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Metastatic spread of cancer cells is the main cause of death of breast cancer patients, and elucidation of the molecular mechanisms underlying this process is a major focus in cancer research. The identification of appropriate therapeutic targets and proof-of-concept experimentation involves an increasing number of experimental mouse models, including spontaneous and chemically induced carcinogenesis, tumor transplantation, and transgenic and/or knockout mice. Here we give a progress report on how mouse models have contributed to our understanding of the molecular processes underlying breast cancer metastasis and on how such experimentation can open new avenues to the development of innovative cancer therapy.
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Breast cancer is the most frequently diagnosed form of cancer and the second leading cause of death in Western women [1]. Death, and most of the complications associated with breast cancer, are due to metastasis developing in regional lymph nodes and in distant organs, including bone, lung, liver, and brain [1, 2]. As in many other metastatic cancer types, specific molecular changes occurring within both the tumor cells and the tumor microenvironment contribute to the detachment of tumor cells from the primary tumor mass, invasion into the tumor stroma, intravasation into nearby blood vessels or lymphatics, survival in the bloodstream, extravasation into and colonization of the target organ and, finally, metastatic outgrowth [3, 4].
In the recent past, our understanding of breast cancer progression and metastasis has greatly profited from the use of genetically modified mouse models and advanced transplantation techniques. Here we describe the currently employed mouse models of breast cancer metastasis and how their use has contributed significantly to our understanding of the molecular processes underlying breast cancer metastasis.
Mechanisms contributing to breast cancer metastasis
A critical step towards the generation of mouse models of breast cancer is the understanding of the molecular pathways underlying mammary carcinogenesis. Our knowledge on how breast tumor progression occurs has also been markedly improved by unraveling the dynamics and the key factors of mammary gland development.
Mammary gland development
Mouse breast tissue undergoes continuous changes throughout the lifespan of reproductively active females, mediated mainly by interactions between the mammary epithelium and the surrounding mesenchyme (Figure 1). The mammary bud develops by forming a network of branched ducts invading into the mammary fat pad [5]. With the release of ovarian hormones, terminal end buds are formed. They represent the invading front of the ducts and they are able to proliferate, to extend into the fat pad, and to form branches. During pregnancy and lactation, hormone-induced terminal differentiation of the mammary epithelium into milk-secreting lobular alveoli takes place. After weaning, the secretory epithelium of the mammary gland involutes into an adult nulliparous-like state by apoptosis and redifferentiation. During these processes, the developing mammary gland has the ability to induce angiogenesis to adjust for blood supply and is protected against premature involution; it is therefore resistant to apoptosis [6]. Interestingly, proliferation, invasion, angio-genesis, and resistance to apoptosis are all features that are abused during the etiology of breast carcinogenesis.
Transformation and metastasis
Mammary gland morphogenesis and branching involve the regulatory function of several signaling pathways, including signaling by Wnt family members [7], transforming growth factor-β (TGF-β) [8], insulin-like growth factor-I (IGF-I) [9], and epidermal growth factor (EGF) and others [10]. These pathways are frequently activated during the tumorigenic process by mutation or gene amplification, thus allowing the mammary epithelium to expand, proliferate, and invade neighboring tissue. The cross-talk and interactions between tumor cells and the surrounding stroma, the extracellular matrix (ECM), and infiltrating cells of the immune system are constantly modulating tumor development. The mammary stroma, composed of pre-adipocytes, adipocytes, fibroblasts, endo-thelial cells, and inflammatory cells, contributes functionally to mammary gland development [6]. In a similar manner, tumor–stroma interactions, occurring via soluble growth factors, cytokines and chemokines, remodeling of the extra-cellular matrix, or direct cell–cell adhesion, are critical for tumor growth, migration, and metastasis. Alteration of the expression or function of adhesion molecules responsible for the adhesion of breast cancer cells to themselves, to stromal cells, or to tumor matrix, including integrin family members, immunoglobulin-domain cell adhesion molecules (such as L1 and NCAM), cadherin family members, or other cell surface receptors (such as CD44), contributes predominantly to late-stage tumor progression and metastatic dissemination of cancer cells [11, 12].
The formation of new blood vessels (angiogenesis) is crucial for the growth and persistence of primary solid tumors and their metastases, and it has been assumed that angiogenesis is also required for metastatic dissemination, because an increase in vascular density will allow easier access of tumor cells to the circulation. Induction of angiogenesis precedes the formation of malignant tumors, and increased vascularization seems to correlate with the invasive properties of tumors and thus with the malignant tumor phenotype [13]. In fact, angiogenesis indicates poor prognosis and increased risk of metastasis in many cancer types, including breast cancer [14]. With the recent identification of lymphangiogenic factors and their receptors it has also been possible to investigate the causal role of lymphangiogenesis in the metastatic process (reviewed in [15]). It is therefore not surprising that molecules essential for mammary gland development, many of them stromal factors, are also critical participants in breast carcinogenesis.
The knowledge gained on the several mechanisms contributing to tumor progression can be used to design and generate better mouse models. At the same time, such models allow a thorough investigation of all different aspects of multistage breast carcinogenesis, including the genetic alterations leading to tumor onset, neovascularization, tumor progression, and formation of metastasis in secondary organs.
Breast cancer metastasis models
Tumor transplantation
There are various ways to mimic breast cancer growth and metastasis in tumor transplantation experiments. The site of injection, together with the specific tropism of the chosen breast cancer cell line used, largely defines primary and secondary metastatic growth. Orthotopic or ectotopic implantation of cancer cells in the skin or mammary fat pad, with the formation of primary tumors and the subsequent formation of metastasis, in part resembles the multiple stages involved in malignant breast cancer development in patients [16]. In contrast, tail vein injection results mainly in lung metastasis, whereas portal vein injection provokes colonization of the liver, and intracardiac infusion gives rise to a broader target organ spectrum, including bone. Notably, the direct introduction of cancer cells into the blood circulation should be considered an assay of organ colonization and not a true metastatic process.
