A mouse model of human breast cancer metastasis to human bone

Currently, an in vivo model of human breast cancer metastasizing from the orthotopic site to bone does not exist, making it difficult to study the many steps of skeletal metastasis. Moreover, models used to identify the mechanisms by which breast cancer metastasizes to bone are limited to intracardiac injection, which seeds the cancer cells directly into the circulation, thus bypassing the early steps in the metastatic process. Such models do not reflect the full process of metastasis occurring in patients. We have developed an animal model of breast cancer metastasis in which the breast cancer cells and the bone target of osteotropic metastasis are both of human origin. The engrafted human bone is functional, based on finding human IgG in the mouse bloodstream, human B cells in the mouse spleen, and normal bone histology. Furthermore, orthotopic injection of a specific human breast cancer cell line, SUM1315 (derived from a metastatic nodule in a patient), later resulted in both bone and lung metastases. In the case of bone, metastasis was to the human implant and not the mouse skeleton, indicating a species-specific osteotropism. This model replicates the events observed in patients with breast cancer skeletal metastases and serves as a useful and relevant model for studying the disease.     

Bone morphogenetic protein 7 in the development and treatment of bone metastases from breast cancer

Bone morphogenetic protein 7 (BMP7) counteracts the physiological epithelial-to-mesenchymal transition (EMT), a process that is indicative of epithelial plasticity. Because EMT is involved in cancer, we investigated whether BMP7 plays a role in breast cancer growth and metastasis. In this study, we show that decreased BMP7 expression in primary breast cancer is significantly associated with the formation of clinically overt bone metastases in patients with > or = 10 years of follow-up. In line with these clinical observations, BMP7 expression is inversely related to tumorigenicity and invasive behavior of human breast cancer cell lines. Moreover, BMP7 decreased the expression of vimentin, a mesenchymal marker associated with invasiveness and poor prognosis, in human MDA-MB-231 (MDA-231)-B/Luc(+) breast cancer cells under basal and transforming growth factor-beta (TGF-beta)-stimulated conditions. In addition, exogenous addition of BMP7 to TGF-beta-stimulated MDA-231 cells inhibited Smad-mediated TGF-beta signaling. Furthermore, in a well-established bone metastasis model using whole-body bioluminescent reporter imaging, stable overexpression of BMP7 in MDA-231 cells inhibited de novo formation and progression of osteolytic bone metastases and, hence, their metastatic capability. In line with these observations, daily i.v. administration of BMP7 (100 mug/kg/d) significantly inhibited orthotopic and intrabone growth of MDA-231-B/Luc(+) cells in nude mice. Our data suggest that decreased BMP7 expression during carcinogenesis in the human breast contributes to the acquisition of a bone metastatic phenotype. Because exogenous BMP7 can still counteract the breast cancer growth at the primary site and in bone, BMP7 may represent a novel therapeutic molecule for repression of local and bone metastatic growth of breast cancer.     

Bone sialoprotein mediates the tumor cell-targeted prometastatic activity of transforming growth factor beta in a mouse model of breast cancer

Transforming growth factor betas (TGF-beta) play a dual role in carcinogenesis, functioning as tumor suppressors early in the process, and then switching to act as prometastatic factors in late-stage disease. We have previously shown that high molecular weight TGF-beta antagonists can suppress metastasis without the predicted toxicities. To address the underlying mechanisms, we have used the 4T1 syngeneic mouse model of metastatic breast cancer. Treatment of mice with a monoclonal anti-TGF-beta antibody (1D11) significantly suppressed metastasis of 4T1 cells to the lungs. When metastatic 4T1 cells were recovered from lungs of 1D11-treated and control mice, the most differentially expressed gene was found to be bone sialoprotein (Bsp). Immunostaining confirmed the loss of Bsp protein in 1D11-treated lung metastases, and TGF-beta was shown to regulate and correlate with Bsp expression in vitro. Functionally, knockdown of Bsp in 4T1 cells reduced the ability of TGF-beta to induce local collagen degradation and invasion in vitro, and treatment with recombinant Bsp protected 4T1 cells from complement-mediated lysis. Finally, suppression of Bsp in 4T1 cells reduced metastasis in vivo. We conclude that Bsp is a plausible mediator of at least some of the tumor cell-targeted prometastatic activity of TGF-beta in this model and that Bsp expression in metastases can be successfully suppressed by systemic treatment with anti-TGF-beta antibodies.     

