900 An iron-rich subset of macrophages promotes tumor growth through a Bach1-Ednrb axis

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Background Tumor associated macrophages (TAMs) frequently comprise the most abundant leukocyte population within the tumor microenvironment (TME). TAMs are phenotypically diverse and can be polarized towards either pro- or anti-tumor functions by signals within the TME. While microvascular dysfunction with red blood cell (RBC) extravasation and intra-tumoral hemorrhage are hallmarks of solid tumors,1 the role of RBCs and heme in polarizing TAMs within the TME is not known. Methods We performed single cell RNA-seq (scRNA-seq) on multiple murine mouse models of sarcoma, including a syngeneic fibrosarcoma (FS) transplant model. Iron-rich TAMs were isolated for downstream analysis using magnetic column-based fractionation. FS flank tumors were also generated in LysMCre:Ednrbflox/flox and Bach1-/- mice. Results scRNA-seq of murine sarcomas identified a population of TAMs with significant enrichment for heme and iron metabolism pathways (figure 1). Sequencing of iTAMs isolated via magnetic column fractionation revealed significant upregulation of angiogenesis pathways and downregulation of antigen presentation machinery (figure 2). Co-transplantation of FS tumor cells with iTAMs resulted in increased tumor growth, decreased T cell infiltration, and increased vascular density (figure 3). Differential expression analysis identified the endothelin B receptor (Ednrb) as one of the top iTAM markers (figure 4). Deletion of Ednrb within myeloid cells led to significant reductions in FS tumor growth, increased TAM surface MHCII expression, and reductions in tumor vasculature (figure 4). Combining an endothelin receptor antagonist with anti-PD1 led to significant reductions in tumor volume and improved survival (figure 4). Transcription factor motif analysis demonstrated enrichment of Bach and Nrf2 transcription factor motifs in iTAMs (figure 5). Bach1 is a transcriptional repressor that is degraded by heme and controls the development of iron-recycling macrophages in the spleen.2 FS tumors transplanted into Bach1-/- mice demonstrated increased tumor growth, increased numbers of iTAMs, upregulation of pathways involved in heme metabolism and angiogenesis with downregulation of inflammatory pathways (figure 5). To investigate the relationship between intra-tumoral hemorrhage and iTAM development, we injected FS flank tumors with autologous RBCs and found increased numbers of iTAMs, with upregulation of genes involved in heme and iron metabolism and downregulation of genes involved in antigen presentation and anti-tumor immune responses (figure 6). Conclusions Solid tumors harbor a unique population of iron-rich TAMs that promote tumor growth and angiogenesis and contribute to a suppressive TME. iTAM development and function are driven by extravascular RBCs and heme-dependent degradation of the transcriptional repressor Bach1. Targeting iTAM Ednrb represents a novel immunotherapeutic approach. Acknowledgements We thank Dr. Ronald DeMatteo, MD for his valuable scientific input. We also thank the flow cytometry cores at UPenn and CHOP, CAG Core at CHOP, and HIC core at UPenn. References Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144:646–74. Haldar M, Kohyama M, So AY-L, Kc W, Wu X, Briseño CG, et al. Heme-mediated SPI-C induction promotes monocyte differentiation into iron-recycling macrophages. Cell 2014;156:1223–34. Ethics Approval All animal experiments were approved by the IACUC at the University of Pennsylvania (Protocol no. 805728). No human subjects were included in the current study.

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