The transition from the normal to a malignant state is a multistep process involving genetic and epigenetic changes. Alterations in cellular regulatory circuits have been identified during this progression 28
. Many tumor cells produce growth factors and/or their receptors, such as platelet-derived and vascular endothelial growth factor and TGF-α produced by glioblastomas and sarcomas 2930
, fibroblast growth factor 2 by melanomas 31
, and epidermal growth factor (EGF) receptor or HER-2/neu overexpressed in breast carcinomas 32
. Signaling pathways that are involved in the transduction of mitogenic stimuli, such as the mitogen-activated protein (MAP) kinase and the phosphatidylinositol (PI)-3 kinase pathways, are often constitutively activated in tumor cells 3334
, while upregulated expression of antiapoptotic genes also occurs frequently 35
. All of these alterations enable tumor cells to elude the commitment to terminal differentiation and quiescence that normally regulate tissue homeostasis. However, little is known about how these cells exhibiting uncontrolled proliferation or increased life span become malignant and able to metastasize to a distant organ, a feature that differentiates them from benign tumor cells. Many genes specifically expressed in malignant tumors have been identified. However, only a very few of these have been shown to influence the acquisition of invasiveness and metastatic potential 36
. In this paper we demonstrate that CSF-1 is one such regulatory factor that has no effect on mammary tumor initiation and growth but promotes tumor progression to malignancy.
Clinically, overexpression of CSF-1 and CSF-1R has been found in a large percentage of breast cancers where expression is correlated with poor prognosis 6
. In these tumors, CSF-1R was expressed in both tumor cells and infiltrating macrophages 8
. A marked leukocytic infiltration in these tumors also correlated with poor prognosis with the majority of these cells being macrophages 837
. CSF-1 is the major growth factor for the mononuclear phagocytic lineage and is a chemoattractant for these cells 538
. Consequently we proposed that CSF-1 might not only act as an autocrine factor for tumor cells but also to recruit macrophages to the tumor site where they promote the progression of the tumor 39
To test whether CSF-1 has a causal role in mammary tumorigenesis, we established a mouse model whereby a tumor-prone mouse strain was made homozygous or heterozygous for a recessive null mutation in the CSF-1 gene, and the effect of the absence of CSF-1 on tumor progression analyzed. This model more likely mimics the situation in humans, as the tumor develops within the natural mammary environment and all of the factors involved in tumor progression and metastasis are present in the environment. In contrast, previous studies have mainly focused on direct effects of CSF-1 on cultured tumor cells. In these studies, or in tumor cells transplanted into immuocompromised mice models, both positive and negative effects of CSF-1 have been reported 40414243
. However, our data clearly indicates that the absence of CSF-1, although not affecting the incidence or growth of the primary tumor, dramatically reduced its progression to malignancy as assessed by tumor morphology and lung metastasis. A caveat to this interpretation is that the necessary breeding results in a mixing of genetic backgrounds that could potentially expose a modifier gene that might affect the rate of tumor progression. However, our closed colony was randomly bred to ensure that independently segregating genes would be randomly assorted to each genotype. Thus, although modifier genes might increase the data variance, they would not affect the final conclusion, as this breeding strategy ensures that such genes would not be enriched in a particular genotype. Furthermore, restoration of CSF-1 specifically in the mammary gland but not elsewhere except the salivary gland, accelerated mammary tumor progression and increased metastasis. This treatment did not restore serum CSF-1 concentrations in the null mutant mice, or rescue other phenotypes including the extended estrous cycles, osteopetrosis, nor tissue populations of macrophages other than in the mammary gland (unpublished observations). Furthermore, overexpression of CSF-1 in +/Csf1op
mice also accelerated mammary gland progression to the metastatic state despite the normal CSF-1 serum and tissue concentrations in these mice. These data argues strongly that the effects of CSF-1 on tumor progression are not secondary to some other phenotype in the mutant mice but are directly due to a requirement in the tumor. They also argue strongly that CSF-1 will have a causal role in tumor progression in humans and that this explains the correlation between CSF-1 overexpression and poor prognosis.