Silencing of transforming growth factor-beta1 in situ by RNA interference for breast cancer: implications for proliferation and migration in vitro and metastasis in vivo

PURPOSE: Overexpression of transforming growth factor (TGF)-beta has been implicated in promoting immune suppression, tumor angiogenesis, tumor cell migration, and invasion in many cancers, including carcinoma of the breast. Thus, targeted down-regulation of TGF-beta1 expression in breast cancer in situ and determination of its implications would provide new treatment approaches for disease management. EXPERIMENTAL DESIGN: Small interfering RNA constructs targeting TGF-beta1 were validated and used to develop clonal derivatives of the metastatic breast cancer cell line MDA-MB-435. The cells were used in several in vitro analyses, including migration, invasion, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, apoptosis, and signaling assays. A wound-healing assay was used to determine migration of the cells in culture and a Boyden chamber transwell assay was used for invasion. Further, the clones were used in an in vivo mouse model for the kinetics of tumor growth and gene expression in the primary site and in lungs upon metastasis. RESULTS: Inhibition of TGF-beta1 expression in MDA-MB-435 cells showed a 35% decrease in migration and a 55% decrease in invasion in vitro, with a 50% increase in proliferation and no effect on apoptosis. In vivo analysis indicated a 90% decrease in the number of mice bearing macroscopic lung metastases; however, the primary tumors did not show any difference in the growth kinetics when compared with the parental MDA-MB-435 cells. Analysis of TGF-beta signaling pathways in the clonal derivatives showed a decrease in Smad2 activation and an increase in AKT and extracellular signal-regulated kinase activation. Interestingly, analysis of TGF-beta receptor expression showed a decrease in both receptor I and II expression in TGF-beta1 silenced cells. These results suggest that inhibition of TGF-beta1 ligand may act as a negative feedback loop to disrupt the function of all TGF-beta isoforms. CONCLUSIONS: Therapies targeting the TGF-beta signaling pathway may be more effective in late-stage disease to prevent organ metastasis but not primary tumor formation and may be combined with other tumor-targeted therapies normally limited by increased circulating TGF-beta levels.     

Development and gene expression profiling of a metastatic variant of the human breast cancer MDA-MB-435 cells

We have developed a model system of late stage metastatic progression by isolating a highly malignant variant of human breast cancer cells from the parental MDA-MB-435 cell line. These cells, isolated from early lung metastasis, displayed increased anchorage independent growth in vitro and when transplanted ortho-topically into nude mice showed accelerated tumor growth rate and early lung spontaneous metastasis when compared to its parental counterpart. These cells, designated as MDA-MB-435-F-L, also showed intense wide spread early skeletal metastasis in vertebrae, mandible, femur, tibia and skull as detected by fluorescence imaging in an experimental bone metastasis model. Gene expression profiles from cDNA microarray showed up or downregulation of the expression of several significant genes regulating angiogenesis, apoptosis, ECM remodeling and metastasis in the MDA-MB-435-F-L cells in comparison to the parental cells. Among the up or downregulated genes, some have also been implicated in the survival of breast cancer patients. As such, the candidate genes selected in this breast cancer progression model system may serve as biomarkers of metastatic progression and also as potential tumor targets for breast cancer therapy.     

