A tractable microscopy- and flow cytometry-based method to measure natural killer cell-mediated killing and infiltration of tumor spheroids.

Understanding the anti-tumor activity of immune cells and testing cancer immunotherapies requires conditions that are as life-like as possible. The tumor microenvironment (TME) describes a complex sum of cellular and acellular actors that influence both immune cells and tumor cells as well as their interplay. Yet in development phases of new immunotherapies, the screening of drugs and adoptive cell products benefits from reproducible and controlled conditions. Two-dimensional (2D) cell cultures cannot simultaneously meet these two challenges therefore lacking considerably predictive power owing to their artificial nature. Various 3D tumor models have therefore been implemented to mimic the architecture and intrinsic heterogeneity of a microtumor. This protocol provides an easy-to-follow, time-efficient, material-limited method for live cell killing and infiltration of single tumor spheroids. It uses multicellular tumor spheroids grown scaffold-free and allows co-culture with immune cells. This protocol is optimized for natural killer (NK) cell functionality assays. However, it can be transferred to other immune cells, in particular cytotoxic T cells. This assay can be analysed using life cell imaging (here with the IncuCyte S3 system) and/or flow cytometry.

The mitochondrial outer membrane protein SYNJ2BP interacts with the cell adhesion molecule TMIGD1 and can recruit it to mitochondria.

BACKGROUND: Transmembrane and immunoglobulin domain-containing protein 1 (TMIGD1) is a recently identified cell adhesion molecule which is predominantly expressed by epithelial cells of the intestine and the kidney. Its expression is downregulated in both colon and renal cancer suggesting a tumor suppressive activity. The function of TMIGD1 at the cellular level is largely unclear. Published work suggests a protective role of TMIGD1 during oxidative stress in kidney epithelial cells, but the underlying molecular mechanisms are unknown. RESULTS: In this study, we address the subcellular localization of TMIGD1 in renal epithelial cells and identify a cytoplasmic scaffold protein as interaction partner of TMIGD1. We find that TMIGD1 localizes to different compartments in renal epithelial cells and that this localization is regulated by cell confluency. Whereas it localizes to mitochondria in subconfluent cells it is localized at cell-cell contacts in confluent cells. We find that cell-cell contact localization is regulated by N-glycosylation and that both the extracellular and the cytoplasmic domain contribute to this localization. We identify Synaptojanin 2-binding protein (SYNJ2BP), a PDZ domain-containing cytoplasmic protein, which localizes to both mitochondria and the plasma membrane, as interaction partner of TMIGD1. The interaction of TMIGD1 and SYNJ2BP is mediated by the PDZ domain of SYNJ2BP and the C-terminal PDZ domain-binding motif of TMIGD1. We also find that SYNJ2BP can actively recruit TMIGD1 to mitochondria providing a potential mechanism for the localization of TMIGD1 at mitochondria. CONCLUSIONS: This study describes TMIGD1 as an adhesion receptor that can localize to both mitochondria and cell-cell junctions in renal epithelial cells. It identifies SYNJ2BP as an interaction partner of TMIGD1 providing a potential mechanism underlying the localization of TMIGD1 at mitochondria. The study thus lays the basis for a better understanding of the molecular function of TMIGD1 during oxidative stress regulation.

Physiological functions of junctional adhesion molecules (JAMs) in tight junctions.

Junctional adhesion molecules (JAMs) comprise a small subfamily of the immunoglobulin superfamily of adhesion receptors with a multitude of physiological functions in vertebrate development and homeostasis. Several members of the JAM family localize at tight junctions of epithelial and endothelial cells where they interact with PDZ domain-containing scaffolding proteins. For some JAM family members, molecular mechanisms have been elaborated through which they regulate cell-cell contact maturation and tight junction formation. For other members of this family our knowledge on their role in barrier-forming epithelia is still fragmentary. Here, we review our current understanding of the contribution of JAM family proteins to the barrier function of epithelial and endothelial cells with a major focus on epithelial tight junctions.