Macrophage clusters extend multiple pseudopods to disrupt cell junctions between cohesive targets

Introduction: Among immune cell types, macrophages are relatively unique in aggregating and even fusing to syncytia with specialized functions that include the foreign body response, bone resorption, and granulomatous reactions. New contexts of macrophage clustering continue to be identified such as in wound healing, but relevance to cancer is unclear. We recently found that phagocytosis of cohesive melanoma tumoroids is cooperative with respect to macrophage number and that phagocytic macrophages form clusters in tumoroids and solid tumors [1]. Tumoroid elimination and clustering were both enhanced with M1 macrophages polarized with interferon-γ (IFNγ) and abrogated with M2 macrophages polarized with interleukin-4 (IL-4). Here, we aimed to 1) identify molecular changes that underlie clustering of macrophages and 2) observe how nearby macrophages interact with phagocytic targets that adhere to one another. We identify roles for integrin αLβ2 and reduced cellular contractility in promoting macrophage aggregation and describe intrusive pseudopods extended from clustered macrophages that appear to disrupt cell-cell junctions between phagocytic targets.

Materials and

Methods: Macrophages were differentiated from primary mouse bone marrow cells or Hoxb8-conditionally immortalized macrophage (CIM) progenitors [2]. CIM progenitors lacking the αL, αM, αX, or β2 integrin subunits or intercellular adhesion molecule-1 (Icam1) were engineered by CRISPR/Cas9 gene knockout. Low-adhesion surfaces were prepared by coating 96-well U-bottom plates with a thin layer of poly(2-hydroxyethyl methacrylate). Macrophages were polarized with IFNγ or IL-4 as appropriate, labeled with CellTracker Deep Red fluorescent dye, and added to passivated plates in a suspension of 1000 cells per well. Some aggregation experiments were performed in the presence of cycloheximide, latrunculin A, or p-amino-blebbistatin. Melanoma spheroids were generated by culturing B16 cells in media containing methylcellulose for 2 days. B16 cells were engineered to lack the macrophage checkpoint ligand CD47 in order to maximize phagocytosis as recently described [1]. Spheroids were washed to remove methylcellulose and transferred via a wide bore pipette to a chamber slide. Macrophages were added together with an opsonizing IgG antibody and the spheroids were imaged on a confocal microscope for up to 6 hours. Animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania.

Results, Conclusions, and Discussions: We found that M1 macrophages on their own form compact clusters on low-adhesion surfaces while M2 macrophages disperse as single cells, which indicates differences in cell-cell and cell-substrate interaction strength among macrophage subsets. We identified changes in adhesion receptor repertoires and cytoskeletal organization that might explain such differences and investigated the functional consequences with pharmacological and genetic perturbations. Aggregation induced by M1 polarization required de novo protein synthesis and actin polymerization, whereas myosin-II inhibitor enhanced aggregation of unpolarized macrophages. Knockout of αL or β2 integrin subunits or Icam1 partially abrogated M1 aggregation.
B16 melanoma cells adhere to each other through calcium-dependent interactions, and likely via N-cadherin. Upon addition of macrophages to B16 spheroids, we observed clusters of macrophages extending multiple protrusions in between cohesive B16 cells (Figure 1). Such cooperating pseudopods preceded phagocytosis of a melanoma cell. Pseudopods from lone macrophages sometimes meandered between B16 cells rather than encircling a single B16 as needed for engulfment.
Macrophage clustering during tumoroid phagocytosis can enable cooperativity between pseudopods from different cells and thereby seems distinct from the other physiological and pathological examples of macrophage aggregation. We found that integrin αLβ2 binding to Icam1 is at least partially responsible for the tight compaction of macrophages on the low adhesion surfaces used for tumoroids, but roles for other pathways including actomyosin is suggested by incomplete effects of knockout of the adhesion proteins. Macrophage β2 integrins have been implicated in FcR-mediated phagocytosis that occurs via extension of a pseudopod around an IgG-opsonized target. Pseudopod-driven disruption of cell-cell junctions in tumoroids and solid tumors might require the multiple ‘intrusive pseudopods’ or ‘intrudopods’ observed here from clustered macrophages, thus providing a key advantage for cooperative phagocytosis.

Acknowledgements (Optional): References Cited:
[1] Dooling, L.J., Andrechak, J.C. et al. Cooperative phagocytosis of solid tumours by macrophages triggers durable anti-tumour responses. Nat. Biomed. Eng (2023). https://doi.org/10.1038/s41551-023-01031-3
[2] Roberts, A.W. et al. Cas9+ conditionally-immortalized macrophages as a tool for bacterial pathogenesis and beyond. eLife (2019). https://doi.org/10.7554/eLife.45957