Depending on the species or genetic background of donor and host, syngeneic or xenograft tumor transplantations need to be distinguished. Transplantation of cancer cells from one mouse into another mouse with identical genetic backgrounds (syngeneic transplantation) bypasses the immunologic host-versus-graft reaction and concomitantly allows the investigation of the contribution of an intact immune system to malignant tumor progression [17, 18]. Syngeneic mouse models have been employed to establish organ-specific metastasis models by several rounds of transplantation/metastasis formation and the selection of metastatic cell lines in vivo [19]. For example, 4T1 cells, which originally derive from a spontaneous mouse mammary tumor of a BALB/C mouse, grow rapidly when injected into the fat pad of a syngeneic animal and metastasize to lungs, liver, bone, and brain [19, 20]. Sublines of 4T1 cells, which exhibit various degrees of metastatic dissemination, have been employed recently to generate distinct gene expression signatures for each stage of tumor progression, namely primary tumor formation, lymph node colonization, metastatic outgrowth in the lymph node, and distant organ metastasis. These experiments led to the identification of the transcriptional repressor Twist, some members of the cadherin family of cell–cell adhesion molecules, and various chemokines as critical factors in the distinct stages of metastatic tumor progression [20]. This and other syngeneic mouse models have also been successfully employed for the testing of experimental drugs designed to interfere with tumor malignancy [18, 21].
To investigate the growth and metastasis of human breast cancer cell lines in vivo, xenograft transplantation experiments are performed in immunocompromised mice [22]. Human breast cancer cells can be injected subcutaneously, intravenously, intracardially, or orthotopically into the fat pad of the mouse [23]. For example, MDA-MB-231 cells, an estrogen-independent breast cancer cell line derived from the pleural effusion of a cancer patient, is able to colonize bone, liver, lung, adrenal glands, ovary, and brain after intravenous injection [24]. This cell line and organ-specific metastatic variants thereof have recently been used to identify and functionally implicate a number of genes in organ-specific metastasis, including IL-11, osteopontin and the connective tissue growth factor (CTGF) in osteolytic metastasis [25, 26], and epiregulin, CXCL1, matrix metalloproteinase-1 (MMP-1), cyclo-oxygenase-2 (COX-2), inhibitor of differentiation-1 (Id1) and others in lung metastasis [27] (see below).
The implantation of established cell lines derived from human breast cancer is relatively simple and allows the genetic or pharmacological manipulation of the implanted cells. However, there are clear limitations to xenograft models. First, immune responses, which have a key role during tumor development, are impaired in immunocompromised mice. Second, stromal components are not of tumor origin. For example, carcinoma-associated fibroblasts derived from a breast cancer patient support the growth of a breast carcinoma cell line better than the normal tissue in a xenograft mouse co-implantation model. Carcinoma-associated fibroblasts seem to activate and sustain CXCR4/stromal cell-derived factor (SDF-1)-mediated chemokine signaling and to recruit endothelial progenitors to the growing tumor, thereby promoting angiogenesis [28, 29]. Last, human cells are apparently not fully adapted to grow in a murine environment. For example, breast cancer metastasis to bone has recently been investigated in an experimental mouse system in which both the breast cancer cells and the metastatic target organ, the bone, are of human origin [30]. After orthotopic injection, cancer cells predominantly colonize the bone of human origin, thus exhibiting a species-specific osteotropism.
Genetically modified mice
Several promoters can be used to drive the expression of transgenes in the mammary epithelium (Table 1), and many known oncogenes have been expressed under their control to initiate or modulate breast carcinogenesis in mice, including ErbB2/Neu, polyoma middle T antigen (PyMT), simian virus 40 (SV40) T antigen, Ha-Ras, Wnt-1, TGF-α, and c-Myc. MMTV-Neu and MMTV-PyMT transgenic mice (in which the expression of the oncogene is driven by the Mouse Mammary Tumor Virus promoter) develop metastasis in lung and lymph nodes, mainly after their first pregnancy, while other transgenic mice have to be combined to generate double-transgenic mice that efficiently develop malignant cancers [31–35]. C3(1)-SV40 T-antigen transgenic mice develop invasive mammary carcinomas independently of hormone supplementation or pregnancy, with a 15% incidence of lung metastasis. This model recapitulates the loss of estrogen receptor-α expression that is frequently observed in human breast cancer [36]. The most commonly used transgenic mouse models that develop metastatic mammary cancer are summarized in Table 2.
Investigating the functional role of distinct genes during the multiple stages of breast carcinogenesis requires the ability to modulate their function in time and space [37]. Inducible transgene expression can be obtained by the use of the bacteria-derived tetracycline-inducible system permitting the switching on or off (Tet-On/Tet-Off system) of a gene of interest in a tissue- and time-specific manner [38]. In contrast, mice are modified by the genetic ablation of a gene of interest in an inducible manner to generate conditional knockouts with the use of the Cre/loxP phage recombinase system, for example [39]. To ablate a gene at a certain time point in mammary epithelial cells, recombinase activity can be controlled by the expression of a tamoxifen-inducible version of Cre (MMTV-ER™-Cre) or by using the tetracycline-inducible system to drive Cre expression [40].
First comparisons of gene expression profiles obtained from mammary gland tumor models initiated by different oncogenes have revealed several common and oncogene-specific targets and similarities with human molecular breast cancer pathology [41]. The challenge now is to test whether genes identified in gene expression profiling experiments with patient samples are able to modulate breast carcinogenesis in transgenic mouse models, for example in the well-characterized MMTV-Neu and MMTV-PyMT mouse models of breast carcinogenesis or in improved versions of these.
Amplification of the gene encoding ErbB2, a member of the EGF receptor gene family, is associated with 15 to 20% of human breast cancers, and in about 30% of cases the increased expression of an activated form of ErbB2 is detected. Consistent with this notion is the observation that transgenic expression of an activated form of the rat homolog of ErbB2 (Neu) in MMTV-Neu transgenic mice results in the development of multifocal adenocarcinomas with lung metastases at about 15 weeks after pregnancy [42]. Transgenic expression of wild-type ErbB2 in mammary gland also provokes tumor formation and metastatic dissemination, yet with longer latency.
