Macrophages Promote Tumor Cell Extravasation across an Endothelial Barrier through Thin Membranous Connections

Abstract

Macrophages are important players involved in the progression of breast cancer, including in seeding the metastatic niche. However, the mechanism by which macrophages in the lung parenchyma interact with tumor cells in the vasculature to promote tumor cell extravasation at metastatic sites is not clear. To mimic macrophage-driven tumor cell extravasation, we used an in vitro assay (eTEM) in which an endothelial monolayer and a matrigel-coated filter separated tumor cells and macrophages from each other. The presence of macrophages promoted tumor cell extravasation, while macrophage conditioned media was insufficient to stimulate tumor cell extravasation in vitro. This finding is consistent with a requirement for direct contact between macrophages and tumor cells. We observed the presence of Thin Membranous Connections (TMCs) resembling similar structures formed between macrophages and tumor cells called tunneling nanotubes, which we previously demonstrated to be important in tumor cell invasion in vitro and in vivo. To determine if TMCs are important for tumor cell extravasation, we used macrophages with reduced levels of endogenous M-Sec (TNFAIP2), which causes a defect in tunneling nanotube formation. As predicted, these macrophages showed reduced macrophage-tumor cell TMCs. In both, human and murine breast cancer cell lines, there was also a concomitant reduction in tumor cell extravasation in vitro when co-cultured with M-Sec deficient macrophages compared to control macrophages. We also detected TMCs formed between macrophages and tumor cells through the endothelial layer in the eTEM assay. Furthermore, tumor cells were more frequently found in pores under the endothelium that contain macrophage protrusions. To determine the role of macrophage-tumor cell TMCs in vivo, we generated an M-Sec deficient mouse. Using an in vivo model of experimental metastasis, we detected a significant reduction in the number of metastatic lesions in M-Sec deficient mice compared to wild type mice. There was no difference in the size of the metastases, consistent with a defect specific to tumor cell extravasation and not metastatic outgrowth. Additionally, with an examination of time-lapse intravital-imaging (IVI) data sets of breast cancer cell extravasation in the lungs, we could detect the presence of TMCs between extravascular macrophages and vascular tumor cells. Overall, our data indicate that macrophage TMCs play an important role in promoting the extravasation of circulating tumor cells in the lungs.