O22 - Spatial Organization of Anti-Tumor Macrophages Enhances Solid Tumor Phagocytosis

Introduction: : Single-cell sequencing of tumors has revealed significant heterogeneity among tumor-associated macrophages (TAMs), and spatial technologies indicate that different TAM subtypes might reside in distinct tumor niches. Anti-tumor TAMs that associate with favorable patient outcomes have been described qualitatively as forming ‘clusters’ near tumor cells and T cells [1], but the factors promoting this spatial organization and any effects on macrophage functions including the defining macrophage function – phagocytosis – are unknown. Here, we identified similar expression of adhesion and cytoskeleton proteins between anti-tumor TAMs in human cancers and macrophages activated in vitro with interferon-γ, and we implicate these factors in macrophage clustering. We further show in spheroid tumor models that clustered macrophages have potential advantages over single macrophages in phagocytosing cohesive solid tumor cells via coordination of intrusive pseudopodia or ‘intrudopodia’.

Materials and

Methods: : Transcriptional profiles and spatial distributions of TAMs from human tumors were analyzed using data from reference [1]. Transcriptional profiles of mouse bone marrow-derived macrophages (BMDMs) polarized in vitro to an M1 state with interferon-γ and lipopolysaccharide or to an M2 state with interleukin-4 were analyzed using data associated with references [2-4]. As a reductionist approach to solid tumor phagocytosis, GFP+ B16 mouse melanoma cells deficient for the phagocytic checkpoint ligand CD47 were cultured in media containing methylcellulose to promote spheroid formation. Spheroids were co-cultured with fluorescently labeled BMDMs and an opsonizing IgG antibody that targets a B16 surface antigen. Phagocytosis of B16 cells in spheroids by BMDMs was imaged by confocal microscopy for several hours. Image analysis including three-dimensional rendering was performed in FIJI/ImageJ. Mouse bone marrow cells were harvested in accordance with protocols approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania.

Results, Conclusions, and Discussions:: In multiple human cancer types, anti-tumor CXCL9+ TAMs were quantified as partitioning to the interface between tumor nests and stroma whereas other macrophage subtypes were not enriched at the interface relative to the distal stroma (Fig. 1A). Macrophage expression of ITGAL and genes encoding surface receptors associated with phagocytosis (FCGR3A, SLAMF7) positively correlated with the abundance of CXCL9+ TAMs across different patient tumors. Conversely, macrophage expression of genes associated with actin bundling and actomyosin contractility anti-correlated with CXCL9+ TAMs. Mouse macrophages polarized in vitro with IFNγ likewise had high expression of Itgal and low expression of genes relating to actomyosin contractility. They also formed compact clusters on low-adhesion surfaces used to prepare spheroids. In melanoma spheroids, macrophage phagocytosis of cohesive tumor cell targets occurred at the periphery (i.e. macrophage-tumor cell interface) and frequently involved clusters of macrophages. Phagocytic macrophages extended intrusive pseudopodia (or ‘intrudopodia’) between target cells to disrupt cancer cell-cell junctions, and macrophages that were part of the same cluster extended intrudopodia in a spatially coordinated manner (Fig. 1B).

In many cell mixtures, multicellular organization reflects differences in cell adhesion molecules and cytoskeletal organization. The spatial organization of macrophages in tumors is incompletely understood, but differences in gene expression among different TAM subtypes are consistent with potential roles for both cell-cell adhesion and cytoskeleton. CXCL9+ TAMs that cluster near tumor cells are likely to be important in macrophage-based immunotherapies including reprogramming or repolarization, selective depletion of macrophage subtypes, and macrophage adoptive transfer. We continue to investigate the functional significance of clustering of anti-tumor macrophages, but our spheroid results suggest that clustering promotes coordination of phagocytosis by neighboring macrophages and facilitates the key step of disrupting adhesions between cohesive solid tumor target cells for subsequent engulfment. Our results therefore position CXCL9+ TAM clusters as potential effectors in cancer immunotherapy.

Acknowledgements and/or References (Optional):: We gratefully acknowledge funding from the National Institutes of Health National Cancer Institute (U01 CA254886 and P01 CA265794) and the National Science Foundation Center for Engineering Mechanobiology (CMMI 15-48571). The views expressed are those of the authors and do not necessarily reflect the views of the National Institutes of Health or the National Science Foundation.

References:
[1] R. Bill, et al., CXCL9:SPP1 macrophage polarity identifies a network of cellular programs that control human cancers. Science 381, 515–524 (2023).
[2] S. X. Liu, et. al., Trajectory analysis quantifies transcriptional plasticity during macrophage polarization. Sci Rep 10, 12273 (2020).
[3] K. A. Jablonski, et al., Novel markers to delineate murine M1 and M2 macrophages. PLOS ONE 10, e0145342 (2015).
[4] M. A. Hoeksema, et al., IFN-γ priming of macrophages represses a part of the inflammatory program and attenuates neutrophil recruitment. The Journal of Immunology 194, 3909–3916 (2015).