Assistant Professor University of Houston Houston, Texas, United States
Introduction: : Phagocytosis is the defining effector function of macrophages responsible for clearing aged and dying cells as well as pathogens. Targeting tumor cells for phagocytosis is the basis for emerging immunotherapies including chimeric antigen receptor macrophages and blockade of “don’t eat me” immune checkpoints such as the CD47−SIRPα axis. However, the mechanisms elucidated using conventional phagocytosis assays, in which adherent macrophages engulf targets from suspension, fail to account for the biophysical barriers posed by cell−cell adhesions within solid tumors. To address this, we have developed a new three-dimensional spheroid phagocytosis assay that recapitulates tumor cohesion. Using this platform, we discovered that macrophages employ disruptive protrusions – which we term “intrudopodia”, or intrusive pseudopodia – to facilitate tumor cell engulfment.
Materials and
Methods: : We co-cultured fluorescently labeled macrophages with melanoma spheroids and visualized interactions between the two cell types using time-lapse confocal microscopy. To produce spheroids, we cultured B16 mouse melanoma cell suspensions on low-attachment surfaces in medium containing methylcellulose. To promote phagocytosis, we knocked out CD47 in the melanoma cells and opsonized the spheroids with anti-Tyrp1 IgG. In parallel, we modulated macrophage clustering with cytokine activation and by genetic or pharmacological perturbations of adhesion proteins and cytoskeleton organization.
Results, Conclusions, and Discussions:: Results and
Discussion: We observed that macrophages extend dynamic cellular intrudopodia between melanoma cells within spheroids. Intrudopodia included both nascent wedges forming at cancer cell−cell junctions and larger, lamellipodia-like protrusions. Additionally, macrophages were frequently observed to engage the spheroid periphery as cell clusters. The simultaneous extension of intrudopodia from clustered macrophages suggests spatial coordination and provides a potential physical basis for cooperativity. Clustering of macrophages in the context of cytokine activation depended in part on integrin receptors and decreased contractility, whereas phagocytosis of cohesive melanoma cells occurred independently of myosin-II contractility and select proteases.
Conclusions: This study introduces intrudopodia as macrophage structures involved in phagocytosis of cohesive cellular targets and reveals a possible function for macrophage clusters in solid tumors. The spheroid model described here will enable further dissection of intrudopodia structure and dynamics to uncover the molecular mechanisms by which they disrupt cell adhesions. The mechanobiological insights from such studies will inform macrophage engineering and design of solid tumor immunotherapies.
Acknowledgements and/or References (Optional): : Thank you to Alişya Anlaş and Michael Tobin for their contributions developing and imaging spheroids and to Dennis Discher for supervising this work.
