Confinement by liquid-liquid interface replicatesin vivoneutrophil deformations and elicits bleb based migration

Abstract

Cell migration plays an essential role in the immune response and is tightly regulated by both chemical and mechanical cues. After exiting circulation, immune cells navigate through tissues while mechanically confined by extracellular matrix components and tissue-resident cells. Common in vitro systems enable modeling of leukocyte migration through collagen-based hydrogels (individual fiber stiffness ~ 300 MPa) or in confined polymer-based microchannels (stiffness > MPa), however these existing systems fail to replicate the bidirectional mechanical interactions between migrating leukocytes and resident cells that typically exhibit a stiffness between 200-3000 Pa. Here, we utilize live-imaging of a dual-transgenic zebrafish to capture mechanical deformations of epidermal keratinocytes induced by interaction with migrating neutrophils. Subsequently, we engineer in vitro migration channels bound by a deformable liquid-liquid interface with tunable mechanical properties that replicate single cell keratinocyte deformations during neutrophil migration in vivo. We find that controlling the mechanical properties of the interface modulates neutrophil motility. This work introduces an in vitro controlled material interface that closely mimics the mechanical interactions between neutrophils and surrounding cells in vivo to provide a biologically relevant platform for exploring the influence of mechanical forces on cell migration.

Naive primary neutrophils play a dual role in the tumor microenvironment

“…The UOM platform is based on the principle of exclusive liquid repellency (ELR) under silicone oil to create a 3D microenvironment, that can include an extracellular matrix (type 1 collagen) coating ( 22 ). While this UOM platform has previously been used to demonstrate neutrophil migration during mechanical constriction ( 13 ) here, we demonstrate how coordinated migration in neutrophils restricts tumor invasion in real time. The UOM migration design is an open, fluid, walled platform that is made up of two spots each with a diameter of 100µm; the cell loading spot (CLS), and the chemoattractant spot (CAS) which are connected by an ECM bridge channel of 500µm in length (Figure 1A).…”

“…To investigate the dynamics of neutrophil coordinated migration toward tumor cells, we first investigated the ability of tumor cells to recruit neutrophils in vitro . The UOM platform is based on the principle of exclusive liquid repellency (ELR) under silicone oil to create a 3D microenvironment, that can include an extracellular matrix (type 1 collagen) coating ( 13 ). The UOM migration design is an open, fluid, walled platform that is made up of two spots each with a diameter of 100µm; the cell loading spot (CLS), and the chemoattractant spot (CAS) which are connected by an ECM bridge channel of 500µm in length (Figure 1A).…”

“…Channels were constructed by sweeping hanging droplets of collagen solution +/- cells across O2-plasma patterned areas. For full details of the channel preparation process, see Schrope et al ( 13 ).…”

“…Here, we use both standard well plate culture and an under oil microfluidic (UOM) assay (12,13) to conduct ex vivo studies of primary neutrophil functional responses when challenged with patient derived HNSCC tumor cells, a HNSCC tumor cell line and a breast cancer cell line (MDA-MB-231). These cell types were investigated because these cancer types are associated with increased neutrophil infiltration into the TME (14)(15)(16).…”