Breast cancer initiation, progression and metastasis rely on a complex interplay between tumor cells and their surrounding microenvironment. Infiltrating immune cells, including macrophages, promote mammary tumor progression and metastasis; however, less is known about the role of macrophages in early stage lesions. In this study, we utilized a transplantable p53-null model of early progression to characterize the immune cell components of early stage lesions. We show that macrophages are recruited to ductal hyperplasias with a high tumor-forming potential where they are differentiated and polarized toward a tumor-promoting phenotype. These macrophages are a unique subset of macrophages, characterized by pro-inflammatory, anti-inflammatory and immunosuppressive factors. Macrophage ablation studies showed that macrophages are required for both early stage progression and primary tumor formation. These studies suggest that therapeutic targeting of tumor-promoting macrophages may not only be an effective strategy to block tumor progression and metastasis, but may also have critical implications for breast cancer prevention.

Infiltrating inflammatory cells, including tumor-associated macrophages (TAMs), have been shown to promote breast cancer cell invasion and have been correlated with metastasis and poor prognosis. While it is well-established that macrophages are recruited to the invasive fronts of established tumors to exert pro-tumor signals, their role in premalignancy remains poorly understood. Using a novel p53−/− syngeneic transplantable model of premalignancy, we show that inflammatory cells, including macrophages, are indeed recruited to early lesions prior to invasion. Microarray analysis was performed on two different pre-invasive lines, termed PN1a, (high tumor-forming potential) and PN1b (low tumor-forming potential). Interestingly, several pro-inflammatory chemokines, macrophages markers, and the pro-inflammatory cytokine growth arrest specific 6 (Gas6) were increased in PN1a lesions as compared to PN1bs. In a 3-D co-culture system, macrophages were recruited to PN1a acini and induced invasion, while PN1b cells remained non-invasive, supporting our in vivo data. Depletion of macrophages in vivo with clodronate containing liposomes significantly delayed progression of PN1a lesions to invasive cancer, indicating a critical role for macrophages in early progression. Gas6 and its receptor tyrosine kinase, Axl, were highly expressed in PN1a preinvasive lesions, but decline in invasive tumors, suggesting that this pathway is a key driver of the transition from pre-invasive to invasive cancer. Further studies using our 3-D co-culture system with Gas6−/− macrophages demonstrated that macrophages promote the formation of non-polar disorganized structures by activating Axl. Finally, transplantation of PN1a cells into Gas6 +/− mice resulted in a delay in tumor formation, indicating that stromal-derived Gas6 (and potentially macrophage derived) may promote the progression of early stage lesions through the paracrine activation of Axl. As a plethora of Axl inhibitors have been developed and are currently in clinical trial, these studies have critical implications for the prevention and treatment of invasive breast cancer. Citation Format: Emily C. Carron, Heather L. Machado, Samuel Homra. Macrophages promote the progression of premalignant mammary gland lesions through activation of the Axl signaling cascade. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4074.