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Macrophages comprise an essential component of the mammary microenvironment necessary for normal gland development. However, there is no viable  in vivo  model to study their role in normal human breast function. We hypothesized that adding primary human macrophages to the murine mammary gland would enhance and provide a novel approach to examine immune-stromal cell interactions during the humanization process.    Methods  Primary human macrophages, in the presence or absence of ectopic estrogen stimulation, were used to humanize mouse mammary glands. Mechanisms of enhanced humanization were identified by cytokine/chemokine ELISAs, zymography, western analysis, invasion and proliferation assays; results were confirmed with immunohistological analysis.    Results  The combined treatment of macrophages and estrogen stimulation significantly enhanced the percentage of the total gland humanized and the engraftment/outgrowth success rate. Timecourse analysis revealed the disappearance of the human macrophages by two weeks post-injection, suggesting that the improved overall growth and invasiveness of the fibroblasts provided a larger stromal bed for epithelial cell proliferation and structure formation. Confirming their promotion of fibroblasts humanization, estrogen-stimulated macrophages significantly enhanced fibroblast proliferation and invasion  in vitro , as well as significantly increased proliferating cell nuclear antigen (PCNA) positive cells in humanized glands. Cytokine/chemokine ELISAs, zymography and western analyses identified TNFα and MMP9 as potential mechanisms by which estrogen-stimulated macrophages enhanced humanization. Specific inhibitors to TNFα and MMP9 validated the effects of these molecules on fibroblast behavior  in vitro , as well as by immunohistochemical analysis of humanized glands for human-specific MMP9 expression. Lastly, glands humanized with macrophages had enhanced engraftment and tumor growth compared to glands humanized with fibroblasts alone.    Conclusions  Herein, we demonstrate intricate immune and stromal cell paracrine interactions in a humanized  in vivo  model system. We confirmed our  in vivo  results with  in vitro  analyses, highlighting the value of this model to interchangeably substantiate  in vitro  and  in vivo  results. It is critical to understand the signaling networks that drive paracrine cell interactions, for tumor cells exploit these signaling mechanisms to support their growth and invasive properties. This report presents a dynamic  in vivo  model to study primary human immune/fibroblast/epithelial interactions and to advance our knowledge of the stromal-derived signals that promote tumorigenesis.

Paracrine interactions between primary human macrophages and human fibroblasts enhance murine mammary gland humanization in vivo