Metastasis is the most common cause of breast cancer mortality. Of the breast cancer subtypes, triple-negative breast cancer (TNBC) is the deadliest due to its increased likelihood to metastasize. Tumor cell extravasation is a critical step of metastasis and allows circulating tumor cells to exit the vasculature and seed distant tissues. Clear understanding of the major regulators of tumor cell extravasation will provide insights into the progression of TNBC metastasis. One potential regulator of TNBC cell extravasation is ACKR1. In many contexts, ACKR1 expression is required in endothelial cells (EC) for leukocyte extravasation. Endothelial ACKR1 binds CXCL2, a promigratory chemokine, and localizes it to EC junctions to guide neutrophils through leukocyte extravasation. CXCL2 expression in TNBC cells is also necessary for tumor cell extravasation from lung microvasculature and for tumor metastasis. Our preliminary data show that ACKR1 is required in at least one stromal cell type for TNBC metastasis from the primary tumor to the lung. These data suggest that endothelial ACKR1-CXCL2 interactions may mediate tumor cell extravasation in TNBC metastasis. Therefore, we hypothesize that endothelial ACKR1 promotes metastasis via retention of CXCL2 at EC junctions, increasing tumor cell chemotaxis and extravasation. To address this hypothesis, we will examine the in vivo significance of endothelial ACKR1 expression using our validated ACKR1 endothelial cell-specific knockout mouse model. We will test the requirement for endothelial ACKR1 for metastasis of orthotopically implanted TNBC tumors to distant sites in the lung and for extravasation of circulating tumor cells into lung tissue. We will determine which steps of extravasation require ACKR1 by evaluating ACKR1-low and ACKR1-overexpressing ECs using an Ibidi flow co-culture system that recapitulates the shear stress conditions of pulmonary microvasculature. We will examine whether these steps are dependent on CXCL2 by introducing CXCL2-neutralizing antibodies to the Ibidi flow system and observing their effects on each extravasation step. Our proposed studies will establish the role of endothelial ACKR1 in TNBC metastatic progression and determine the specific steps of tumor cell extravasation in which endothelial ACKR1 and CXCL2 function. Understanding these processes may guide development of ACKR1 as a prognostic marker for metastasis and can provide mechanistic insight into candidate chemokine and chemokine receptor inhibitors under evaluation for treatment of breast cancer. Citation Format: Samuel Tanner Roach, Chinwe Ewenighi-Amankwah, J Dufraine, L.A. Naiche, Jan K. Kitajewski. The role of endothelial ACKR1 in triple-negative breast cancer metastasis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3618.
Triple-negative breast cancer (TNBC), a highly aggressive and metastatic subtype of breast cancer, lacks expression of the estrogen and progesterone receptors and amplification of HER2 and comprises 15-20% of all breast cancer. Incidence and mortality of TNBC are higher in Black Americans than in other racial/ethnic groups. Conversely, Black patients are underrepresented in clinical breast cancer drug trials, limiting our understanding of differential treatment responses. Leukocyte infiltration is a key modulator of TNBC outcome, and Black patients show immunological differences that may contribute to differential outcomes in TNBC. About 60-80% of Black Americans are homozygous for a polymorphism in the Atypical Chemokine Receptor 1 (ACKR1) promoter region that causes loss of ACKR1 expression on red blood cells, also known as the “Duffy-null” serotype. ACKR1 is expressed on tumor cells, red blood cells, and endothelial cells, and can act as a chemokine sink or chemokine concentrator, depending on the context. Whole-tumor RNA analysis shows that overall loss of ACKR1 expression in TNBC tumors correlates strongly with decreased survival, but it is unclear which ACKR1-expressing cell type(s) contribute to this effect. Under inflammatory conditions, ACKR1 expression in endothelial cells localizes chemotactic cytokines at the endothelial junction, and loss of endothelial ACKR1 blocks the ability of leukocytes to extravasate through the endothelial barrier into surrounding tissues. We hypothesized that loss of endothelial/stromal ACKR1 reduces immune infiltration and activation in TNBC and racial/ethnic differences in ACKR1 expression may contribute to health disparities. To determine the role of ACKR1 in tumor immune infiltration, we chose a syngeneic immunocompetent mouse model to incorporate species-matched stromal-tumor interactions. We orthotopically inoculated E0771.LMB cells, a C57BL6/J syngeneic TNBC cell line, into ACKR1null and ACKR1het female mice. Tumor growth was measured triweekly, and tumor mass was measured at the 28-day endpoint. To measure immune infiltration, we fluorescently stained representative tumor cryosections with CD45 to detect all classes of infiltrating hematopoietic cells involved in immune response. There was no significant difference in tumor volume and mass among the ACKR1null and ACKR1het controls. Tumors implanted in ACKR1het controls showed broadly infiltrated leukocytes clusters, while tumors in ACKR1nullhosts showed sparse and infrequent leukocyte infiltration. Conclusion: Stromal ACKR1 does not have a strong effect on primary tumor growth. Loss of stromal ACKR1 reduces infiltration of leukocytes into the tumor microenvironment even when ACKR1 is not manipulated in the tumor cells, suggesting that ACKR1 in endothelial or other stromal cells regulates immune response in TNBC. Impact: Understanding the immunogenic functions of ACKR1 allelic variation will elucidate mechanisms of racially distinct therapeutic response in TNBC, enhance precision oncology, and promote cancer health equity. Citation Format: Chinwe Obianuju Ewenighi-Amankwah, Tanner Roach, Joseph Dufraine, Naiche Adler, Jan Kitajewski. ACKR1 expression in stromal cells regulates immune infiltration in triple negative breast cancer [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C035.
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