{
  "abstract": "Background Macrophages pervade solid tumors where they predominantly contribute to tumor progression but also have roles in antitumor immunity. Spatial analyses indicate that different tumor-associated macrophage (TAM) subtypes might occupy distinct niches, and some TAMs that associate with increased patient survival were described qualitatively as forming ‘clusters’ adjacent to tumor cells. 1 In mouse tumors and reductionist tumoroid models, we recently observed clustering among macrophages that facilitates coordinated phagocytosis of tumor cells.2 Understanding this spatial organization and any unique functions of macrophage clusters could improve macrophage engineering and reprogramming for immunotherapies including chimeric antigen receptor-macrophages and blockade of the ‘don’t eat me’ CD47-SIRPα checkpoint to enhance tumor phagocytosis.Methods Macrophages were differentiated from mouse bone marrow cells or Hoxb8-conditionally immortalized macrophage (CIM) progenitors, labeled with CellTracker dyes, and added to low-adhesion well-plates with interferon-γ (IFNγ). Clustering was monitored by fluorescence microscopy for hours to days. Lentiviral transduction of CIMs was used to introduce synthetic cell adhesion molecules. Transcriptomic analyses were performed on published macrophage datasets. 3 Results Based on the IFNγ signaling signature of antitumor clusters of CXCL9 + TAMs 1 and the increased clustering of IFNγ-primed BMDMs in tumoroids,2 we analyzed gene expression in BMDMs activated with IFNγ (with or without lipopolysaccharide). We found that Itgal encoding integrin αL and Icam1 encoding ICAM-1 are both strongly upregulated relative to nonactivated macrophages with kinetic profiles that are similar to Cxcl9 and other inflammatory genes based on unsupervised clustering. Genes associated with actomyosin contractility are also coregulated based on unsupervised clustering but have more complex kinetics marked by modest early upregulation followed by significant downregulation. We previously showed that knockout of any αLβ2−ICAM-1 component partially abrogates macrophage clustering mediated by IFNγ whereas pharmacological inhibition of contractility enhanced clustering.2 To interrogate these adhesion receptors and their linkage to the cytoskeleton independently of inflammatory activation, synthetic adhesion molecules comprising associative extracellular domains and intracellular domains from integrin αLβ2 and ICAM-1 were introduced into macrophages.Conclusions Here, we identified adhesion receptors and cytoskeletal organization underlying clustering of pro-inflammatory, highly phagocytic macrophages in tumoroid models. These features are potentially shared by recently described antitumor TAM clusters at the tumor nest-stroma interface in human cancers. Together with our finding that neighboring macrophages cooperate to disrupt cancer cell adhesions for subsequent phagocytosis, these results position macrophage clusters as potential effectors in cancer immunotherapies.References Bill, R, Wirapati, P, Messemaker, M, Roh, W, Zitti, B, Duval, F, Kiss, M, Park, JC, Saal, TM, Hoelzl, J, Tarussio, D, Benedetti, F, Tissot, S, Kandalaft, L, Varrone, M, Ciriello, G, McKee, TA, Monnier, Y, Mermod, M, Blaum, EM, Gushterova, I, Gonye, ALK, Hacohen, N, Getz, G, Mempel, TR, Klein, AM, Weissleder, R, Faquin, WC, Sadow, PM, Lin, D, Pai, SI, Sade-Feldman, M, Pittet, MJ. CXCL9:SPP1 macrophage polarity identifies a network of cellular programs that control human cancers. Science. 2023;381(6657):515–524.Dooling, LJ, Anlaş, AA, Tobin, MP, Ontko, NM, Marchena, T, Wang, M, Andrechak, JC, Discher, DE, Clustered macrophages cooperate to eliminate tumors via coordinated intrudopodia. Proc Natl Acad Sci USA in press.Liu, SX, Gustafson, HH, Jackson, DL, Pun, SH, Trapnell, C,Trajectory analysis quantifies transcriptional plasticity during macrophage polarization. Sci Rep. 2020;10(1):12273.Ethics Approval Mouse bone marrow cells were harvested in accordance with protocols approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania under protocols #803177 and #804455.",
  "authors": [
    {
      "affiliations": [
        "University of Houston, Houston, TX, USA"
      ],
      "name": "Lawrence J Dooling"
    },
    {
      "affiliations": [
        "University of Houston, Houston, TX, USA"
      ],
      "name": "Johncarlo Ruvalcaba"
    },
    {
      "affiliations": [
        "University of Houston, Houston, TX, USA"
      ],
      "name": "Afra Azim"
    }
  ],
  "title": "789 Spatial organization of antitumor macrophages to enhance solid tumor phagocytosis",
  "uid": "66fe5572-00f5-580f-9953-9196389a3cfe"
}