Distant metastasis, which results in >90% of cancer-related deaths, is enabled by hematogenous dissemination of tumor cells via the circulation. This requires the completion of a sequence of complex ...steps including transit, initial arrest, extravasation, survival and proliferation. Increased understanding of the cellular and molecular players enabling each of these steps is key to uncovering new opportunities for therapeutic intervention during early metastatic dissemination. As a protocol extension, this article describes an adaptation to our existing protocol describing a microfluidic platform that offers additional applications. This protocol describes an in vitro model of the human microcirculation with the potential to recapitulate discrete steps of early metastatic seeding, including arrest, transendothelial migration and early micrometastases formation. The microdevice features self-organized human microvascular networks formed over 4-5 d, after which the tumor can be perfused and extravasation events are easily tracked over 72 h via standard confocal microscopy. Contrary to most in vivo and in vitro extravasation assays, robust and rapid scoring of extravascular cells, combined with high-resolution imaging, can be easily achieved because of the confinement of the vascular network to one plane close to the surface of the device. This renders extravascular cells clearly distinct and allows tumor cells of interest to be identified quickly as compared with those in thick tissues. The ability to generate large numbers of devices (∼36) per experiment further allows for highly parametric studies, which are required when testing multiple genetic or pharmacological perturbations. This is coupled with the capability for live tracking of single-cell extravasation events, allowing both tumor and endothelial morphological dynamics to be observed in high detail with a moderate number of data points.
Brain endothelial cells (BECs) are key constituents of the blood-brain barrier (BBB), protecting the brain from pathogens and restricting access of circulatory factors. Yet, because circulatory ...proteins have prominent age-related effects on adult neurogenesis, neuroinflammation, and cognitive function in mice, we wondered whether BECs receive and potentially relay signals between the blood and brain. Using single-cell RNA sequencing of hippocampal BECs, we discover that capillary BECs—compared with arterial and venous BECs—undergo the greatest transcriptional changes in normal aging, upregulating innate immunity and oxidative stress response pathways. Short-term infusions of aged plasma into young mice recapitulate key aspects of this aging transcriptome, and remarkably, infusions of young plasma into aged mice exert rejuvenation effects on the capillary transcriptome. Together, these findings suggest that the transcriptional age of BECs is exquisitely sensitive to age-related circulatory cues and pinpoint the BBB itself as a promising therapeutic target to treat brain disease.
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•Brain capillary, arterial, and venous cells age differently at the transcriptome level•Aged capillaries upregulate innate immunity and oxidative stress pathways•Capillary transcriptomes respond to inflammatory and age-related circulatory cues•Aged and young plasma mimics and reverses signatures of normal aging, respectively
Through scRNA-seq, Chen et al. demonstrate how mouse hippocampal capillary cells undergo significant alterations with age, including the upregulation of innate immunity and oxidative stress pathways, and that components of these aging signatures can be recapitulated or rejuvenated with acute exposure to aged or young mouse plasma, respectively.
SARS‐CoV‐2 and other respiratory viruses spread via aerosols generated by infected people. Face masks can limit transmission. However, widespread use of disposable masks consumes tremendous resources ...and generates waste. Here, a novel material for treating blown polypropylene filtration media used in medical‐grade masks to impart antimicrobial activity is reported. To produce thin copper@ZIF‐8 core‐shell nanowires (Cu@ZIF‐8 NWs), Cu NWs are stabilized using a pluronic F‐127 block copolymer, followed by growth of ZIF‐8 to obtain uniform core‐shell structures. The Cu@ZIF‐8 NWs are applied to filtration media by dip coating. Aerosol filtration efficiency decreases upon exposure to ethanol (solvent for dip‐coating), but increases with addition of Cu@ZIF‐8 NWs. Cu@ZIF‐8 NWs shows enhanced antibacterial activity, compared to Cu NWs or ZIF‐8 alone, against Streptococcus mutans and Escherichia coli. Antiviral activity against SARS‐CoV‐2 is assayed using virus‐infected Vero E6 cells, demonstrating 55% inhibition of virus replication after 48 h by 1 µg of Cu@ZIF‐8 NWs per well. Cu@ZIF‐8 NWs’ cytotoxicity is tested against four cell lines, and their effect on inflammatory response in A549 cells is examined, demonstrating good biocompatibility. This low‐cost, scalable synthesis and straightforward deposition of Cu@ZIF‐8 NWs onto filter media has great potential to reduce disease transmission, resource consumption, and environmental impact of waste.
Antimicrobial copper nanowires coated with ZIF‐8 metal organic framework (Cu@ZIF‐8 NWs) are prepared and applied to blown polypropylene filter media typically used in face masks. The Cu@ZIF‐8 NWs and treated mask material show enhanced antimicrobial activity that can enable their use in self‐sanitizing reusable face masks and other medical textiles to reduce transmission of disease, including COVID‐19.
Systemic inflammation occurring around the course of tumor progression and treatment are often correlatedwith adverse oncological outcomes. As such, it is suspected that neutrophils, the first line ...of defense against infection, may play important roles in linking inflammation and metastatic seeding. To decipher the dynamic roles of inflamed neutrophils during hematogenous dissemination, we employ a multiplexed microfluidic model of the human microvasculature enabling physiologically relevant transport of circulating cells combined with real-time, high spatial resolution observation of heterotypic cell–cell interactions. LPS-stimulated neutrophils (PMNs) and tumor cells (TCs) form heterotypic aggregates under flow, and arrest due to both mechanical trapping and neutrophil–endothelial adhesions. Surprisingly, PMNs are not static following aggregation, but exhibit a confined migration pattern near TC–PMN clusters. We discover that PMNs are chemotactically confined by self-secreted IL-8 and tumor-derived CXCL-1, which are immobilized by the endothelial glycocalyx. This results in significant neutrophil sequestration with arrested tumor cells, leading to the spatial localization of neutrophil-derived IL-8, which also contributes to increasing the extravasation potential of adjacent tumor cells through modulation of the endothelial barrier. Strikingly similar migration patterns and extravasation behaviors were also observed in an in vivo zebrafish model upon PMN–tumor cell coinjection into the embryo vasculature. These insights into the temporal dynamics of intravascular tumor–PMN interactions elucidate the mechanisms through which inflamed neutrophils can exert proextravasation effects at the distant metastatic site.
