and hydrophobic subunits (CP monomer) and evaluated their suitability for in vitro siRNA delivery into glioblastoma cells. DM_0.4/CP_0.6 and NM_0.4/CP_0.6 polymers with similar subunit ratios were synthesized to compare the effect of different cationic subunits. Additionally, we utilized NM_0.2/CP_0.8 polymers to evaluate the impact of the different hydrophobic content in the polymer chain. The siRNA condensation ability and polymer–siRNA complex stability was evaluated by unmodified and modified SYBR gold assays, respectively. Further physicochemical characteristics, e.g., particle size and surface charge, were evaluated by dynamic light scattering and laser Doppler anemometry, whereas a relatively new method for polyplex size distribution analysis—tunable resistive pulse sensing—was additionally developed and compared to DLS measurements. Transfection efficiencies, the route of cell internalization, and protein knockdown abilities in glioblastoma cells were investigated by flow cytometry. Furthermore, cellular tolerability was evaluated by MTT and LDH assays. All the polymers efficiently condensed siRNA at N/P ratios of three, whereas polymers with NM cationic subunits demonstrated smaller particle size and lower polyplex stability. Furthermore, NM_0.2/CP_0.8 polyplexes with the highest hydrophobic content displayed significantly higher cellular internalization in comparison to more cationic formulations and successful knockdown capabilities. Detailed investigations of the cellular uptake route demonstrated that these polyplexes mainly follow clathrin-mediated endocytotic uptake mechanisms, implying high interaction capacity with cellular membranes. Taken together with conducive toxicity profiles, highly hydrophobic nylon-3 polymers provide an appropriate siRNA delivery agent for the potential treatment of glioblastoma.