Doxycycline-inducible expression of ErbB2 in mammary epithelial cells of transgenic mice also results in invasive mammary carcinoma and extensive metastasis, yet the tumors regress with the loss of ErbB2 expression upon the withdrawal of doxycycline. However, most mice exhibit recurrences of the tumors [43]. These recurrent tumors exhibit epithelial-mesenchymal transition (EMT), which seems to be mediated by the upregulated expression of the transcriptional repressor Snail, a molecular process that seems to have a high prognostic value in predicting human breast cancer recurrence. Expression of oncogenic versions of ErbB2 that bind either Grb-2 or Shc demonstrate that focal mammary tumors with a high rate of lung metastasis require Grb-2-mediated signaling, whereas low metastatic multifocal mammary tumors rely on Shc function [44].
Mammary gland-specific expression of PyMT under the control of the MMTV promoter/enhancer in transgenic mice (MMTV-PyMT) results in widespread transformation of the mammary epithelium and in the development of multifocal mammary adenocarcinomas and metastatic lesions in the lymph nodes and in the lungs [45]. Tumor formation and progression in these mice is characterized by four stages: hyperplasia, adenoma/mammary intra-epithelial neoplasia, and early and late carcinoma [46]. The close similarity of this model to human breast cancer is also exemplified by the fact that in these mice a gradual loss of steroid hormone receptors (estrogen and progesterone) and β1-integrin is associated with overexpression of ErbB2 and cyclin D1 in late-stage metastatic cancer [47]. The MMTV-PyMT mouse model of breast cancer is furthermore characterized by short latency, high penetrance, and a high incidence of lung metastasis occurring independently of pregnancy and with a reproducible kinetics of progression.
In MMTV-PyMT transgenic mice, increased metastatic potential has been shown to depend on the presence of macrophages in primary tumors and on the establishment of a chemoattractant paracrine loop of colony-stimulating factor-1 (CSF-1) and EGF ligands between macrophages and tumor cells [48, 49]. In MMTV-PyMT/CSF-1-/- mice, tumor progression and metastasis are significantly delayed but restored on the overexpression of CSF-1 in the mammary gland [48, 50]. The crucial role of macrophages in sustaining tumor progression was further shown by depletion of plasminogen, a downstream effector of CSF-1, either by its genetic ablation or by affecting the expression of its activator uPA, resulting in significantly reduced metastasis in the MMTV-PyMT mouse model without affecting primary tumor growth [51, 52]. The uPA/plasminogen system may contribute to metastasis mainly by ECM degradation. The relevance of this mechanism is further supported by experiments with MEKK1-deficient MMTV-PyMT mice, which show a significant delay in lung metastasis, whereas no differences are observed in the primary tumor growth. MEKK1 signaling is involved in cell adhesion and controls uPA induction. Accordingly, MEKK1-deficient mice display decreased levels of uPA, which result in reduced levels of activated plasminogen and impaired tumor cell migration and invasiveness [53].
The role of adhesion molecules during mammary gland tumor progression has also been addressed with the use of MMTV-PyMT mice. Specifically, loss of CD44 promotes lung metastasis in these mice, highlighting the role of tumor–stroma interaction for adhesion and invasion [12]. CD44 expression on tumor cells mediates their interaction with hyaluronan-expressing stromal cells and results in increased cancer progression. Loss of another adhesion molecule, Muc-1, in the MMTV-Wnt1 tumor model results in a delayed onset of tumorigenesis as well as delayed metastasis to lungs. Muc-1 seems to form complexes with β-catenin at the cell membrane and in the cytoplasm of cells at the tumor's invading front [54].
Recent results indicate that changes in cell adhesion have a critical function in tumor progression [11]. For example, the epithelial adherens junction molecule E-cadherin is considered a tumor and invasion suppressor. Forced expression of E-cadherin prevents tumor cell migration and invasion, whereas inhibition of E-cadherin function enhances tumor cell invasion and metastatic dissemination. E-cadherin is irreversibly lost in more than 85% of invasive lobular breast cancer associated with an invasive phenotype, and in the remaining 15% the retention of E-cadherin is associated with dysfunctional adhesion. Interestingly, a transgenic mouse model of epithelial loss of both E-cadherin and p53 develops metastatic mammary carcinoma resembling human invasive lobular breast cancer (J. Jonkers, personal communication). Taken together, these examples indicate that transgenic mouse models of breast cancer metastasis are essential to understanding the role of several molecules in modulating key steps during malignant progression.
In vivoimaging
Non-invasive in vivo imaging techniques have been developed to reveal metastatic mammary tumors in experimental systems. Cell lines and transgenic mice can be engineered to express luminescent or fluorescent markers, permitting the visualization of primary tumor growth and the formation of metastatic nodes in live animals over time. MMTV-enhanced green fluorescent protein (eGFP) mice or mice in which expression of eGFP or luciferase marker genes is 'switched on' in the mammary gland in a Cre-dependent way upon crossing with either WAP-Cre or MMTV-Cre mice have been generated [55–57]. Tumor growth and metastasis formation can be easily monitored in composite transgenic animals after crossing of these mice with breast cancer mouse models [58]. Moreover, tumor progression and the actual metastatic mobility of tumor cells can be detected in live animals by multiphoton microscopy, positron-enhanced tomography scans, and magnetic resonance analysis [59–61]. Furthermore, the newest technologies, including intravital microscopy [62, 63], in vivo flow cytometry [64], and multicolor fluorescent-based approaches, provide the possibility of quantitatively detecting individual tumor cells in living animals and documenting their clearance, motility, and migration to or retention in target organs.