An aged circulatory environment can activate microglia, reduce neural precursor cell activity and impair cognition in mice. We hypothesized that brain endothelial cells (BECs) mediate at least some ...of these effects. We observe that BECs in the aged mouse hippocampus express an inflammatory transcriptional profile with focal upregulation of vascular cell adhesion molecule 1 (VCAM1), a protein that facilitates vascular-immune cell interactions. Concomitantly, levels of the shed, soluble form of VCAM1 are prominently increased in the plasma of aged humans and mice, and their plasma is sufficient to increase VCAM1 expression in cultured BECs and the hippocampi of young mice. Systemic administration of anti-VCAM1 antibody or genetic ablation of Vcam1 in BECs counteracts the detrimental effects of plasma from aged individuals on young brains and reverses aging aspects, including microglial reactivity and cognitive deficits, in the brains of aged mice. Together, these findings establish brain endothelial VCAM1 at the blood-brain barrier as a possible target to treat age-related neurodegeneration.
The vascular interface of the brain, known as the blood-brain barrier (BBB), is understood to maintain brain function in part via its low transcellular permeability
. Yet, recent studies have ...demonstrated that brain ageing is sensitive to circulatory proteins
. Thus, it is unclear whether permeability to individually injected exogenous tracers-as is standard in BBB studies-fully represents blood-to-brain transport. Here we label hundreds of proteins constituting the mouse blood plasma proteome, and upon their systemic administration, study the BBB with its physiological ligand. We find that plasma proteins readily permeate the healthy brain parenchyma, with transport maintained by BBB-specific transcriptional programmes. Unlike IgG antibody, plasma protein uptake diminishes in the aged brain, driven by an age-related shift in transport from ligand-specific receptor-mediated to non-specific caveolar transcytosis. This age-related shift occurs alongside a specific loss of pericyte coverage. Pharmacological inhibition of the age-upregulated phosphatase ALPL, a predicted negative regulator of transport, enhances brain uptake of therapeutically relevant transferrin, transferrin receptor antibody and plasma. These findings reveal the extent of physiological protein transcytosis to the healthy brain, a mechanism of widespread BBB dysfunction with age and a strategy for enhanced drug delivery.
It is now evident that the cell nucleus undergoes dramatic shape changes during important cellular processes such as cell transmigration through extracellular matrix and endothelium. Recent ...experimental data suggest that during cell transmigration the deformability of the nucleus could be a limiting factor, and the morphological and structural alterations that the nucleus encounters can perturb genomic organization that in turn influences cellular behavior. Despite its importance, a biophysical model that connects the experimentally observed nuclear morphological changes to the underlying biophysical factors during transmigration through small constrictions is still lacking. Here, we developed a universal chemomechanical model that describes nuclear strains and shapes and predicts thresholds for the rupture of the nuclear envelope and for nuclear plastic deformation during transmigration through small constrictions. The model includes actin contraction and cytosolic back pressure that squeeze the nucleus through constrictions and overcome the mechanical resistance from deformation of the nucleus and the constrictions. The nucleus is treated as an elastic shell encompassing a poroelastic material representing the nuclear envelope and inner nucleoplasm, respectively. Tuning the chemomechanical parameters of different components such as cell contractility and nuclear and matrix stiffnesses, our model predicts the lower bounds of constriction size for successful transmigration. Furthermore, treating the chromatin as a plastic material, our model faithfully reproduced the experimentally observed irreversible nuclear deformations after transmigration in lamin-A/C-deficient cells, whereas the wild-type cells show much less plastic deformation. Along with making testable predictions, which are in accord with our experiments and existing literature, our work provides a realistic framework to assess the biophysical modulators of nuclear deformation during cell transmigration.
The mechanical and biochemical microenvironment influences the morphological characteristics of microvascular networks (MVNs) formed by endothelial cells (ECs) undergoing the process of ...vasculogenesis. The objective of this study was to quantify the role of individual factors in determining key network parameters in an effort to construct a set of design principles for engineering vascular networks with prescribed morphologies. To achieve this goal, we developed a multiculture microfluidic platform enabling precise control over paracrine signaling, cell-seeding densities, and hydrogel mechanical properties. Human umbilical vein endothelial cells (HUVECs) were seeded in fibrin gels and cultured alongside human lung fibroblasts (HLFs). The engineered vessels formed in our device contained patent, perfusable lumens. Communication between the two cell types was found to be critical in avoiding network regression and maintaining stable morphology beyond 4 days. The number of branches, average branch length, percent vascularized area, and average vessel diameter were found to depend uniquely on several input parameters. Importantly, multiple inputs were found to control any given output network parameter. For example, the vessel diameter can be decreased either by applying angiogenic growth factors--vascular endothelial growth factor (VEGF) and sphingosine-1-phsophate (S1P)--or by increasing the fibrinogen concentration in the hydrogel. These findings introduce control into the design of MVNs with specified morphological properties for tissue-specific engineering applications.
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