Abstract 5279: Utilizing intravascular MHC-I antibody labeling to investigate regulation of tumor cell extravasation by ACKR1

Abstract Background: Endothelial Ackr1 is a major regulator of immune cell extravasation. Tumor cells can co-opt mechanisms of leukocyte extravasation to exit the vasculature at distant metastatic sites. Detection of intravascular vs. extravasated tumor cells in the lung has previously relied on time-consuming confocal imaging with 3-D reconstruction. We optimized an intravascular labeling procedure to easily and quantitatively detect tumor cell extravasation via flow cytometry, and used it to investigate the effect of endothelial Ackr1 expression on tumor cell extravasation into the lung metastatic niche. Methods: To investigate the effect of endothelial Ackr1 on tumor cell extravasation, we employed an experimental metastasis model. Metastatic breast cancer cells (E0771.LMB) expressing mCherry were intravenously injected into Control or endothelial-specific Ackr1 knock out (Ackr1 ECKO) mice and allowed to circulate and extravasate into lung tissue over 24 hours. 3 minutes before sacrificing the mice, we intravenously injected a fluorescently labeled anti-MHC I antibody to label tumor cells remaining within the vascular compartment. Harvested lungs were dissociated, and tumor (mCherry+) cells were classified as intravascular or extravascular depending on the presence of the MHC I antibody signal. To confirm that inflammation from the tumor did not create extravascular antibody leakage, we treated wildtype mice intratracheally with either PBS (control) or 300 ng of TNF-α. After 24 hours, we administered 15 μg of anti-Type IV Collagen (Col4) antibody, which does not bind within the vascular lumen but will bind endothelial basement membrane if extravasated. Antibody leakage was evaluated via Col4 staining in lung tissue and in the liver, which served as a positive control due to its fenestrated endothelial barrier. Results: We were successfully able classify intravascular versus extravascular tumor cells in the lung metastatic niche via flow cytometry using intravascular MHC I antibody labeling. We detected no leakage of antibody from the vascular compartment of the lung in naïve or inflamed settings. Despite the well-characterized role of Ackr1 in extravasation of other cell types, we observed that endothelial Ackr1 expression does not affect the ability of tumor cells to extravasate into lung tissue after 24 hours in circulation in an experimentally induced metastasis model. Conclusions/Future Directions: Our results highlight an efficient intravascular antibody labeling technique to assess tumor cell extravasation at distant metastatic sites. While endothelial Ackr1 did not impact tumor cell extravasation in the lung metastatic niche, we are using this labeling technique to evaluate the impact of endothelial Ackr1 expression on neutrophil extravasation into the lung pre-metastatic niche. Citation Format: Rishi Patel, Samuel Tanner Roach, Serena Thomas, Braulio Aguilar, Hsun Wang, L.A. Naiche, Jan Kitajewski. Utilizing intravascular MHC-I antibody labeling to investigate regulation of tumor cell extravasation by ACKR1 [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 5279.