Molecular pathways dissected using breast cancer mouse models
Transforming growth factor-β
TGF-β exerts a dual role during tumor progression: by inducing the expression of cell cycle inhibitors, it acts as a tumor suppressor during the initial phases of tumor progression. Yet it promotes metastasis and invasion in the later stages by inducing EMT [8]. The role of TGF-β in breast cancer metastasis is still under investigation. One of its major functions, beside the induction of EMT, is inducing the migration and intravasation of breast cancer cells into the circulation, thereby promoting osteolytic metastasis [65]. Expression of TGF-β1 in double-transgenic MMTV-Neu/MMTV-TGF-β1 mice increased the number of cancer cells circulating in the blood as well as the lung metastases, whereas primary tumors developed at unchanged frequency [66, 67]. Inducible expression of TGF-β1 in mammary glands of MMTV-PyMT transgenic mice also demonstrated the pro-metastatic function of TGF-β1 [68]. Transgenic mice expressing TGF-βRI or a dominant-negative version of TGF-βRII under the control of the MMTV promoter crossed with MMTV-Neu mice promoted and repressed, respectively, tumor metastasis [44]. Surprisingly, conditional knockout of TGF-βRII in the mammary epithelium of the MMTV-PyMT mouse resulted in increased metastasis formation [69]. Together, these experiments in mouse models demonstrate the pivotal role of TGF-β signaling in breast carcinogenesis. These observations have implications for the development of anti-metastatic therapies. For example, long-term treatment of MMTV-Neu mice with a soluble version of TGF-βRII protects MMTV-Neu mice from metastasis without increasing primary tumor growth, hence selectively blocking the metastatic effects of TGF-β while not affecting its functions in early tumor stages [70]. Chronic exposure to the soluble TGF-βRII in these mice did not cause any unwanted side effects, suggesting a potential avenue for the development of therapy. Small inhibitors of the TGF-β receptor kinase activity and agents specifically blocking TGF-β-mediated signaling pathways are currently in clinical trials [71].
EGF family members
The importance of TGF-α, an EGF family member, in mammary tumor onset has been demonstrated by the transgenic expression of TGF-α under the control of several mammary epithelium-specific promoters. Such tissue-specific expression has led to distorted mammary gland development. However, primary tumors and pulmonary metastasis formed only after the combination of several additional tumor-promoting factors, such as crossing TGF-α transgenic mice with MMTV-Myc transgenic mice or treating MMTV-TGF-α mice with chemical carcinogens. In double-transgenic MMTV-TGF-α; MMTV-TGF-β mice, tumor development is, however, suppressed [72].
We have already introduced the importance of ErbB2 in breast carcinogenesis. In addition, amplification of the gene encoding EGFR correlates with increased metastasis and is a bad prognosis factor in breast cancer [73]. MMTV-Neu mice have also been extensively employed to investigate the functional contribution of EGFR to mammary carcinogenesis. EGFR-mediated signaling contributes to invasion, intra-vasation and metastasis, along with the mitogenic signaling in this model [49, 74, 75]. Moreover, EGFR contribution to metastasis was shown by using MTLn3 rat mammary adeno-carcinoma cells injected into the fat pad of mice. By quantifying the number of tumor cells in the blood as a direct measure of cell intravasation it was possible to show that EGFR acts via increased cell motility and intravasation rather then by affecting cell proliferation [76]. A neutralizing antibody against ErbB2 (Herceptin) has been developed to repress the tumorigenic stimuli of ErB2 and has been approved for clinical use (reviewed in [10]). Together with newly developed inhibitors of EGFR signaling, combinatorial repression of EGFR and ErbB2 activity may therefore be an efficient way to combat breast cancer.
Wnt signaling
Wnt family members were the first proto-oncogenes to be discovered by an MMTV-mediated insertion–activation mechanism. Transgenic expression of Wnt-1 in the mammary gland of transgenic mice results in mammary adeno-carcinomas with metastasis to lymph nodes and lungs [7]. Moreover, Wnt-1 collaborates with fibroblast growth factor-3, another MMTV-insertion-activated gene, in tumor onset. Surprisingly, in double-transgenic MMTV-Wnt-1;MMTV-TGF-β animals, tumor cell proliferation is not repressed by TGF-β expression, showing an opposite effect to that observed for MMTV-TGF-α; MMTV-TGF-β mice (see above) [77].
Genes involved in organ-specific metastasis
Cancers developing in a certain organ usually exhibit particular patterns of organ-specific metastasis. Breast cancer predominantly colonizes bone, followed by axillary and other lymph nodes, lung, liver, brain, and (rarely) adrenal glands. A combination of physical factors, such as lymphatic and blood vessel capillary networks encountered by disseminating tumor cells, and environmental factors, such as chemo-attractive cytokines or chemokines and the presence of 'vasculature addresses', contribute to the specific dissemination of metastastic cancer cells [78, 79]. One possible underlying mechanism is that breast cancer cells follow a cytokine gradient by co-opting immune cells' strategies to arrive at target organs [80].
Xenograft transplantation experiments using the MDA-MB-231 cell line have been instrumental in demonstrating the functional role of certain genes in organ-specific breast cancer metastasis. For instance, prevention of CXCR4 expression by using short interfering RNA technology or blocking its function with specific antibodies or synthetic peptides repressed the formation of lung metastasis, indicating that the CXCR4 ligand, SDF-1, expressed by metastatic target organs, is recruiting tumor cells via CXCR4, which is expressed on breast cancer cells [80–82]. Orthotopic, intracardiac, and tail vein injections of MDA-MB-231 cells have also been performed to identify genes modulating organ-specific metastasis, for example to bone or lung [25, 27]. Gene expression profiling experiments with sublines of MDA-MB-231 selected for organ-specific metastasis have identified specific gene expression signatures for different organ-specific metastases. The functional involvement of these genes and factors in directing organ-specific metastasis was demonstrated subsequently. Genes involved in lung metastasis include those encoding the EGF-like factor epiregulin, CXCL1, MMP-1 and MMP-2, SPARC, vascular cell adhesion molecule-1 (VCAM-1), Id1, and COX-2, and genes promoting bone metastasis include those encoding IL-11, osteopontin, CTGF, CXCR4, and MMP-1 [25, 27].
Overexpression of osteopontin induces metastasis of poorly metastatic MDA-MB-231 cells, whereas its downregulation is correlated with reduced osteolytic metastasis [26]. Osteo-pontin upregulates uPA plasminogen activator, which, upon binding to integrins and surface receptors, provokes the activation of both the hepatocyte growth factor (HGF) and EGF pathways [83]. Xenograft transplantation of MT2994 primary breast cancer cells has shown that the expression of osteopontin was associated with a constitutive activation of the phosphoinositide 3-kinase pathway and a metastatic phenotype of tumor cells [74]. Moreover, osteopontin can induce the expression of alternatively spliced isoforms v6 and v9 of CD44 in breast cancer cells, leading to an increase in cell migration [84].