CIP4 promotes metastasis in triple-negative breast cancer and is associated with poor patient prognosis

Signaling via epidermal growth factor receptor (EGFR) and Src kinase pathways promote triple-negative breast cancer (TNBC) cell invasion and tumor metastasis. Here, we address the role of Cdc42-interacting protein-4 (CIP4) in TNBC metastasis in vivo, and profile CIP4 expression in human breast cancer patients. In human TNBC cells, CIP4 knock-down (KD) led to less sustained activation of Erk kinase and impaired cell motility compared to control cells. This correlated with significant defects in 3D invasion of surrounding extracellular matrix by CIP4 KD TNBC cells when grown as spheroid colonies. In mammary orthotopic xenograft assays using both human TNBC cells (MDA-MB-231, HCC 1806) and rat MTLn3 cells, CIP4 silencing had no overt effect on tumor growth, but significantly reduced the incidence of lung metastases in each tumor model. In human invasive breast cancers, high CIP4 levels was significantly associated with high tumor stage, TNBC and HER2 subtypes, and risk of progression to metastatic disease. Together, these results implicate CIP4 in promoting metastasis in TNBCs.     

Consequences of altered TGF-beta expression and responsiveness in breast cancer: evidence for autocrine and paracrine effects.

To characterize the impact of increased production of TGF-beta in a xenograft model of human breast cancer, TGF-beta-responsive MDA-231 cells were genetically modified by stable transfection so as to increase their production of active TGF-beta1. Compared with control cells, cells that produced increased amounts of TGF-beta proliferated in vitro more slowly. In vivo, however, tumors derived from these cells exhibited increased proliferation and grew at an accelerated pace. To evaluate the role of autocrine TGF-beta signaling, cells were also transfected with a dominant-negative truncated type II TGF-beta receptor (TbetaRII). Disruption of autocrine TGF-beta signaling in the TGF-beta-overexpressing cells reduced their in vivo growth rate. Co-inoculation of Matrigel with the TGF-beta-overexpressing cells expressing the truncated TbetaRII compensated for their diminished in vivo growth capacity, compared with the TGF-beta-overexpressing cells with an intact autocrine loop. Tissue invasion by the tumor was a distinctive feature of the TGF-beta-overexpressing cells, whether or not the autocrine loop was intact. Furthermore, tumors derived from TGF-beta-overexpressing cells, irrespective of the status of the autocrine TGF-beta-signaling pathway, had a higher incidence of lung metastasis. Consistent with the suggestion that TGF-beta's enhancement of invasion and metastasis is paracrine-based, we observed no significant differences among the cell clones in an in vitro invasion assay. Thus, in this experimental model system in vitro assays of cell proliferation and invasion do not accurately reflect in vivo observations, perhaps due to autocrine and paracrine effects of TGF-beta that influence the important in vivo-based phenomena of tumor growth, invasion, and metastasis.     

New metastatic model of human small‐cell lung cancer by orthotopic transplantation in mice

Small‐cell lung cancer (SCLC) is an aggressive cancer with high metastatic ability and novel strategies against the metastasis are urgently needed to improve SCLC treatment. However, the mechanism of metastasis of SCLC remains largely to be elucidated. For further studies of SCLC metastasis, we developed a new orthotopic transplantation model in mice. We established a GFP‐labeled subline from the human SCLC cell line DMS273 and transplanted them orthotopically into the lung of nude mice with Matrigel. The GFP‐labeled cells showed significant metastatic activity and formed metastatic foci in distant tissues such as bone, kidney, and brain, as observed in SCLC patients. From a bone metastasis focus of the mouse, we isolated another subline, termed G3H, with enhanced metastatic potential and higher hepatocyte growth factor (HGF) expression than the parental line. Further studies indicated that the HGF/MET signaling pathway was involved in in vitro motility and invasion activities of the G3H cells and treatments with MET inhibitors decreased formation of distant metastases in our orthotopic model using G3H cells. These data indicated that our model mimics the clinical aspect of SCLC such as metastatic tropism and autocrine of HGF/MET signaling. Compared with other orthotopic SCLC models, our model has a superior ability to form distant metastases. Therefore, our model will provide a valuable tool for the study of SCLC metastasis.     

Suppression of lung tumor growth and metastasis in mice by adeno-associated virus-mediated expression of vasostatin.

