Metastasis is a multi-step process that requires increased motility of the tumor cells inside the primary tumor and invasion of surrounding tissues and blood vessels. The tumor microenvironment has an essential role in promoting these steps of motility and invasion in tumor cells, through either secretion of chemotactic factors or direct interactions with stromal cells. In rat and mouse mammary tumors, tumor- associated macrophages are essential for promoting angiogenesis, matrix remodeling and chemotactic motility of the tumor cells (1
). In particular, a paracrine interaction between macrophages and tumor cells, that involves epidermal growth factor (EGF) and colony stimulating factor 1 (CSF-1), is the driving force for relay chemotaxis supporting macrophage-mediated invasion in both transgenic mouse and rat mammary tumors. During relay chemotaxis, tumor cells secrete CSF-1 and sense EGF, while the macrophages secrete EGF and sense CSF-1. This phenomenon has been studied extensively both by intravital imaging in living animals as well as by reconstituting the interaction of the two cell types in vitro (2
). Additional studies support the importance of macrophages in invasion and metastasis of the tumor cells. Absence of CSF-1 in the mammary cancer-susceptible PyMT mice retarded tumor progression and metastasis but did not affect primary tumor development (5
), directly implicating macrophages and CSF-1 as important regulators of invasion and metastasis. In later work with xenogeneic tumors in mice, blockade of CSF-1 through antisense oligonucleotides or neutralizing anti-CSF-1 antibodies also reduced primary tumor growth and angiogenesis and prolonged long-term survival (6
In humans, patient sample data has suggested that CSF-1 and its receptor might play critical roles during progression of tumors of the female reproductive system and other solid tumors. Expression of the CSF-1 receptor (CSF-1R) has been associated with adverse clinicopathological prognostic outcome in ovarian, endometrial and breast carcinomas (8
). Expression of CSF-1R has also been detected in prostate cancer cells in tumors with elevated Gleason scores (11
). Interestingly, CSF-1 is also expressed in ovarian and endometrial tumors and cell lines (12
) and in breast cancer (10
) and CSF-1 and the CSF-1R are co-expressed in greater than 50% of mammary tumors (14
). In addition, increased circulating CSF-1 levels are a prognostic marker for epithelial ovarian cancer (15
) and are elevated in a large proportion of endometrial cancers (16
). Elevated circulating CSF-1 was also suggested to be an indicator of early metastatic relapse in breast cancer patients (13
). These observations have led to the hypothesis that there may be an autocrine loop involving tumor cells expressing both CSF-1 and CSF-1R, which contributes to invasion and metastasis in human tumors (14
). Several in vitro studies now support this hypothesis. Human lung cell lines and breast cell lines that express CSF-1R, but not the ligand, show increased invasion in vitro into an amniotic basement membrane upon stimulation with CSF-1 (18
). Similarly, when the normal non-invasive mouse mammary cell line HC11, that expresses CSF-1 but not the receptor, is forced to overexpress CSF-1R, it shows rapid growth and colony formation in soft agar, increased invasion through matrigel, and a higher incidence of lung tumors after tail vein injections into BALB/c mice, compared to the parental line (19
). Additionally, when CSF-1 and its receptor are both stably overexpressed in the MCF10A human mammary cell line, the cells exhibit abnormal acinar morphogenesis and increased motility in an in vitro wound-healing assay (20
These latter in vitro studies were performed with cells in which CSF-1 or its receptor, or both, were overexpressed, in order to mimic hypothesized autocrine signaling and they did not assess the contribution of the tumor microenvironment to the onset of autocrine signaling. Indeed, there have been no direct in vivo studies to determine whether a CSF-1 autocrine signaling loop is induced in the microenvironment of the primary tumor and if it contributes to invasion in human breast cancer. Moreover, the contribution of autocrine CSF-1 signaling to invasion cannot be investigated in the popular transgenic mouse models, because neither the mouse epithelial cells nor the mouse tumor cells express the CSF-1R. Epithelial expression of the CSF-1R transcript in human mammary epithelium is hormonally regulated via a glucocorticoid response element that is absent from the murine locus (21
). Therefore, additional models and studies are needed to test the hypothesis that CSF-1/CSF-1R autocrine signaling contributes to invasion of human breast cancer cells in vivo.
Based on the rodent models and the patient data described above, the CSF-1R could be involved in human breast tumor invasion in at least two ways. The rodent studies point to macrophage-assisted invasion involving an EGF/CSF-1 paracrine loop and the patient studies suggest that tumor cell invasion is regulated in an autocrine manner by CSF-1, provided the tumor cells express both CSF-1 and CSF-1R. In this study, we directly address these two possibilities in vivo.
We used the human breast tumor cell line MDA-MB-231 as a model for our analysis, because its triple-negative status (estrogen/progesterone receptor negative, HER2 negative) categorizes it in the basal-like subtype of breast cancers (22
). These are generally the most aggressive and highly metastatic breast tumors. As we show that MDA-MB-231 cells express both CSF-1 and the CSF-1R, this cell line, without further manipulation, is an appropriate one in which to study the role of autocrine signaling in aggressive breast tumors.