The crosstalk between macrophages and 4T1 mammary tumor cells promotes metastasis in a novel intraductal model for breast cancer

Immunotherapeutics that target the immune responses in mammary tumors are a promising alternative treatment for breast cancer, but require additional preclinical validation. We recently developed an innovative intraductal mouse breast cancer model that faithfully reproduces breast tumor progression and provides a preclinical tool for evaluating candidate immunomodulatory drugs. This screening model relies on the orthotopic injection of 4T1 mammary tumor cells in the mammary ducts of immunocompetent, lactating mice. We previously described that intraductal breast tumors develop at a slower rate, but metastasize at a similar rate compared to classical fat pad inoculated tumors. We now aimed to characterize the tumor-associated immune responses underlying intraductal tumor progression, focusing on macrophage-tumor cell interactions since these play a key role in the establishment of those responses. RAW 264.7 macrophages were intraductally co-injected with 4T1 cells and the augmented crosstalk between macrophages and tumor cells did not affect mammary tumor growth kinetics. However, these co-injected mice suffered from severe splenomegaly and more aggressive metastasis compared to 4T1-only injected mice. Profiling of cytokine and chemokine levels in serum and mammary tumors provided evidence that intraductal transfer of naive macrophages provokes a malignant immune response switch in support of metastasis. More specifically, the transferred macrophages mediated a stronger pro-inflammatory response in the early phases of intraductal tumor progression compared to normal 4T1 tumors. This was overruled by an anti-inflammatory response when metastasis became apparent. Co-injection of macrophages and 4T1 tumor cells also significantly induced systemic levels of the innate immune-related proteins chitinase 3-like 1 and lipocalin 2, confirming both proteins as biomarkers for breast cancer prognosis and tumor-associated immune responses. Based on our results, we identified a macrophage-mediated immune program that stimulates metastatic breakthrough of 4T1 tumor cells inoculated in the mouse mammary ducts and can be explored to identify druggable targets or test immunotherapeutics