Elaine Y. Lin

A Paracrine Loop between Tumor Cells and Macrophages Is Required for Tumor Cell Migration in Mammary Tumors

Invasion of tumor cells into the surrounding connective tissue and blood vessels is a key step in the metastatic spread of breast tumors. Although the presence of macrophages in primary tumors is associated with increased metastatic potential, the mechanistic basis for this observation is unknown. Using a chemotaxis-based in vivo invasion assay and multiphoton-based intravital imaging, we show that the interaction between macrophages and tumor cells facilitates the migration of carcinoma cells in the primary tumor. Gradients of either epidermal growth factor (EGF) or colony-stimulating factor 1 (CSF-1) stimulate collection into microneedles of tumor cells and macrophages even though tumor cells express only EGF receptor and macrophages express only CSF-1 receptor. Intravital imaging shows that macrophages and tumor cells migrate toward microneedles containing either EGF or CSF-1. Inhibition of either CSF-1– or EGF-stimulated signaling reduces the migration of both cell types. This work provides the first direct evidence for a synergistic interaction between macrophages and tumor cells during cell migration in vivo and indicates a mechanism for how macrophages may contribute to metastasis.

Macrophages Regulate the Angiogenic Switch in a Mouse Model of Breast Cancer

The development of a tumor vasculature or access to the host vasculature is a crucial step for the survival and metastasis of malignant tumors. Although therapeutic strategies attempting to inhibit this step during tumor development are being developed, the biological regulation of this process is still largely unknown. Using a transgenic mouse susceptible to mammary cancer, PyMT mice, we have characterized the development of the vasculature in mammary tumors during their progression to malignancy. We show that the onset of the angiogenic switch, identified as the formation of a high-density vessel network, is closely associated with the transition to malignancy. More importantly, both the angiogenic switch and the progression to malignancy are regulated by infiltrated macrophages in the primary mammary tumors. Inhibition of the macrophage infiltration into the tumor delayed the angiogenic switch and malignant transition whereas genetic restoration of the macrophage population specifically in these tumors rescued the vessel phenotype. Furthermore, premature induction of macrophage infiltration into premalignant lesions promoted an early onset of the angiogenic switch independent of tumor progression. Taken together, this study shows that tumor-associated macrophages play a key role in promoting tumor angiogenesis, an essential step in the tumor progression to malignancy.

Direct visualization of macrophage assisted tumor cell intravasation in mammary tumors

Although the presence of macrophages in tumors has been correlated with poor prognosis, until now there was no direct observation of how macrophages are involved in hematogenous metastasis. In this study, we use multiphoton microscopy to show, for the first time, that tumor cell intravasation occurs in association with perivascular macrophages in mammary tumors. Furthermore, we show that perivascular macrophages of the mammary tumor are associated with tumor cell intravasation in the absence of local angiogenesis. These results show that the interaction between macrophages and tumor cells lying in close proximity defines a microenvironment that is directly involved in the intravasation of cancer cells in mammary tumors.

Progression to Malignancy in the Polyoma Middle T Oncoprotein Mouse Breast Cancer Model Provides a Reliable Model for Human Diseases

Animal models are powerful tools to analyze the mechanism of the induction of human breast cancer. Here we report a detailed analysis of mammary tumor progression in one mouse model of breast cancer caused by expression of the polyoma middle T oncoprotein (PyMT) in the mammary epithelium, and its comparison to human breast tumors. In PyMT mice, four distinctly identifiable stages of tumor progression from premalignant to malignant stages occur in a single primary tumor focus and this malignant transition is followed by a high frequency of distant metastasis. These stages are comparable to human breast diseases classified as benign or in situ proliferative lesions to invasive carcinomas. In addition to the morphological similarities with human breast cancer, the expression of biomarkers in PyMT-induced tumors is also consistent with those associated with poor outcome in humans. These include a loss of estrogen and progesterone receptors as well as integrin-beta1 expression and the persistent expression of ErbB2/Neu and cyclinD1 in PyMT-induced tumors as they progress to the malignant stage. An increased leukocytic infiltration was also closely associated with the malignant transition. This study demonstrates that the PyMT mouse model is an excellent one to understand the biology of tumor progression in humans.

Tumor-associated macrophages press the angiogenic switch in breast cancer.

The development of a supportive vasculature is essential for tumor progression. In a mouse model of breast cancer, we found that tumor-associated macrophages that are recruited to the tumor just before malignant conversion are essential for the angiogenic switch. These findings establish a causal linkage to explain well-documented clinical correlations between macrophages, microvessel density, and poor prognosis in breast tumors.

The macrophage growth factor CSF-1 in mammary gland development and tumor progression.

Colony stimulating factor 1 (CSF-1), a major regulator of the mononuclear phagocytic lineage, is expressed in more than 70% of human breast cancers and its expression is correlated with poor prognosis. Studies of CSF-1 null mutant mice demonstrated that CSF-1 plays an important role in normal mammary ductal development as well as in mammary tumor progression to metastasis. CSF-1 regulates these processes through the recruitment and regulation of macrophages, cells that become associated with mammary tumors and the terminal end buds at the end of the growing ducts. This phenomenon suggests that the tumors subvert normal developmental processes to allow invasion into the surrounding stroma, a process that gives the tumor access to the vasculature and consequently the promotion of metastasis. In addition, soluble CSF-1 secreted from the tumor acts to divert antitumor macrophage responses and suppresses the differentiation of mature tumor-antigen-presenting dendritic cells. This review discusses these observations in detail and attempts to fit them into a larger picture of CSF-1 and macrophage action in the regulation of normal mammary gland development and tumor progression.

