Abstract 5281: Analysis of tumor cell and neutrophil distribution relative to ACKR1 expressing venules during breast cancer metastasis

Abstract Background: Metastasis is the leading cause of breast cancer-related mortality. However, the success of metastatic colonization is not solely determined by tumor cells’ ability to disseminate; it is also influenced by the establishment of premetastatic niches within distant organs. Neutrophils promote lung metastasis by priming the premetastatic niche to make it a more hospitable environment for disseminating tumor cells. Atypical Chemokine Receptor 1 (ACKR1) is expressed on endothelial cells and is critical for neutrophil migration in inflammatory conditions, but its role has not previously been characterized in pre-metastatic niche formation. Methods: To study the role of endothelial ACKR1 in tumor cell and neutrophil extravasation, we analyzed the distribution of disseminating tumor cells and neutrophils relative to ACKR1-positive versus ACKR1-negative vessels in the lungs of orthotopic mammary tumor-bearing mice. To analyze disseminating tumor cells and extravasated neutrophils, we performed immunofluorescent staining of lung vasculature from immunocompetent mice implanted with E0771.LMB mammary tumor cells 28 days post-tumor injection. Lung samples were stained with ACKR1, CD31, and either Pan-CK (to label tumor cells) or Ly6G (to label neutrophils). We developed a semi-automated workflow to analyze the distance between disseminated tumor cells, neutrophils, and the closest venule within the lung. Images were blinded and target cells (tumor cells and neutrophils), ACKR1-positive venules, and ACKR1-negative venules were manually annotated in QuPath. A custom script was developed to outline venules and calculate the minimum distance between each target cell and the nearest vessel wall within 150 microns of a venule. These results were binned to categorize target cells based on their proximity within 50, 100, or 150 microns of an ACKR1-positive or -negative venule. Results: Our analysis revealed a significant increase in the average number of tumor cells within 50 microns of ACKR1-positive venules compared to ACKR1-negative venules. Similarly, neutrophil analysis showed a significant increase of neutrophils within 50 microns of ACKR1-positive venules. In lung tissue more than 50 microns from a venule, no differences in the number of tumor cells or neutrophils were detected. Conclusion/Future Directions: These findings demonstrate that both disseminated tumor cells and neutrophils preferentially localize near ACKR1-positive lung venules during breast cancer metastasis, suggesting that both cell types extravasate preferentially through ACKR1-expressing venules. Ongoing studies aim to define the role of ACKR1 in regulating neutrophil and tumor cell extravasation, phenotype, and survival in the premetastatic niche. Citation Format: Serena Thomas, S Tanner Roach, Rishi Patel, Lauren Raasch, L A. Naiche, Jan K. Kitajewski. Analysis of tumor cell and neutrophil distribution relative to ACKR1 expressing venules during breast cancer metastasis [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 5281.