In +/Csf1op PyMT mice, a transition point of the primary mammary tumor to malignancy was identified at 10 wk of age when there was a significant increase in the number of mice that developed late carcinomas, followed by pulmonary metastases at 18 wk of age. This transition to carcinoma was preceded by recruitment of abundant macrophages to the tumor. In contrast, in CSF-1 null mutant PyMT mice, macrophage infiltration at the tumor site was not observed and both malignant transition and pulmonary metastasis were markedly delayed. In addition, increased macrophage density at the tumor site was associated with accelerated tumor progression and metastasis in both Csf1op/Csf1opand +/Csf1op PyMT mice expressing the CSF-1 Tg in the mammary gland. In this mouse model, in contrast to human tumors, CSF-1R expression could only be detected in macrophages and not in the tumor cells. Although it cannot be completely ruled out that tumor cells could also express CSF-1R mRNA at a level below detection in our experiments, our data strongly argues that macrophages are the only target for CSF-1 action. In humans, CSF-1 is also expressed in the tumor cells. However, in mice, analysis of CSF-1 transcripts in the mammary gland and their tumors showed a low level of expression that was not enriched as the tumors grew (data not shown). This suggests that the lack of macrophages in the tumors of Csf1op/Csf1opmice is due primarily to the systemic loss of CSF-1 leading to few circulating monocytes, and that in mice there are chemoattractants other than CSF-1 which recruit macrophages to the tumor site. Nevertheless, as CSF-1 is found in serum and mammary tissue under normal circumstance, these macrophages will be exposed to CSF-1.
Overall, our data suggests a role for infiltrated macrophages in the progression to malignancy. The biological basis of this is unknown. However, we observed high densities of macrophages at sites where the basement membrane of the acini had lost its integrity. Macrophages are potent producers of proteases and, at least, plasminogen activator is CSF-1–regulated in these cells 44
. Macrophages also produce many angiogenic factors, including thymidine phosphorylase and vascular endothelial–derived growth factor (VEGF), suggesting that these could also promote this essential process for metastasis 45
. Macrophages are also major producers of growth factors 46
such as epidermal growth factor that could act as a paracrine factor on tumor cells promoting their migration and invasion through the acinar basement membrane and into blood vessels 15
. However, given the extraordinary range of product that macrophages can synthesize, it seems likely that macrophages play multifunctional roles in the progression of mammary tumors.
The dramatic reduction in tumor progression and metastasis in the absence of CSF-1 represents the first occasion in which histological progression and metastasis have been coordinately modulated in the PyMT breast cancer model. Pulmonary metastasis was found to be reduced in Mgat5-deficient PyMT mice. However, the effect on metastasis could be attributed to an inhibitory effect of Mgat5 deficiency on the growth of the primary tumor 47
. Modified tumor organization and increased infiltration of monocytes/macrophages at the tumor site were observed in tenascin-C–deficient PyMT mice; however, neither metastasis nor primary tumor progression were altered in the mice 48
. Suppression of pulmonary metastasis was observed in plasminogen-deficient PyMT transgenic mice in the absence of effect on the growth of primary mammary tumor 49
. However, no histological difference was detected between tumors in plasminogen-deficient and wild-type mice: all tumors presented as well differentiated adenocarcinomas 49
. The age range (10–20 wk) of mice in the latter study was similar to that in the current report, suggesting that the histological defect observed in Csf1op/Csf1op
PyMT mice is a specific feature of CSF-1 deficiency and may reflect CSF-1–dependent steps that are antecedent to those affected by plasminogen.
In conclusion, this study has identified CSF-1 as an important regulatory factor in mammary tumor progression to metastasis, possibly acting through macrophages. These data in mice have provided a causal explanation for the correlation between poor prognosis in breast cancer patients with CSF-1 overexpression and leukocytic invasion into the tumor. Consequently, therapeutics targeted at CSF-1 or the CSF-1R could potentially benefit patients.