Supported Membrane Platform to Assess Surface Interactions between Extracellular Vesicles and Stromal Cells

Abstract

Extracellular vesicles (EVs) are membrane-encapsulated particles secreted by eukaryotic cells that stimulate cell communication and horizontal cargo exchange. EV interactions with stromal cells can result in molecular changes in the recipient cell and, in some cases, lead to disease progression. However, mechanisms leading to these changes are poorly understood. A few model systems are available for studying the outcomes of surface interactions between EV membranes with stromal cells. Here, we created a hybrid supported bilayer incorporating EVs membrane material, called an extracellular vesicle supported bilayer, EVSB. Using EVSBs, we investigated the surface interactions between breast cancer EVs and adipose-derived stem cells (ADSCs) by culturing ADSCs on EVSBs and analyzing cell adhesion, spreading, viability, vascular endothelial growth factor (VEGF) secretion, and myofibroblast differentiation. Results show that cell viability, adhesion, spreading, and proangiogenic activity were enhanced, conditions that promote oncogenic activity, but cell differentiation was not. This model system could be used to develop therapeutic strategies to limit EV−ADSC interactions and proangiogenic conditions. Finally, this model system is not limited to the study of cancer but can be used to study surface interactions between EVs from any origin and any target cell to investigate EV mechanisms leading to cellular changes in other diseases.

Dual Mode Sensing of Binding and Blocking of Cancer Exosomes to Biomimetic Human Primary Stem Cell Surfaces

“…We chose to study ADSCs because they are an important cell group in the TME with a critical role in cancer progression and aggressiveness. , In particular, ADSCs secrete inflammatory biomarkers, such as VEGF, that promote angiogenesis and a TME with high levels of inflammation, fostering tumorigenesis. , Notably, surface interactions between cEXOs and ADSCs are known to stimulate VEGF secretion in ADSCs contributing to their angiogenic potential . Because of these malignant implications, it is imperative to study cEXO–ADSC binding and to find treatments to block it as a means to decrease ADSC angiogenic properties.…”

“…We previously used an in vitro cEXO-membrane platform (EVSB) to isolate interactions between the cEXO surface and ADSCs and found proangiogenic markers including upregulation of VEGF and cell proliferation to be a direct outcome . Therefore, to validate the mediating role of integrin β1 in cEXO–ASDC binding, found in the previous sections, we investigated its influence on VEGF and cell proliferation upregulation, as binding outcomes.…”

“…ADSCs grown to 80% confluency were treated for 5 days under four conditions: 40 μg of cEXOs (cEXOs), CD29 Ab followed by cEXOs (CD29), transforming growth factor beta (TGFβ) as a positive control, and no treatment (NT) as a negative control (Figure d). ADSCs with no treatment (NT) were expected to secrete less VEGF than the treated counterparts. , Conversely, ADSCs treated with TGFβ were expected to show increased VEGF secretion because TGFβ plays a key role in VEGF regulation and induction of proangiogenic factors. − Secreted VEGF concentration per cell for all conditions was assessed via ELISA at days 2 and 5 of treatment. For easier data interpretation, the results displayed in Figure represent VEGF secretion per cell for all conditions normalized by the no treatment (NT 2 ) values on day 2.…”

“…SLBs are employed in several research fields as cell membrane models − and have been used as cell culture platforms, − as tools to investigate interactions at the cell membrane interface including cell–cell interactions and cell–particle interactions, − , and as strategies to inhibit virus binding. , In the past, our group has expanded SLB platform molecular complexity by developing methods to integrate native components from cell membranes that preserve the natural function and orientation of the proteins in mammalian cell membranes. , Here, we adapted these methods to create a stem cell-supported bilayer from human primary stem cells that maintains much of the authenticity of the plasma membrane of ADSCs, preserving native constituents, molecular complexity, and lateral diffusivity of the membrane. Because this platform is a representative mimic of the plasma membrane that is free of the dynamism of live cells, it is useful for isolating and focusing on cEXO binding and blocking without competing effects of cEXO uptake and cargo delivery.…”

“…The versatility of our “native-like” lipid bilayer platform to be formed on surfaces compatible with multiple sensing modes allows us to use both optical and label-free electrical means to readout membrane-related events. For example, glass surfaces enable optical techniques such as fluorescence recovery after photobleaching (FRAP) for bilayer fluidity characterization ,,− and total internal reflection fluorescence microscopy (TIRFM) to detect interactions such as nanoparticle binding − and blocking virus binding. , Moreover, given the practicality of label-free methods for screening applications, we create these bilayers on customized bioelectronic sensors, to electrically monitor molecular events at the cell membrane. , …”