PURPOSE: Angiogenesis inhibitors have strong therapeutic potential as antitumor agents in suppressing tumor growth and metastatic progression. Vasostatin, the N-terminal domain of calreticulin, is a potent angiogenesis inhibitor. In this study, we determined the effectiveness of vasostatin delivered by recombinant pseudotype adeno-associated virus 2/5 (rAAV2/5-VAS) as a gene therapy approach for lung cancer treatment. EXPERIMENTAL DESIGN: We used rAAV2/5 to deliver vasostatin intratumorally or systemically in different mouse lung tumor models--subcutaneous, orthotopic xenograft, and spontaneous metastasis lung tumor models. The therapeutic efficacy of rAAV2/5-VAS was determined by monitoring tumor volume, survival rate, and degree of neovascularization after treatment in these models. RESULTS: Mice bearing subcutaneous tumor of rAAV2/5-VAS pretreated Lewis lung carcinoma cells showed >50% reduction in primary tumor volume and reduced spontaneous pulmonary metastases. The tumor-suppressive action of rAAV2/5-VAS in subcutaneous human lung tumor A549 xenograft correlated with a reduced number of capillary vessels in tumors. In the orthotopic xenograft model, rAAV2/5-VAS suppressed metastasis of A549 tumors to mediastinal lymph nodes and contralateral lung. Furthermore, treatment of immunocompetent mice in the spontaneous lung metastases model with rAAV2/5-VAS after primary tumor excision prolonged their median survival from 21 to 51.5 days. CONCLUSION: Our results show the effectiveness of rAAV2/5-VAS as an angiogenesis inhibitor in suppressing tumor growth during different stages of tumor progression, validating the application of rAAV2/5-VAS gene therapy in treatment against lung cancer.     

Stimulation of cyclooxygenase-2 expression by bone-derived transforming growth factor-beta enhances bone metastases in breast cancer

Cyclooxygenase-2 (COX-2), the rate-limiting enzyme of prostaglandin synthesis, has been implicated in invasiveness and distant metastases of cancer. Bone is one of the most common target sites of cancer metastasis. However, the role of COX-2 in bone metastasis is unclear. We examined the surgical specimens of bone metastases from patients with various types of cancers by using immunohistochemistry and observed evident COX-2 expression in these bone metastases. In a nude mouse model of bone metastasis, the MDA-MB-231 human breast cancer cells showed no COX-2 expression at orthotopic sites, whereas these cells, when metastasized to bone, intensely expressed COX-2, suggesting that the bone microenvironment induced COX-2 expression. Consistent with this notion, inhibition of bone resorption by the bisphosphonate ibandronate reduced COX-2 expression in MDA-MB-231 cells in bone. Transforming growth factor-beta (TGFbeta), one of the most abundant growth factors stored in bone, increased COX-2 expression and prostaglandin E2 production in MDA-MB-231 cells in culture. MDA-MB-231 cells overexpressing dominant-negative TGFbeta type II receptors showed decreased bone metastases and reduced osteoclastic bone resorption with impaired COX-2 expression. The COX-2 inhibitors, NS-398 and nimesulide, significantly suppressed bone metastases with decreased osteoclast number and increased apoptosis in MDA-MB-231 cells. These results suggest that bone-derived TGFbeta up-regulates COX-2 expression in breast cancer cells, thereby increasing prostaglandin E2 production, which in turn, stimulates osteoclastic bone destruction, leading to the progression of bone metastases. Our results also suggest that COX-2 is a potential therapeutic target for bone metastases in breast cancer.     

COX-2 involvement in breast cancer metastasis to bone

Cyclooxygenase-2 (COX-2) is expressed in 40% of human invasive breast cancers. Bone is the predominant site of metastasis in case of breast cancer. We investigated the role of COX-2 in a suitable mouse model of breast cancer metastasis to bone using the whole-body luciferase imaging of cancer cells. We provide several lines of evidence that COX-2 produced in breast cancer cells is important for bone metastasis in this model including (1) COX-2 transfection enhanced the bone metastasis of MDA-435S cells and (2) breast cancer cells isolated and cultured from the bone metastases produced significantly more prostaglandin E(2) (an important mediator of COX-2) than the parental injected cell populations of breast cancer cells. Next, we found that a COX-2 inhibitor, MF-tricyclic, inhibited bone metastasis caused by a bone-seeking clone both in prevention regimen (in which case mice started receiving MF-tricyclic 1 week before the injection of cancer cells) and in treatment regimen (in which case mice received MF-tricyclic after the development of bone metastasis). These studies indicate that COX-2 produced in breast cancer cells may be vital to the development of osteolytic bone metastases in patients with breast cancer, and that COX-2 inhibitors may be useful in halting this process.     