In a similar approach, sublines of the breast cancer cell line MDA-MB-435 have been selected for specific colonization of lung, lymph node, and thorax. Several adhesion and matrix molecules are correlated with lymph node metastasis, including CD73, a cell surface protein previously implicated in lymphocyte homing to lymph nodes [85]. Moreover, MDA-MB-468 variant metastatic cells with tropism to lymph nodes may use differential expression of adhesion molecules and may mimic angiogenesis pathways to reach lymph nodes [86]. Notably, these cells express α9β1 integrin, an integrin that is specifically expressed on lymphatic endothelial cells and can bind many ligands previously implicated in metastasis, including osteopontin, tenascin C, VCAM-1 and the lymphangiogenic factors vascular endothelial growth factor (VEGF)-C and VEGF-D.
Recent work has documented a role for RANK/RANK ligand (RANKL) signaling together with parathyroid hormone-related protein (PTHrP) and osteoprotegerin in bone metastasis. Treatment with a humanized antibody against PTHrP significantly suppressed osteolytic metastasis in mice injected with a subline of MDA-MB-231 that showed high metastatic ability to bone and expressed high levels of PTHrP, IL-8, IL-6, and MMP-1 [87]. The importance of the role of RANK/RANKL signaling in the regulation of tumor cell migration has also been reported for melanoma cells in vivo [88], whereas experiments performed with MDA-MB-231 breast cancer cells have shown that the RANK soluble receptor, osteoprotegerin, is effective in specifically decreasing bone metastasis by preventing the signaling that mediates the differentiation and activation of osteoclasts [89]. However, in an intra-tibial ectotopic injection model of osteoprotegerin and PTHrP overexpression by MCF-7 breast cancer cells it was revealed that overexpression of osteoprotegerin by tumor cells actually supports tumor growth [90].
Upregulated expression of VEGF-C, and to a smaller extent that of VEGF-D, is highly correlated with lymphangiogenesis and lymph node metastasis in cancer patients. Moreover, forced expression of VEGF-C or VEGF-D in tumor cell lines or in transgenic mouse models of tumorigenesis results in upregulated lymphangiogenesis and in the formation of lymph node metastasis [15]. The role of lymphangiogenesis and angiogenesis in breast cancer metastasis is a major focus of current research. Mammary overexpression of the blood vessel angiogenic factor VEGF-A markedly accelerates the formation of lung metastasis in MMTV-PyMT mice, not only by promoting tumor angiogenesis but also by sustaining tumor proliferation and survival [91]. In a xenograft tumor transplantation model using MDA-MB-231 breast cancer cell line variants with brain tropism, the formation of brain metastases seems highly dependent on the presence of VEGF-A [92]. Moreover, in orthotopic xenograft transplantation of human breast cancer cells with high or low metastatic ability (MDA-MB-435 and MCF-7, respectively), overexpression of VEGF-C induces intra-tumoral lymphoangiogenesis and the subsequent formation of lymph node and lung metastasis [93, 94]. Blockade of VEGF receptor-3 signaling by specific antibodies inhibits regional and distant lymph node metastasis in these models, whereas VEGF receptor-2 inhibition results in a suppression of angiogenesis and tumor growth. Notably, a combination of the two treatments suppresses the formation of metastases better than single treatments [95]. These results indicate that angiogenic and lymphangiogenic factors may have central roles in defining organ-specific breast cancer metastasis.
Elucidation of the molecular mechanisms underlying breast cancer progression and metastasis has gained tremendously from mouse models in which the multiple stages of tumor progression are recapitulated. However, despite their obvious convenience in basic cancer research and in the testing of experimental therapies, the use of mouse models carries several limitations. There are obvious differences between human and mouse tumorigenesis, among which are the kinetics of carcinogenesis and the final size of tumors, differences in cell intrinsic features such as the requirements to transform cells, and differences in organ-specific gene expression, in physiology, metabolism, pathology, and in the immune system. Moreover, metastatic dissemination occurs mainly via hematogenous spreading to lungs and lymph nodes in MMTV-PyMT and MMTV-Neu mice, as opposed to the initial spreading of cancer cells to local lymph nodes via the lymphatics in human breast cancer.
Another important aspect to the understanding of breast cancer metastasis is the role of different subpopulations of breast cells, including cancer stem cells. A great effort is put into their isolation by means of molecular markers or functional assays. The use of transplanted breast cancer stem cells isolated from mice harboring different genetic modifications thereby offers a valuable tool not only in the unraveling of breast cancer development but also in designing effective therapeutic strategies.
Recent technological advances have greatly improved the use of animal models in breast cancer research, such as the use of bioluminescence and fluorescence systems, magnetic resonance, positron-enhanced tomography scans or in vivo confocal analysis to image tumor development in live animals, also allowing observation for long periods. Moreover, extended time-lapse observation of labeled tumor cells in vivo provides new insights into the actual dynamics of tumor growth, extravasation, cell migration, and organ colonization, as well as the contribution of the tumor stroma and subsets of immune cells. Finally, gene expression analysis of tumor samples matched with normal tissue from patients will provide gene signatures that will have to be tested in vivo by proof-of-concept experiments in reliable mouse models of breast cancer metastasis.
In the future it will be necessary to generate mouse models that more accurately recapitulate human breast carcino-genesis, while offering the advantages of model systems, such as easy genetic or pharmacological manipulation and imaging. The quest for such improved models has just begun.
colony-stimulating factor
connective tissue growth factor
extracellular matrix
epidermal growth factor
epithelial–mesenchymal transition
insulin-like growth factor
MAP kinase/ERK kinase kinase
matrix metalloproteinase
murine mammary tumour virus
- PTHrP:
parathyroid hormone-related protein
- PyMT:
polyoma middle T antigen
stromal cell-derived factor
transforming growth factor
vascular cell adhesion molecule
vascular endothe-lial growth factor.
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We are grateful to Dr Miguel Cabrita and Dr François Lehembre for critical comments on the manuscript, and to Dr Jos Jonkers for sharing unpublished results. Research in the laboratory of the authors is supported by the Krebsliga Beider Basel, Novartis Pharma Inc., NCCR Molecular Oncology, the Swiss National Science Foundation and the EU-FP6 framework programs LYMPHANGIOGENOMICS LSHG-CT-2004-503573 and BRECOSM LSHC-CT-2004-503224.
