Sites of direct contact between a macrophage, a tumor cell and endothelial cell [Tumor MicroEnvironment of Metastasis (TMEM)], correlates with metastasis in breast cancer patients independently of other clinical prognostic indicators suggesting a direct role for TMEM function in hematogenous dissemination. Here we show, using intravital high-resolution two-photon microscopy, that tumor cell intravasation occurs only at TMEM. Tumor cell intravasation occurs concurrently with transient, local vascular permeability at TMEM in an autochthonous mouse mammary carcinoma model and a human patient-derived xenograft model. Ablation of the presence or activity of the TMEM associated macrophages blocks tumor cell intravasation at TMEM demonstrating an essential role of perivascular macrophages in TMEM function. A subset of TMEM macrophages are identified as Tie2-expressing macrophages that are characterized by F4/80+/CD11b+/CD68+/MRC1+/Tie2Hi/VEGFAHi/CD11c-. VEGFA signaling from Tie2Hi TMEM-associated macrophages causes the local loss of vascular junctions resulting in transient vascular permeability and tumor cell intravasation at TMEM. Macrophage-specific ablation of VEGFA results in increased vascular junction stability and inhibition of intravasation, demonstrating that vascular junction dissolution at VEGFAHi/Tie2Hi TMEM-associated macrophages leads to vascular permeability and tumor cell intravasation. Inhibition of Tie2 with the first in class small molecular inhibitor rebastinib impairs TMEM function leading to a reduction in vascular permeability, tumor cell dissemination and metastasis. Further, rebastinib inhibition of Tie2 blocks tumor cell extravasation and metastatic growth in the lungs.
Together, the findings that TMEM macrophages mediate vascular permeability and tumor cell intravasation demonstrate an essential role for TMEM within the primary mammary tumor as sites of tumor cell dissemination. These data reveal the mechanism of tumor cell intravasation in breast cancer, explain the prognostic value of TMEM density in predicting distant metastatic recurrence in breast cancer patients and document a strategy for inhibition of dissemination.
This research is supported by the Department of Defense Breast Cancer Research Program under award number BC120227 (ASH), NIH CA100324 (JSC) and the Integrated Imaging Program.
Citation Format: Allison S. Harney, Esther N. Arwert, David Entenberg, Yarong Wang, Peng Guo, Bin-Zhi Qian, Bryan D. Smith, Jeffrey W. Pollard, Joan G. Jones, Daniel L. Flynn, John S. Condeelis. Imaging the tumor microenvironment of metastasis reveals the mechanism of transient blood vessel permeability and tumor cell intravasation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5125. doi:10.1158/1538-7445.AM2015-5125