RESUMO
Extracellular vesicles (EVs) influence a host of normal and pathophysiological processes in vivo. Compared to soluble mediators, EVs can traffic a wide range of proteins on their surface including extracellular matrix (ECM) binding proteins, and their large size (â¼30-150 nm) limits diffusion. We isolated EVs from the MCF10 series-a model human cell line of breast cancer progression-and demonstrated increasing presence of laminin-binding integrins α3ß1 and α6ß1 on the EVs as the malignant potential of the MCF10 cells increased. Transport of the EVs within a microfluidic device under controlled physiological interstitial flow (0.15-0.75 µm/s) demonstrated that convection was the dominant mechanism of transport. Binding of the EVs to the ECM enhanced the spatial concentration and gradient, which was mitigated by blocking integrins α3ß1 and α6ß1. Our studies demonstrate that convection and ECM binding are the dominant mechanisms controlling EV interstitial transport and should be leveraged in nanotherapeutic design.
Assuntos
Vesículas Extracelulares , Laminina , Humanos , Laminina/metabolismo , Convecção , Integrina alfa6beta1/metabolismo , Vesículas Extracelulares/metabolismo , Integrina alfa3beta1/metabolismo , Matriz Extracelular/metabolismoRESUMO
Bone marrow niches (endosteal and perivascular) play important roles in both normal bone marrow function and pathological processes such as cancer cell dormancy. Unraveling the mechanisms underlying these events in humans has been severely limited by models that cannot dissect dynamic events at the niche level. Utilizing microfluidic and stem cell technologies, we present a 3D in vitro model of human bone marrow that contains both the perivascular and endosteal niches, complete with dynamic, perfusable vascular networks. We demonstrate that our model can replicate in vivo bone marrow function, including maintenance and differentiation of CD34+ hematopoietic stem/progenitor cells, egress of neutrophils (CD66b+), and niche-specific responses to doxorubicin and granulocyte-colony stimulating factor. Our platform provides opportunities to accelerate current understanding of human bone marrow function and drug response with high spatial and temporal resolution.
Assuntos
Medula Óssea , Dispositivos Lab-On-A-Chip , Células da Medula Óssea , Osso e Ossos , Diferenciação Celular/fisiologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas , Humanos , Nicho de Células-TroncoRESUMO
Hypoxia, or low oxygen (O2) tension, is a central feature of important disease processes including wound healing and cancer. Subtle temporal and spatial variations (≤1% change) in the concentration of O2 can profoundly impact gene expression and cellular functions. Sodium sulfite reacts rapidly with O2 and can be used to lower the O2 concentrations in PDMS-based tissue culture systems without exposing the cell culture to the chemical reaction. By carefully considering the mass transfer and reaction kinetics of sodium sulfite and O2, we developed a flexible theoretical framework to design an experimental microfluidic system that provides fine spatial and temporal control of O2 tension. The framework packages the dimensions, fluid flow, reaction rates, concentrations, and material properties of the fluidic lines and device into dimensionless groups that facilitate scaling and design. We validated the theoretical results by experimentally measuring O2 tension throughout the experimental system using phosphorescence lifetime imaging. We then tested the system by examining the impact of hypoxia inducible factor-1α (HIF-1α) on the proliferation and migration of MDA-MB-231 breast cancer cells. Using this system, we demonstrate that mild constant hypoxia (≤4%) induces HIF-1α mediated functional changes in the tumor cells. Furthermore, slow (>12 hours), but not rapid (<1 hour), fluctuations in O2 tension impact HIF-1α mediated proliferation and migration. Our results provide a generalized framework for fine temporal and spatial control of O2 and emphasize the need to consider mild spatial and temporal changes in O2 tension as potentially important factors in disease processes such as cancer.
Assuntos
Microfluídica , Oxigênio , Técnicas de Cultura de Células , Hipóxia Celular , Humanos , HipóxiaRESUMO
Vascular endothelial growth factor (VEGF) is known to activate proliferation, migration, and survival pathways in endothelial cells through phosphorylation of VEGF receptor-2 (VEGFR-2). VEGF has been incorporated into biomaterials through encapsulation, electrostatic sequestration, and covalent attachment, but the effect of these immobilization strategies on VEGF signaling has not been thoroughly investigated. Further, although growth factor internalization along with the receptor generally occurs in a physiological setting, whether this internalization is needed for receptor phosphorylation is not entirely clear. Here we show that VEGF covalently bound through a modified heparin molecule elicits an extended response of pVEGFR-2 in human umbilical vein endothelial cells (HUVECs) and that the covalent linkage reduces internalization of the growth factor during receptor endocytosis. Optical tweezer measurements show that the rupture force required to disrupt the heparin-VEGF-VEGFR-2 interaction increases from 3-8 pN to 6-12 pN when a covalent bond is introduced between VEGF and heparin. Importantly, by covalently binding VEGF to a heparin substrate, the stability (half-life) of VEGF is extended over three-fold. Here, mathematical models support the biological conclusions, further suggesting that VEGF internalization is significantly reduced when covalently bound, and indicating that VEGF is available for repeated phosphorylation events.