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Fantozzi, A., Christofori, G. Mouse models of breast cancer metastasis. Breast Cancer Res 8, 212 (2006). https://doi.org/10.1186/bcr1530
DOI: https://doi.org/10.1186/bcr1530

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Day 361-367, 7-14 Jul 09: Mums in Mexico pt.2: Playa, more ruins & Isla Cozumel
September 28, 2009 by Harry,
Filed under Trip reports, North America, Mexico, Yucatan Peninsula
As mentioned in the previous post, we have parked our bikes for a few weeks as both our mums are visiting us on the Yucatan Peninsula. After the ruins of Chichen Itza & Ek Balam, it is now time for the beach!
Playa del Carmen, overcrowded and overrated
"Don't go to Cancun, move to Playa de Carmen!", was the advice of friends, websites and guidebooks. "More European, more relaxed, less commerce and more affordable than Cancun.", were the reasons given.
We arrived in our rental car and first could not park anywhere to find a hostel.
Finally we found a spot, only to find much more expensive hotels than in downtown Cancun. Once we found a decent room (3 beds, Ivana & I can share), we checked out the main street. It was filled to the top with souvenir shops and overpriced restaurants and hundreds of semi-relaxed tourists, many of them, yes, European. Large clubs and uber-cool lounge bars were promoted, while Guatemalan art was being sold for western art gallery prices.
Finally we discovered some real food for almost downtown Cancun prices, sold on the streets close to the Central Plaza. Great juice and tortas, the Mexican sandwiches with a choice of meat and/or vegetables and different types of very spicy and tasty sauces.
Just a bit ahead were the local fruit ladies selling nice big bags of mixed cut fruits for less than half of the price of a glass of water in a restaurant of the main street.
The beach is very nice at Playa and the water is green-blue as in the brochures, but it's like that all along the Costa Maya.
The girls were happy with the sea and sand, but I rather wanted to use the last day we had a car to see one more ancient Maya City and took off alone.
More ruins, Coba solo
Coba has the same charm that Ek Balam has and that Chichen Itza is lacking: the 'Indiana Jones' sense that you are discovering the ancient hidden cities yourself while strolling through the lush Jungle.
The structures of Coba are not as neatly organised and lined up as in Chichen Itza. From the first group, which contains the large 'Templo de las Iglesias', the temple of the Churches, it is more than a kilometre walk through the jungle if you want to see some of the other big ones.
Several dozen sacbe's, ancient Mayan road crossed the surrounding jungle to get to Coba, an important hub in times gone by. Only a few percent of the estimated 6500 (!) structures of Coba have been excavated, and many of these not even fully and the jungle has remained intact, which is good news for the many different animals living there.
It is hot in Coba, even in the shade of the trees and for those who do not want to walk the sacbe's there are many eco-taxis handy: bicycles & rickshaws!
The largest Maya structure (of the entire Peninsula) is called Nohoch Mul, better known as the Great Pyramid and its eroded steps leading to the 42m (140ft) high top can fortunately still be climbed. A thick rope is attached to help the brave people down that made it up and realized that it was quite high and the steps narrow and down-sloping 🙂
There will be several persons on the Pyramid, but the views are great: jungle as far as the eye can see. The sweet views are spiced with the knowledge of the thousands of hidden treasures still to be found.
Tulum: busy ruins and empty beach
I was just too late to make a quick visit to the most popular Maya ruins: Tulum. As it is close to Cancun and Playa de Carmen, and the site is open and compact, bus loads of tourists come here every day.
I saw a huge line of them coming out of the exit and caught a glimpse of the famous Castillo, with its postcard location on the edge of the sea. I was not allowed in, but used my time to view some nearby rough beaches, totally deserted.
It was time to return to Playa de Carmen and head over to our next destination: an island!
Isla Cozumel, CouchSurfing and anniversary on the divers and cruise-ship paradise
One part of our way of travelling that we wanted to share with our mums was the use of the CouchSurfing and WarmShowers network, where travellers host other travellers. We managed to find a great host on the Island of Cozumel, that agreed to host all 4 of us.
Ivan not only provided us with a great place to sleep, but also gave us a quick tour of the rough east side of the diver's paradise, with some great swimming beaches and blowholes. The next day he took Ivana, Cristi and myself for a nice little snorkel tour, while my mum relaxed in a hammock near a pool. Life should not get much harder than this 🙂
It was wonderful to have such a perfect example of a great CouchSurfing host to demonstrate to our mums a taste of the hospitality we have encountered all over Mexico, USA and Canada, made possible by the technology and the mentality of our generation, but which has spread far beyond that.
Cozumel is a popular stop for huge cruise-ships and we saw many pass during the few days we were there. But to see further than the shopping tours along the silver-shops and the basic restaurants, you need to spend some days there.
For example it gives you the chance to see the local Sunday dance on the main square, where the local couples dress up and play to the music of a live band, while the sun sets behind a blue and purple sky.
Next and final part of the Mother-ship series coming up: An Island of Women!
Stay tuned, it will be up and running soon as long as the Internet gods are willing 🙂
« Previous: Day 354-360, 30Jun-6Jul 09: Mums in Mexico pt.1: Ruins & Cenotes! «
» Next: Day 368-375, 15-21 Jul 09: Mums in Mexico pt.3: Isla Mujeres »
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Man Crashes Truck Into a FOX 4 TV News Station in Downtown Dallas
Wednesday morning, a man was arrested in downtown Dallas, Texas, after ramming his truck into the Fox 4 television news station.
According to a report by Fox 4 News in Dallas, the man was a middle-aged white male who began ranting after he crashed a silver truck multiple times into the glass windows of the TV station at around 6 a.m. Fortunately, no one was injured in the initial onslaught, but Dallas police found a suspicious duffel bag at the scene that the man had left behind.
Believing the bag could contain an explosive device, FOX 4 news anchor Hanna Battah, in a live video posted to Facebook, said that police evacuated the news building and blocked off several streets until a bomb squad could arrive at the scene to investigate.
