Natural killer (NK) cells can protect from tumor-transformed cells using a fine-tuned machinery of activating and inhibiting receptors. An important activating receptor is Fc gamma receptor IIIa (FcγRIIIA or CD16A), which can trigger antibody-dependent cellular cytotoxicity (ADCC) when recognizing antibody-opsonized target cells. One strategy to boost ADCC responses may be achieved by inhibiting activation-induced shedding of CD16A from the NK cell surface. However, previous preclinical studies have shown contrasting results regarding the effectiveness and limitations of this approach. Here, microchip-based live cell-imaging was used to assess the consequences of CD16A shedding inhibition on the dynamics of NK cell cytotoxicity. The bispecific innate cell engager acimtamig (AFM13) was superior to IgG1 monoclonal antibodies in ADCC and in increasing the fraction of cytotoxic NK cells and serial killers. Under conditions where CD16A shedding was inhibited, acimtamig still triggered ADCC; however, the ability to promote serial killing was reduced and associated with impaired NK cell detachment from target cells. These results demonstrate that CD16A shedding represents an intrinsic feature of NK cell biology that is critical to sustain the antitumoral cytotoxicity of NK cells. This has implications for CD16A engineering of NK cell products and their combination with CD16A-directed NK cell engagers.

Several factors present in the extracellular environment regulate epithelial cell adhesion and dynamics. Among them, growth factors such as EGF, upon binding to their receptors at the cell surface, get internalized and directly activate the acto-myosin machinery. In this study we present the effects of EGF on the contractility of epithelial cancer cell colonies in confined geometry of different sizes. We show that the extent to which EGF triggers contractility scales with the cluster size and thus the number of cells. Moreover, the collective contractility results in a radial distribution of traction forces, which are dependent on integrin beta 1 peripheral adhesions and trans-mitted to neighboring cells through adherens junctions. Taken together, EGF-induced contractility acts on the mechanical crosstalk and linkage between the cell-cell and cell-matrix compartments, regulating collective responses.

Miniaturization of cell culture substrates enables controlled analysis of living cells in confined micro-scale environments. This is particularly suitable for imaging individual cells over time, as they can be monitored without escaping the imaging field-of-view (FoV). Glass materials are ideal for most microscopy applications. However, with current methods used in life sciences, glass microfabrication is limited in terms of either freedom of design, quality, or throughput. In this work, we introduce laser-induced deep etching (LIDE) as a method for producing glass microwell arrays for live single cell imaging assays. We demonstrate novel microwell arrays with deep, high-aspect ratio wells that have rounded, dimpled or flat bottom profiles in either single-layer or double-layer glass chips. The microwells are evaluated for microscopy-based analysis of long-term cell culture, clonal expansion, laterally organized cell seeding, subcellular mechanics during migration and immune cell cytotoxicity assays of both adherent and suspension cells. It is shown that all types of microwells can support viable cell cultures and imaging with single cell resolution, and we highlight specific benefits of each microwell design for different applications. We believe that high-quality glass microwell arrays enabled by LIDE provide a great option for high-content and high-resolution imaging-based live cell assays with a broad range of potential applications within life sciences. 

Glass materials have excellent optical and chemical properties for microscopy-based live cell assays but state-of-the-art methods for microfabrication of Lab-on-Chip (LoC) devices are often limited by either complex manufacturing and/or low quality results. In this work, we have evaluated glass microwell array chips produced using a recently introduced laser-based microfabrication method. Three different types of microwell designs have been tested for imaging and screening of on-chip cell cultures and live cell assays.