Tumorigenicity and metastasis of human breast carcinoma cell lines in nude mice

There are few reports describing experimental models of the growth and metastasis of human breast carcinomas. This article discusses the tumorigenic and metastatic properties of two estrogen receptor-negative breast carcinomas injected into nude mice. Tumor growth in the mammary fatpad (m.f.p.) and the subcutis was compared in female nude mice. The injection of 10(5) viable cells of two human breast carcinoma cell lines (MDA-MB-231 and MDA-MB-435) gave a 100% tumor take rate in the m.f.p., whereas only 40% of the s.c. injections produced tumors and these occurred several weeks after the appearance of the m.f.p. tumors. Thus, the m.f.p. of nude mice is a favorable site for the growth of human breast carcinomas. MDA-MB-435 tumors produced distant metastases in 80% to 100% of recipients. The most common sites for metastasis were the lymph nodes and lungs, with a lower incidence of metastases in muscle (chest wall and thigh), heart, and brain. New variant cell lines were isolated from metastases in the lungs, brain, and heart. All the cell lines were tumorigenic in the m.f.p., and the lung- and heart-derived metastasis lines produced slightly more lung metastases than the original cell line. However, the brain metastasis variant produced significantly fewer lung metastases. Intravenous inoculation of the spontaneous metastasis-derived cell lines produced few lung colonies. Only cell variants isolated from experimental lung metastases showed enhanced lung colonization potential when reinjected i.v. Our results suggest that the estrogen receptor-negative MDA-MB-435 cell line injected in the m.f.p. of nude mice could be a valuable tool for analysis of the cellular and molecular basis of the metastasis of advanced breast cancer.     

Identification of VEGF-regulated genes associated with increased lung metastatic potential: functional involvement of tenascin-C in tumor growth and lung metastasis

Metastasis is the primary cause of death in patients with breast cancer. Overexpression of c-myc in humans correlates with metastases, but transgenic mice only show low rates of micrometastases. We have generated transgenic mice that overexpress both c-myc and vascular endothelial growth factor (VEGF) (Myc/VEGF) in the mammary gland, which develop high rates of pulmonary macrometastases. Gene expression profiling revealed a set of deregulated genes in Myc/VEGF tumors compared to Myc tumors associated with the increased metastatic phenotype. Cross-comparisons between this set of genes with a human breast cancer lung metastasis gene signature identified five common targets: tenascin-C(TNC), matrix metalloprotease-2, collagen-6-A1, mannosidase-alpha-1A and HLA-DPA1. Signaling blockade or knockdown of TNC in MDA-MB-435 cells resulted in a significant impairment of cell migration and anchorage-independent cell proliferation. Mice injected with clonal MDA-MB-435 cells with reduced expression of TNC demonstrated a significant decrease (P<0.05) in (1) primary tumor growth; (2) tumor relapse after surgical removal of the primary tumor and (3) incidence of lung metastasis. Our results demonstrate that VEGF induces complex alterations in tissue architecture and gene expression. The TNC signaling pathway plays an important role in mammary tumor growth and metastases, suggesting that TNC may be a relevant target for therapy against metastatic breast cancer.     

The effect of the bisphosphonate ibandronate on breast cancer metastasis to visceral organs