The suspect in the incident has since been identified as 34-year-old Michael Chadwick Fry. This isn't the first time he's been arrested. Fry has past convictions in Denton County that include assault, disorderly conduct, public intoxication, and burglary.
Regarding the suspect now in custody, Battah said: "...he was apparently screaming about some injustice, saying there was some sort of conspiracy. He made it sound like someone had been killed and he was seeking justice, wanting the media's help at first...when police took him, when they took him into custody, a photographer said that they saw them actually loading him onto an ambulance."
Fry reportedly left multiple cardboard boxes at the scene that contained hundreds of flyers that he had made himself. Dallas police have not yet determined a specific motive for the assault, but say that the man's ramblings may be connected to an unspecified officer-involved shooting.
Regardless, police say that Fry will be charged with criminal mischief.


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Search the attorney directory
Choose an area of law to find top-rated attorneys near you.
Lawyers (also called attorneys or counsel) serve as advocates for people and organizations. They represent clients both to the court and to opposing parties. Lawyers can represent clients in criminal cases, where a law has been broken, and in civil cases, in which one party is suing another.
Lawyers are also important partners in situations that don't involve the courtroom, advising clients about their legal rights and obligations for personal or business issues. They are trained to interpret complicated systems of laws and navigate the court system.
First, a lawyer will advise you as to whether or not you actually need legal help. It's not always obvious whether an issue is a legal matter or something that can be resolved without involving the court system. A lawyer will help you answer these initial questions.
Once you've decided to hire a lawyer, then they have the power to represent you to the court and to opposing parties. Your lawyer is your most important ally in any legal matter. They provide advice on how to proceed with your legal issue, and prepare important documents for you.
If your issue involves going to court, your lawyer will accompany you, and can speak for you. If your legal matter involves mediation or arbitration, your lawyer will negotiate with the opposing party on your behalf.
Many lawyers tend to focus only on one area, or several related areas of law. In your search for the right lawyer, you should look for a lawyer who understands and has experience in the specific area of law for your issue. For example, a divorce lawyer may not be able to help you if you are considering declaring bankruptcy for your business, but a bankruptcy lawyer who understands bankruptcy and business law would be a perfect match. Reputable lawyers will be upfront about their experience and specific focus, but it will save you some time by narrowing your search beforehand.
Avvo attorneys handle cases in over 120 practice areas, including divorce, criminal defense, and bankruptcy.
Practice size is definitely something you should take into account. Some firms consist of only one lawyer, while some firms retain hundreds. There are many different types of law firms, some of which focus on very specific issues, while others may be more general. For instance, a large corporate firm may not help if you only need a lawyer to review a contract or a few documents, but may be the right choice if you are filing a personal injury suit against a large company. If your legal issue involves only small sums of money, or a few documents, a small firm may be more cost-effective. The size of a practice may also determine how quickly you will be able to actually speak to your potential lawyer; larger offices may require you to schedule your initial consultation through a lawyer's assistant or secretary.
Location is an important factor in looking for a lawyer to represent you. Since your issue will likely depend on state or local laws and regulations, you will want an attorney who understands the regulations for your location. On the practical side, you will also want a lawyer who has an office that is convenient for you to travel to. Some lawyers are willing to travel to meet you, but may also charge for travel time. It's usually a good idea to find a lawyer located in your geographical area. Besides legal knowledge of state and local laws, lawyers also have practical local knowledge. Lawyers in specific cities or towns will also have connections to helpful agencies in that city, which will expedite the process of resolving your issue.
One of the best ways to know if an attorney is right for you are reviews. Read what previous clients have said about specific attorneys and their experiences. Once you choose an attorney to contact, read all the reviews about that particular lawyer to get an idea of how they may handle your case and interact with you.
Most lawyers accept standard payment methods, such as cash, check, and credit cards. Sometimes the method of payment will involve part of the amount of a settlement, so be sure to discuss payment options with your lawyer before signing a fee agreement. Most lawyers are also willing to set up a payment plan, if necessary.
A fee agreement, or representation agreement, is a payment agreement between a lawyer and a client. It can consist of several pages, or simply one page outlining the agreement. This document covers key issues such as how much you will be expected to pay for services rendered, how the lawyer will be paid, the timeline of payment, and which lawyer (if a multi-lawyer firm) will work on the case. Be sure to read the fee agreement carefully before signing it, so you know exactly what is expected of you and your lawyer.
This is an important question that a reputable attorney should have no problem answering. You want to find a lawyer that has experience dealing with your specific issue, and this question will help you determine whether a lawyer is right for you. Feel free to ask for specific statistics, and for their average verdict/settlement numbers in cases similar to yours.
Lawyers, like all professionals, will have varying degrees of experience. Understanding a lawyer's previous experience is essential to understanding how they can handle your case. You'll want to ask how many years the lawyer has been practicing. A lack of experience doesn't necessarily make for a bad lawyer, though, especially if they have experienced lawyers around them. This is why you'll want to ask about their workload: do they handle many cases? How much of each case do they personally oversee, and how much do they delegate to assistants?
Make sure you and your attorney are on the same page about communication. Ask about their preferred communication methods, and whether they are willing to use your preferred communication methods. Does the lawyer you are considering hiring accept (and respond to) communication via email? Will they respond if you send a text? Make sure they respond promptly (within 24 hours) to your initial inquiries--you don't want to hire a lawyer who won't return your calls or avoids prompt communication about your case.
It's especially important to understand a potential lawyer's availability. Will they be going on a three-week vacation in the middle of your case? Do they take phone calls after business hours? Do their legal assistants have a way to contact them if they are not in the office? You should have a good sense of how and when your lawyer will be available to communicate with you.


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TechRadar Verdict
- +
Strong AV performance
- +
Powerful EPG
- +
Linux-powered networkability
- -
No timeshift or blind search
- -
No native support for internal SATA HDD
- -
User interface can be clumsy
Why you can trust TechRadar
The TM-800 HD is Technomate's most affordable Linux-based receiver and our review unit came pre-installed with a variant ('IQ') of the Enigma 2 firmware 'image' that you'll find on many competing Linux-based products like the Dreambox.
