The discovery of a fetal origin for tissue-resident macrophages (trMacs) has inspired an intense search for the mechanisms underlying their development. Here, we performed in vivo lineage tracing of cells with an expression history of IL7Rα, a marker exclusively associated with the lymphoid lineage in adult hematopoiesis. Surprisingly, we found that Il7r-Cre labeled fetalderived, adult trMacs. Labeling was almost complete in some tissues and partial in others. The putative progenitors of trMacs, yolk sac (YS) erythromyeloid progenitors, did not express IL7R, and YS hematopoiesis was unperturbed in IL7R-deficient mice. In contrast, tracking of IL7Rα message levels, surface expression, and Il7r-Cremediated labeling across fetal development revealed dynamic regulation of Il7r mRNA expression and rapid upregulation of IL7Rα surface protein upon transition from monocyte to macrophage within fetal tissues. Fetal monocyte differentiation in vitro produced IL7R + macrophages, supporting a direct progenitor-progeny relationship. Additionally, blockade of IL7R function during late gestation specifically impaired the establishment of fetal-derived trMacs in vivo. These data provide evidence for a distinct function of IL7Rα in fetal myelopoiesis and identify IL7R as a novel regulator of trMac development.

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The bone is the most common site of metastasis and the usual first site of recurrence in metastatic breast cancer. Bone metastasis is incurable and causes severe pain with significant bone loss in breast cancer patients. Current treatments for bone metastasis aim to diminish bone loss; however, most patients still progress on current therapies, making it critical to identify new therapeutic targets. We have identified the receptor Recepteur d'Origine Nantais (RON) as a target for treating metastatic breast cancer. RON is the receptor for the macrophage stimulating protein (MSP) and has both tumor-intrinsic and -extrinsic roles in metastasis. The gene encoding RON (MST1R in human or Stk in mouse) gives rise to two transcripts coding for full-length RON (FL-RON) and short-form RON (SF-RON) isoforms, where SF-RON lacks the ligand-binding domain for MSP and is constitutively active. In mouse breast cancer lung metastasis models, deletion of kinase activity from both isoforms of RON in the host nearly eradicates tumors through increased anti-tumor immunity. More recently, we identified SF-RON as the main isoform mediating tumor-associated immunity in these models, with loss of SF-RON promoting increased anti-tumor T cell activity. In the bone, loss of kinase activity from both isoforms of RON protects against bone loss, but its role in anti-tumor immunity is unknown. We now aim to delineate the role of SF-RON-mediated anti-tumor immune responses in the bone, where immunotherapy has been less effective. In SF-RON knockout mice (RonSF-/-), tumor cells injected into the bone grow for a short time but are undetectable by endpoint. Preliminary analyses suggest the importance of immune-mediated clearance in the loss of tumors in RonSF-/- mice as depletion of CD8+ T cells rescued tumor growth by approximately 50%. To gain insight into the role of immune cell populations in this anti-tumor response, we utilized immunohistochemistry and flow cytometry to characterize specific immune cells in bone metastasis. Initial results suggest increased infiltration of T cells, B cells, and NK cells into the tumors in the bones of RonSF-/- mice compared to wild-type controls. Notably, these immune cell populations are all known to play a significant role in the anti-tumor response in other models. Future work will utilize depletion studies to further investigate the role of T cells, B Cells, and NK cells in SF-RON-mediated tumor clearance in the bone. This work will help elucidate a role for host SF-RON in breast cancer bone metastasis and its potential to be a potent immunotherapy target for the treatment of bone metastasis. Citation Format: Clint H Valencia, Jaime Fornetti, Alana L Welm. Defining the immune milieu in short-form RON-mediated tumor clearance in breast cancer bone metastasis [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A55.

The discovery of a fetal origin for tissue-resident macrophages (trMacs) has inspired an intense search for the mechanisms underlying their development. Here, we performed in vivo lineage tracing of cells with an expression history of IL-7Rα, a marker exclusively associated with the lymphoid lineage in adult hematopoiesis. Surprisingly, we found that IL7R-Cre labeled fetalderived, adult trMacs. Labeling was almost complete in some tissues and partial in other organs.The putative progenitors of trMacs, yolk sac (YS) erythromyeloid progenitors (EMPs), did not express IL-7R, and YS hematopoiesis was unperturbed in IL-7R-deficient mice. In contrast, tracking of IL-7Rα message levels, surface protein expression, and IL7R-Cre-mediated labeling across fetal development revealed dynamic regulation of IL-7Rα mRNA expression and rapid upregulation of IL-7Rα surface protein upon transition from monocyte to macrophage within fetal tissues. Fetal liver monocyte differentiation in vitro produced IL-7R+ macrophages, supporting a direct progenitor-progeny relationship. Additionally, blockade of IL-7R function during late gestation specifically impaired the establishment of fetal-derived tissue macrophages in vivo. These data provide evidence for a distinct function of IL-7Rα in fetal myelopoiesis and identify IL-7R as a novel regulator of tissue-resident macrophage development.

Tissue-resident macrophages (TRMs) play critical roles in tissue homeostasis and disease. Many populations of TRMs derive from fetal progenitors and independently self-maintain across the lifespan through in situ proliferation. Here, we have identified the interleukin-7 receptor (IL7R) as a novel regulator of TRM development. Using an IL7R-Cre lineage tracing model, we observed that adult TRMs in the brain, epidermis, liver, and lung were highly labeled by IL7R-cre, in the absence of IL7Ra mRNA or protein expression. To gain insight into developmental expression of IL7Ra, we profiled surface expression, mRNA expression, and IL7R-cre driven labeling across fetal development. Erythromyeloid progenitors, putative TRM precursors, were barely labeled by IL7Ra-cre, and IL7R deletion did not affect YS hematopoiesis. In contrast, we observed IL7Ra mRNA expression in fetal monocytes, and robust IL7Ra surface expression on developing TRMs during late gestation. Sorted Ly6chi fetal liver monocytes cultured ex vivo with M-CSF differentiated into macrophages expressing IL7R, suggesting a precursor-product relationship. Blockade of the IL7R with a monoclonal antibody during gestation impaired liver, lung, and epidermal TRM cellularity at birth, with a concomitant increase in cellularity of liver monocytes, suggesting that IL7Ra regulates TRM differentiation from fetal monocytes during fetal development. These data reveal dynamic regulation of IL7Ra expression in TRMs and TRM precursors during late gestation, and provide evidence that IL7R signaling regulates fetal TRM development. Ongoing work addresses downstream signaling and the specific developmental processes regulated by IL7R signaling during fetal TRM development.