Bisphosphonate (BPs), specific inhibitors of osteoclastic bone resorption, are widely used therapeutic agents for bone metastases in breast cancer patients. Nevertheless, the effects of BPs on visceral metastases are controversial. Here we specifically studied the effects of the BP ibandronate on visceral metastases of breast cancer using two animal models. In the first set of experiments, we examined the effects of ibandronate on lung metastasis using 4T1 mouse mammary tumor that developed pulmonary and bone metastases following orthotopic inoculation in syngeneic female Balb/c mice. In the second set of experiments, we examined the effects of ibandronate on adrenal metastasis using a clone of the MDA-MB-231 (MDA-231) human breast cancer (MDA-231AD cells) that developed adrenal and bone metastases following intracardiac inoculation in female nude mice. These breast cancer cells were stably transfected with a firefly luciferase cDNA to facilitate quantification of the metastatic tumor burden in visceral organs. Ibandronate (4 µg/day, sc, daily) was given either after metastases were established (therapeutic administration) or at the time of tumor cell inoculation (preventative administration). In both models with each protocol, ibandronate reproducibly reduced bone metastases, establishing that BPs are effective pharmacological agents for the treatment of bone metastases in breast cancer. In the 4T1 model, neither the preventative nor therapeutic administration of ibandronate caused any effects on lung metastases. In the MDA-231 model, the preventative administration of ibandronate significantly increased adrenal metastases. However, no increase in the adrenal metastases was observed when an anti-cancer agent doxorubicin was co-administered. Therapeutic administration of ibandronate showed no effects on the adrenal metastases. Our results suggest that BPs cause no adverse effects on visceral metastases when administered in the manners that breast cancer patients usually receive.     

Direct evidence of the importance of stromal urokinase plasminogen activator (uPA) in the growth of an experimental human breast cancer using a combined uPA gene-disrupted and immunodeficient xenograft model

Several studies have indicated an interaction between tumor cells and infiltrating stromal cells regarding the urokinase plasminogen activation (uPA) system. By developing combined uPA gene-disrupted and immunodeficient mice, we have studied the role of stromal uPA for the growth of the MDA-MB-435 BAG human tumor xenograft. Subcutaneous tumor growth and lung metastasis were compared between wild-type immunodeficient mice and mice with the combined deficiencies. Tumor growth was evaluated by volume measurements and plasma beta-galactosidase activity and metastasis was evaluated by counting lung surface metastases. Although no differences appeared in primary tumor take between the two groups of mice, a significant difference was observed in primary tumor growth, with tumors in uPA-/- mice growing significantly more slowly. In addition, a nonsignificant trend toward fewer lung metastases in uPA-/- mice was observed. The present data points to a critical role of stromal-derived uPA in the primary tumor growth of MDA-MB-435 BAG xenografts, whereas only a trend toward fewer lung metastases in uPA gene-disrupted mice was found.     

Inhibition of apoptosis in human breast cancer cells: role in tumor progression to the metastatic state

Reduction in apoptosis has been associated with tumor metastases and response to chemotherapy in breast cancer. We examine the influence of apoptosis status and the expression of antiapoptotic proteins Bcl-2 and Bcl-x(L) on metastatic progression and response to therapy in an experimental model of breast cancer. We used human breast cancer cells (MDA-MB 435, MDA-MB 468 and MCF-7) to induce orthotopic xenograft tumors in nude mice. The overexpression of Bcl-2 or Bcl-x(L) influenced tumorigenicity, 468 transfectants being less tumorigenic than control (p < 0.0001). Lung metastasis appeared at day 120 in animals injected with 435/Bcl-2 or 435/Bcl-x(L) and they showed higher metastatic activity than control 435/Neo tumors (p = 0.02). In contrast, mice with 468 tumors were followed for 1 year after tumor excision, but they did not develop metastatic foci. 435/Bcl-2 and 435/Bcl-x(L) transfectant cells bound less readily to laminin (ANOVA, p < 0.0001), fibronectin (ANOVA, p < 0.0001) and collagen type-IV (ANOVA, p < 0.0001) than 435/Neo cells. The overexpression of antiapoptotic proteins in 435 transfectants rescued 20-40% of cells from anoikis at 64 hr in rocking conditions. In contrast, at this time only 5-10% of 468 and MCF-7 transfectant cells were rescued. Thus, the overexpression of the Bcl-2 or Bcl-x(L) associated with the loss of apoptosis in breast cancer cells in vivo may account for their metastatic behavior. These genes increase tumor metastasis when the oncogenic background has triggered the metastatic process, in which anoikis might determine tumor progression when the life span of the cells is extended.