At least four other 800 HD-compatible images are also available. Dealers can usually install the one of your choice.
The hardware it runs on, which is based on an NXP 'jungle chip', is well specified. It's HD-capable, the single Sharp tuner supporting DVB-S2 channels. A card slot (capable of emulating various CAMs, with the appropriate Enigma plug-in) is present, although there's no conditional access slot.
Among a healthy sprinkling of connectivity are HDMI, USB and, of course, Ethernet – an internal Wi-Fi upgrade is available as an option. Also optional is PVR functionality. The 800 HD will record onto USB media if you're not prepared to install an internal 2.5-inch drive.
The 800 HD has been available for over a year now, and during that time was plagued by problems (at one point there was a product recall). Although now stable enough to be given the Wotsat treatment, it's still not perfect, although Technomate assures us that a major new firmware revision is in the wings.
Build and connectivity
Other than a huge blue-backlit joypad, the most prominent feature of this pleasantly styled midi-sized receiver's front panel is an informative alphanumeric display. Channel names or status information scroll along merrily, and the display also acts a clock when in standby. Under this are a series of basic controls.
A pull-down flap, meanwhile, conceals a USB port, a card reader and blanked-off CI slot. The remote control has plenty of useful 'shortcut' buttons – available functions include satellite selection, recordings, timer, video modes, multimedia and positioner – but its feel leaves much to be desired.
The buttons have a rather lumpy feel to them and sometimes catch on the casework. Some of the smaller ones are awkward to operate, too.
On the rear panel is a healthy sprinkling of connectivity. Underneath its LNB input you'll find a loopthrough output. Next to these are an aerial input and output for the onboard UHF modulator, which can be surprisingly useful for simple multi-room reception.
The 800 HD is well prepared for better-quality options, though. A pair of Scarts and a HDMI port are complemented by component and composite outputs. Audio outputs are optical digital and stereo analogue phonos. A couple of bungs cover the holes into which sockets for the aerials associated with an optional Wi-Fi adapter (£50 though some dealers are currently bundling it with the receiver) would be mounted. A second USB port, RS232 port and a modem jack round off the data socketry.
Talking of which, there's a catch if self-contained PVR operation is needed. To accommodate an internal HDD, which screws into a bracket located just behind the front panel, you'll require a SATA-to-USB converter. This is wired internally to the main board instead of the front-panel USB socket, which is as a consequence no longer available. It is disappointing that no SATA interface is built in.
The irony is that the beefy internal power supply – designed with DiSEqC mounts in mind – has a SATA power cable trailing from it.
A dedicated system menu is provided for basic receiver configuration. In AV settings the output mode (PAL/50 or NTSC/60) and resolution can be specified. For HDMI, all modes from 480p/576p to 1080p are available.
Another menu configures networking – separate categories cover Wi-Fi (if fitted) and Ethernet. If you're using Wi-Fi, WEP, WPA or WPA2 encryption can be specified. To ease network configuration, DHCP is supported. There's also a network wizard, but attempting to use this always caused the receiver to crash.
Alternative skins, which customise the appearance of the user interface, can be chosen. Other menus look after parental controls and languages and video fine-tuning (aka a test-pattern generator).
Two basic setup modes – simple and advanced – are available. The first is ideal for basic systems – notably those using a single dish/LNB or a DiSEqC 1.0 setup. For the latter, each of the four positions can be assigned to a specific satellite.
In the 'advanced' menu, far more parameters (notably relating to LNB type and DiSEqC) are available. In addition to 1.0, this receiver supports 1.2 and USALS – all of the usual controls for dish movement are supported via the handset's coloured buttons. Hardware blind search is missing, although we might eventually see it in software 'plug-in' form.
Searching can be taking place on single transponders or complete satellites – all channels, or just free ones, can be found and stored. FEC and symbol rate can be specified for manual searches, but you can't enter PIDs.
One of the plug-ins present on the review sample was a satfinder, which displays larger-than-usual strength/quality bar graphs for a user-specified satellite/transponder. It's a shame that signal level isn't represented by an audible tone which could aid dish installation.


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BRISBANE, Australia's third largest city, is a sub-tropical metropolis with a population of 2.2 million.
It offers an all-year-round climate ideal for holidays and tourism. Conveniently, most things to see and do in Brisbane are centrally located either in – or close to – the central business district. South Bank is an artificially-created beach and parklands area along the banks of the Brisbane River, opposite the CBD, which features a protected swimming area complete with lifeguard.
From South Bank you can access the city and its attractions as well as the river ferries and the nearby Brisbane Botanical Gardens.On the other side of the river, further along, is the Riverside Centre, which has a selection of cafes and is the embarkment point for paddlesteamer cruises on the river.
The river is one of the city's main attractions and there are many cruises that pass under under the Story Bridge, on their way to popula destinations like New Farm and Bulimba. The river is also the setting for the annual Riverfire fireworks display. Adjoining the CBD and not far from the Story Bridge is Fortitude Valley and the Brunswick Street Mall with its many cafes and hugely popular Sunday afternoon entertainment. "The Valley" is also known for its night life.
Chinatown, with its eateries and night life, is also situated in Fortitude Valley. For those interested in the city and state's history and architecture, there are historic buildings scattered througout the CBD and other parts of Brisbane.
And speaking of history, what about the Breakfast Creek Hotel, which has its own resident ghost and a fabulous old public bar where journos and politicians used to rub noses in the '60s. Brisbane has some colourful shopping areas. Paddington, in particular, is steeped in history because of all its old workers cottages now revitalised as shops.
For some peace and quiet, Roma Street Parkland is a green area with gardens and rainforest walks, which is adjacent to the Roma Street Railway Station.
If you have the time for day trips, there are any number of fun things to do on Queensland's fabulous Gold Coast, two hours south of Brisbane. There you will find Dreamworld and Movie World, Sea World and Wet and Wild.
Travelling north of the city, there is the fabulous Sunshine Coast, a mecca for beaches and home to Australia Zoo, the creation of the famous Crocodile Hunter, the late Steve Irwin.
We are located at the following address: Level 3, 87 Wickham Terrace, Spring Hill QLD 4000, Australia


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