Abstract B30: Upregulation of fibronectin expression on endothelial cells by tumor-conditioned macrophages facilitates lung metastasis in breast cancer
Authors: Low Pin Yan , Lim Yaw Chyn 
Published: May 2015

Abstract
Metastasis is the major cause of mortality in cancer. Malignant tumors can secrete factors that enter the circulation and influence distant microenvironments to become pre-metastatic niches, which are more receptive to arrest and egress of circulating tumor cells. These factors can mobilize bone marrow-derived progenitor cells to the target organ, or act directly on the stromal cells in the target organ. The aim of this study is to examine whether tumor-secreted factors can condition macrophages to prime endothelial cells from preferred sites of metastasis to enhance tumor extravasation during metastasis.
MDA-MB-231 and MCF-7 were used as representative breast tumor cell lines. Normal macrophages and MDA-MB-231-conditioned macrophages were derived from freshly isolated CD14+ monocytes respectively treated with macrophage colony-stimulating factor and MDA-MB-231 culture supernatant. Phenotypes of the derived macrophages were confirmed by cytokine profiling and cell surface marker expression by ELISA and/or FACS. Endothelial cells from different organ sources were conditioned with culture supernatant harvested from the different macrophage types, and then utilized in in-vitro static binding and transwell assays to determine the endothelial monolayer permissiveness for breast tumor cells adhesion and subsequent transendothelial migration.
MDA-MB-231-secreted factors conditioned macrophages towards developing a M2 phenotype (CD68+/CD206+/CD163+). Compared to lung endothelium stimulated with culture supernatant from normal macrophages (MΦ), these conditioned M2-type macrophages (MDA-MΦ) then secreted factors that significantly enhanced the adhesiveness of lung endothelium towards MDA-MB-231 (2.7±0.6 fold greater than that of untreated lung endothelium, p&lt;0.05) and MCF-7 cells (1.7±0.2 fold greater than that of untreated lung endothelium, p&lt;0.05) and augmented the ability of these tumor cells to transmigrate across the endothelial barrier (MDA-MB-231: 1.2±0.9 fold greater than that of untreated lung endothelium, p&lt;0.05). Such enhanced interactions were not observed with endothelial cells from umbilical cord vein under similar conditions.
We found that the observed enhanced breast tumor cell adhesion involved the binding of heterodimer α5β1 integrins on the tumor cells to fibronectin expressed on the MDA-MΦ-primed lung endothelium. Increased cytoplasmic JNK, nuclear C-JUN and nuclear STAT3 expressions were present in the MDA-MΦ-primed lung endothelium as compared to MΦ-primed or untreated lung endothelium. We identified MDA-MΦ-derived interleukin 6 (IL-6) as the dominant mediator regulating fibronectin expression on the MDA-MΦ-primed lung endothelium surface.
We propose a novel two-step mechanism where breast tumor cells in the primary lesion can indirectly modulate the adhesiveness of the endothelium in the target organ for disseminated tumor cells. Our data suggest that soluble factors secreted by breast cancer cells can transform resident macrophages in target organs to become M2-like with pro-tumor properties; and these M2 macrophages in turn prime the metastatic site. Macrophage-derived IL-6 enhances fibronectin expression in the endothelial cells via the activation of JNK and STAT3 pathways. Fibronectin acts as the tumor binding receptor on the endothelial cells that facilitates the binding and extravasation of disseminated tumor cells into the lung parenchyma.
Citation Format: Pin Yan Low, Yaw Chyn Lim. Upregulation of fibronectin expression on endothelial cells by tumor-conditioned macrophages facilitates lung metastasis in breast cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B30. doi:10.1158/1538-7445.CHTME14-B30