Invasion of human breast cancer cells in vivo requires both paracrine and autocrine loops involving the colony-stimulating factor-1 receptor

Abstract

Colony-stimulating factor-1 (CSF-1) and its receptor (CSF-1R) have been implicated in the pathogenesis and progression of various types of cancer, including breast cancer. This is based on high levels of circulating CSF-1 in patient sera with aggressive disease and increased CSF-1R staining in the tumor tissues. However, there have been no direct in vivo studies to determine whether a CSF-1 autocrine signaling loop functions in human breast cancer cells in vivo and whether it contributes to invasion. Recently, in mouse and rat models, it has been shown that invasion and metastasis are driven by an epidermal growth factor (EGF)/CSF-1 paracrine loop between tumor cells and host macrophages. In this macrophage-dependent invasion, tumor cells secrete CSF-1 and sense EGF, whereas the macrophages secrete EGF and sense CSF-1. Here, we test the hypothesis that in human breast tumors, the expression of both the CSF-1 ligand and its receptor in tumor cells leads to a CSF-1/CSF-1R autocrine loop which contributes to the aggressive phenotype of human breast tumors. Using MDA-MB-231 cell-derived mammary tumors in severe combined immunodeficiency mice, we show here for the first time in vivo that invasion in a human mammary tumor model is dependent on both paracrine signaling with host macrophages as well as autocrine signaling involving the tumor cells themselves. In particular, we show that the autocrine contribution to invasion is specifically amplified in vivo through a tumor microenvironment-induced upregulation of CSF-1R expression via the transforming growth factor-beta1.

Figure 1. Expression of chemotactic factors and their receptors by the human breast tumor cell line MDA-MB-231

A. mRNA expression was analyzed by PCR of total cDNA made from MDA-MB-231 cells in culture. GAPDH expression is shown as a control. A similar analysis for the mRNA expression of the rat MTLn3 cells was previously published in reference . B. Protein expression of CSF-1 receptor was probed by western blot in MDA-MB-231 and MTLn3 cells. Control is a macrophage cell line forced to stably overexpress the human CSF-1R. C. MDA-MB-231 cells’ ability to invade in vitro into a collagen matrix was greatly enhanced in the presence of BAC1.2F5 macrophages. Bars: SEM, *: p<0.05.

Figure 2. MDA-MB-231 cells are invasive in vivo and macrophages are a subpopulation of the invasive cells

A. Invasive cells were collected from primary MDA-MB-231-derived mammary tumors in response to 25nM EGF using the in vivo invasion assay. As a control, some needles contained only matrigel and no additional EGF. B. Proportion of tumor cells and macrophages in the invasive subpopulation of the MDA-MB-231 tumors revealed by staining with cell-type specific antibodies. C. Typing of the invasive cells collected from primary MTLn3-derived mammary tumors. Bars: SEM, *: p<0.05.

Figure 3. Invasion in vivo is a result of both autocrine and paracrine loops involving the CSF- 1 receptor

A. Schematic of the postulated EGF/CSF-1 paracrine and autocrine interactions between human tumor cells and mouse macrophages inside the MDA-MB-231 xenograft. EGFR signaling can be blocked with the specific EGFR kinase inhibitor, Iressa. The species-specificity of the anti-CSF-1R antibodies was exploited in this experiment to block selectively either the autocrine or the paracrine loop with an anti-human or an anti-mouse CSF-1R specific neutralizing antibody, respectively. B. Invasive cells from MDA-MB-231 primary mammary tumors were collected in response to either EGF or CSF-1 with needles containing either the EGFR inhibitor, Iressa, or control DMSO. C. Invasive cells from MDA-MB-231 primary tumors were collected in response to EGF with needles containing the indicated CSF-1R blocking antibodies or control IgG. Bars: SEM, *: p<0.05.

Figure 4. Autocrine regulation via the CSF-1R is less important for tumor cell invasion in vitro than in vivo

A. MDA-MB-231 cells were tested in vitro for their ability to invade through a 3D collagen matrix in the presence and absence of macrophages. A schematic of the assay geometry and the EGF/CSF-1 paracrine and/or autocrine interactions between the two cell populations is shown. B. Quantification of the in vitro invasion of MDA-MB-231 human breast carcinoma cells (MDA) when plated alone or with BAC1.2F5 macrophages (BAC). The co-culture of tumor cells and macrophages was treated with either the EGFR-specific inhibitor, Iressa, or a DMSO control. C. Quantification of the in vitro invasion of MDA-MB-231 cells when co-plated with macrophages (BAC) and treated with either the human or the mouse CSF-1R blocking antibodies or control IgG. Bars: SEM, *: p<0.05.

Figure 5. Expression of CSF-1R mRNA is upregulated in MDA-MB-231 cells in the microenvironment of the primary tumor

A. mRNA expression was analyzed by real-time PCR in MDA-MB-231 cells from cell culture (MDA-231), and compared to tumor cells isolated from primary mammary tumors (the average primary tumor cells or APTC). Results here are plotted as fold- expression relative to the cultured MDA-MB-231 cells. B. MDA-MB-231 cells were stimulated in vitro with EGF, and total RNA was prepared and analyzed for mRNA expression levels of the same genes by real-time PCR. Results are plotted as fold-expression relative to the no EGF control condition. C. MDA-MB-231 cells were stimulated with CSF-1 and analyzed as in panel B. Results are plotted as fold-expression relative to the no CSF-1 control condition. Bars: SEM, *: p<0.05.

Figure 6. TGFβ1 induces CSF-1R expression and in vivo invasion of MDA-MB-231 cells

A. MDA-MB-231 cells were stimulated in vitro with TGFβ1 and total RNA was prepared and analyzed for mRNA expression levels by real-time PCR. Results are plotted as fold-expression relative to no TGFβ1 control condition. B. mRNA expression was analyzed by real-time PCR in MDA-MB-231 cells from cell culture (MDA-231) and tumor cells from xenografts treated with either DMSO or the TGFβ1 receptor specific inhibitor SB431542. Results are plotted as fold-expression relative to the cultured MDA-MB-231 cells. C. Invasive cells from MDA-MB-231 xenograft mice that were treated with either DMSO or the SB431542 inhibitor were collected in response to EGF with the in vivo invasion assay. Bars: SEM, *: p<0.05.