Abstract 5285: Activation of endothelial ACKR1 in the pre-metastatic niche regulates neutrophil recruitment and tumor metastasis in breast cancer

Abstract Background: Extravasation of neutrophils into the pre-metastatic niche promotes metastatic breast cancer cell survival. ACKR1 is a non-signaling cytokine receptor expressed on endothelial and erythroid cells and acts as a critical regulator of neutrophil extravasation in multiple inflammatory contexts. Our work has shown that endothelial ACKR1 is upregulated in the lung pre-metastatic niche during primary breast tumor progression. Therefore, we tested the requirement for lung endothelial ACKR1 expression in neutrophil recruitment to the pre-metastatic niche and for breast cancer metastasis to the lung. Methods: We investigated the role of endothelial ACKR1 in the lung pre-metastatic niche using a novel endothelial-specific ACKR1 knockout mouse model (ACKR1 ECKO). Pre-metastatic niche formation was induced in control and ACKR1 ECKO mice via daily intraperitoneal injections of tumor-conditioned media. Immunohistochemistry and flow cytometry were employed to analyze the percentage of neutrophils in the pre-metastatic niche and quantify neutrophil extravasation into the lung parenchyma. Intravenous tumor cell injections were performed following pre-metastatic niche formation to assess the impact of endothelial ACKR1 on tumor cell extravasation and colonization in the lung. Lung tissues were collected at 24 hours and 2 weeks post-tumor cell injection, and tumor cell extravasation and overall metastatic burden were analyzed using flow cytometry and whole-mount immunofluorescent imaging. The effect of endothelial ACKR1 expression on neutrophil adhesion and transendothelial migration under tumor inflammatory conditions was tested using monolayer transmigration assays in vitro. Results: We found that endothelial ACKR1 expression was activated in the lungs of mice bearing mammary tumors or injected with tumor-conditioned media. After pre-metastatic niche induction, the lung parenchyma of ACKR1 ECKO mice had significantly fewer neutrophils than control mice, with no reduction in circulating neutrophils. Tumor cell extravasation and seeding of the lung 24 hours after tail vein injection were unaffected by endothelial ACKR1. However, at the 2-week timepoint, we found that ACKR1 ECKO mice had significantly decreased lung metastatic nodules compared to controls. Finally, we observed that tumor-conditioned media can induce high ACKR1-expressing HUVEC cells in vitro and promote neutrophil adhesion and transendothelial migration. Conclusion: Our study shows that secreted factors from the primary tumor upregulate endothelial ACKR1 in the lung pre-metastatic niche and facilitate neutrophil recruitment, ultimately promoting lung metastasis. We are now investigating which steps of neutrophil extravasation are regulated by endothelial ACKR1 and identifying the chemokines that allow ACKR1 to regulate this process during metastasis. Citation Format: Samuel Tanner Roach, Serena Thomas, Rishi Patel, Hsun Wang, Braulio Aguilar, Chinwe Ewenighi-Amankwah, Lauren Raasch, William Muller, L.A. Naiche, Jan Kitajewski. Activation of endothelial ACKR1 in the pre-metastatic niche regulates neutrophil recruitment and tumor metastasis in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5285.