Abstract LB274: Targeting Epha2 in primary and metastatic murine breast cancer models reduces the expansion of bone metastasis-associated macrophages and the progression of tumor-induced bone disease

Abstract Bone metastases are present in over 70% of metastatic breast cancer patients and are complicated by the development of tumor-induced bone disease (TIBD) which results in pathological fractures due to the osteolytic nature of the tumor. Moreover, the receptor tyrosine kinase, EphA2, which participates in bone homeostasis and inflammation is overexpressed in a variety of cancers and expression is further increased in bone metastases. Despite this, the role of EphA2 in bone metastasis remains unexplored. We have demonstrated that EphA2 expression is increased in osteolytic breast cancer cell lines, 4T1 and MDA-MB-231, in comparison to non-osteolytic lines, MCF-7 and T47D. In a murine intratibial (IT) tumor model using EphA2 knockout (KO) 4T1 breast cancer cells versus control cells, x-ray images showed significantly lower bone lesion area in the EphA2 KO group. Flow cytometry analysis of the bone marrow demonstrated the expansion of macrophages, particularly CD124+ CD204+ bone metastasis-associated macrophages (BoMAMs) in the control group. To determine if the EphA2-dependent expansion of macrophages was site dependent, we injected control and EphA2 KO 4T1 cells into the mammary fat pad of 5–6-week-old mice and allowed tumors to grow for 21 days. EphA2 KO tumors were significantly smaller, and splenomegaly occurred in the control group. Despite no observable tumors in the bone sections from control and EphA2 KO groups, BoMAMs expanded in the bone marrow and spleens for both groups with a significant reduction in EphA2 KO tumor-bearing mice. Moreover, EphA2 KO mammary tumors had significantly more CD3+ T cells and higher trending CD4+ T cells and macrophage infiltrates suggesting hotter tumors. To determine a mechanism for the recruitment and expansion of BoMAMs we analyzed the expression of the monocyte chemoattractant protein-1 receptor, CCR2, in BoMAMs isolated from control and EphA2-KO tumor-bearing mice. Interestingly, flow cytometry analysis of BoMAMs from intratibial and mammary fat pad studies revealed no differences in CCR2 expression. To determine if pharmacological inhibition of EphA2 could recapitulate our previous studies using EphA2 KO cells, we treated IT WT (wild type) 4T1 mice with either drug or control. We were able to demonstrate that pharmacological inhibition of EphA2 significantly reduces bone destruction by analysis of x-ray images. Thus, highlighting the effectiveness of targeting EphA2 to reduce TIBD. Future studies are required to fully understand how pharmacological inhibition of EphA2 will impact primary breast cancer models and the expansion of BoMAMs. Elucidating the mechanism of EphA2-dependent TIBD and macrophage expansion will uncover novel potential therapeutic targets for bone metastatic breast cancer. Citation Format: Dominique Vanessa Parker, Verra Nawa, Natalie Bennett, Jade Miller, Erik Beadle, Jin Chen, Julie Rhoades. Targeting Epha2 in primary and metastatic murine breast cancer models reduces the expansion of bone metastasis-associated macrophages and the progression of tumor-induced bone disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB274.