Requirement of macrophages and eosinophils and their cytokines/chemokines for mammary gland development.

Epithelial/mesenchymal cell interactions are necessary for proper ductal morphogenesis throughout all stages of mammary gland development. Besides the well-established stromal components, such as adipocytes and fibroblasts, the mammary stroma is also infiltrated with migrating blood cells, mostly macrophages and eosinophils. The focus of this review is on the role of macrophages and their growth factor colony-stimulating factor 1 (CSF-1) in promoting branching morphogenesis during postnatal mammary gland development through to lactation. The more restricted role of eosinophils and their chemoattractant eotaxin during pubertal ductal morphogenesis is also discussed. A possible interaction between macrophages and eosinophils in ductal morphogenesis is considered, along with the roles of other chemokines. This role of macrophages in normal development also appears to be subverted by tumors of the mammary gland to promote the escape of the tumor cells from the local environment and enhance their rate of metastasis. These data emphasize the dual role of macrophages in the promotion of epithelial growth in normal and cancer states.

A Novel Mouse Model of Inflammatory Bowel Disease Links Mammalian Target of Rapamycin-Dependent Hyperproliferation of Colonic Epithelium to Inflammation-Associated Tumorigenesis

Inflammatory bowel disease (IBD) is a high-risk condition for human colorectal cancer. However, our mechanistic understanding of the link between inflammation and tumorigenesis in the colon is limited. Here we established a novel mouse model of colitis-associated cancer by genetically inactivating signal transducer and activator of transcription 3 (Stat3) in macrophages, with partial deletion in other myeloid and lymphoid cells. Inflammation developed in the colon of mutant mice spontaneously, and tumor lesions, including invasive carcinoma, arose in the inflamed region of the intestine with a frequency similar to that observed in human IBD patients. The development of both inflammation and tumors in the mutant mice required the presence of microflora. Indeed, inflammation was associated with disruption of colonic homeostasis, fulminant epithelial/tumor cell proliferation, and activation of the mammalian target of rapamycin (mTOR)-Stat3 pathway in epithelial and tumor cells. The activation of this pathway was essential for both the excess proliferation of epithelial/tumor cells and the disruption of colonic homeostasis in the mutant mice. Notably, a similar abnormal up-regulation of mTOR-Stat3 signaling was consistently observed in the colonic epithelial cells of human IBD patients with active disease. These studies demonstrate a novel mouse model of IBD-colorectal cancer progression in which disrupted immune regulation, mTOR-Stat3 signaling, and epithelial hyperproliferation are integrated and simultaneously linked to the development of malignancy.

Vascular endothelial growth factor restores delayed tumor progression in tumors depleted of macrophages

Genetic depletion of macrophages in Polyoma Middle T oncoprotein (PyMT)‐induced mammary tumors in mice delayed the angiogenic switch and the progression to malignancy. To determine whether vascular endothelial growth factor A (VEGF‐A) produced by tumor‐associated macrophages regulated the onset of the angiogenic switch, a genetic approach was used to restore expression of VEGF‐A into tumors at the benign stages. This stimulated formation of a high‐density vessel network and in macrophage‐depleted mice, was followed by accelerated tumor progression. The expression of VEGF‐A led to a massive infiltration into the tumor of leukocytes that were mostly macrophages. This study suggests that macrophage‐produced VEGF regulates malignant progression through stimulating tumor angiogenesis, leukocytic infiltration and tumor cell invasion.

Positron emission tomography agent 2-deoxy-2-[18F]fluoro-D-glucose has a therapeutic potential in breast cancer.

BackgroundNovel approaches are needed for breast cancer patients in whom standard therapy is not effective. 2-Deoxy-2-[18F]fluoro-D-glucose (18F-FDG) was evaluated as a potential radiomolecular therapy agent in breast cancer animal models and, retrospectively, in patients with metastatic breast cancer.MethodsPolyoma middle T antigen (PyMT) and mouse mammary tumor virus-NeuT transgenic mice with tumors 0.5–1 cm in diameter were imaged with 18F-FDG, and tumor to liver ratios (TLRs) were calculated. The radiotoxicity of 18F-FDG administration was determined in healthy mice. PyMT mice with small (0.15–0.17 cm) and large (more than 1 cm) tumors were treated with 2–4 mCi of 18F-FDG, and control C3H/B6 mice with 3 mCi of 18F-FDG. At 10 days after treatment the tumors and control mammary glands were analyzed for the presence of apoptotic and necrotic cells. Five patients with breast cancer and metastatic disease were evaluated and standardized uptake values (SUVs) in tumors, maximum tolerated dose, and the doses to the tumor were calculated.ResultsDoses up to 5 mCi proved to be non-radiotoxic to normal organs. The 18F-FDG uptake in mouse tumors showed an average TLR of 1.6. The treatment of mice resulted in apoptotic cell death in the small tumors. Cell death through the necrotic pathway was seen in large tumors, and was accompanied by tumor fragmentation and infiltration with leukocytes. Normal mammary tissues were not damaged. A human 18F-FDG dose delivering 200 rad to the red marrow (less than 5% damage) was calculated to be 4.76 Ci for a 70 kg woman, and the dose to the tumors was calculated to be 220, 1100 and 2200 rad for SUVs of 1, 5 and 10, respectively.ConclusionWe have shown that positrons delivered by 18F-FDG to mammary tumors have a tumoricidal effect on cancer cells. The study of breast cancer patients suggests that the tumor and normal organ dosimetry of 18F-FDG makes it suitable for therapy of this malignancy.