Effect of IL-1 on experimental bone/bone-marrow metastases.

Bone metastasis is a common event and a major cause of morbidity in cancer patients. The hematopoietic marrow of the bones, rather than the bone tissue per se, is the target organ in bone metastasis. In the bone marrow, IL-1 induces the release of hematopoietic growth factors that may affect tumor-cell growth. We treated groups of mice with rhuIL-1 alpha to examine its role in the establishment of experimental bone/bone-marrow metastasis. We found that injection of 2 micrograms of rhuIL-1 alpha 24 hr prior to, simultaneously with or 24 hr after the injection of 10(4) B16 melanoma cells into the left cardiac ventricle of mice resulted in a 2-fold increase in the average number of colonized bones per mouse. GM-CSF is produced by bone-marrow stromal cells in response to IL-1, and its receptor has been found on tumor cells, including melanoma cells. However, the administration of rmuGM-CSF to mice by either multiple injections or continuous infusion did not affect the number of colonized bones. Many of the biologic effects of IL-1 are mediated by prostaglandins. Treatment of mice with 100 micrograms of indomethacin, a potent inhibitor of prostaglandin synthesis, prior to the injection of rhuIL-1 alpha, prevented the increase in number of bone metastases. To determine whether constitutive productions of IL-1 and/or prostaglandins are involved in the pathogenesis of bone/bone marrow metastasis, we treated mice with antimouse IL-1 alpha neutralizing antibodies, rhuIRAP (an inhibitor of IL-1 activity) or indomethacin. We found no difference in the average number of colonized bones per mouse between treated and control mice. We conclude that exogenous administration of IL-1 enhances experimental bone/bone-marrow metastases, and that this phenomenon is mediated through prostaglandins. However, neither the constitutive production of IL-1 nor that of prostaglandins appear to play a role in the pathogenesis of bone/bone-marrow metastasis in our murine model system.     

Tumor alphavbeta3 integrin is a therapeutic target for breast cancer bone metastases

In breast cancer bone metastasis, tumor cells stimulate osteoclast-mediated bone resorption, and bone-derived growth factors released from resorbed bone stimulate tumor growth. The alphavbeta3 integrin is an adhesion receptor expressed by breast cancer cells and osteoclasts. It is implicated in tumor cell invasion and osteoclast-mediated bone resorption. Here, we hypothesized that the therapeutic targeting of tumor alphavbeta3 integrin would prevent bone metastasis formation. We first showed that, compared with mock-transfected cells, the i.v. inoculation of alphavbeta3-overexpressing MDA-MB-231 breast cancer cells in animals increased bone metastasis incidence and promoted both skeletal tumor burden and bone destruction. The direct inoculation of alphavbeta3-overexpressing transfectants into the tibial bone marrow cavity did not however enhance skeletal tumor burden and bone destruction, suggesting that alphavbeta3 controls earlier events during bone metastasis formation. We next examined whether a nonpeptide antagonist of alphavbeta3 (PSK1404) exhibits meaningful antitumor effects in experimental breast and ovarian cancer bone metastasis. A continuous PSK1404 treatment, which inhibited osteoclast-mediated bone resorption in an animal model of bone loss, substantially reduced bone destruction and decreased skeletal tumor burden. Importantly, a short-term PSK1404 treatment that did not inhibit osteoclast activity also decreased skeletal tumor burden and bone destruction. This dosing regimen caused a profound and specific inhibition of bone marrow colonization by green fluorescent protein, alphavbeta3-expressing tumor cells in vivo and blocked tumor cell invasion in vitro. Overall, our data show that tumor alphavbeta3 integrin stands as a therapeutic target for the prevention of skeletal metastases.     

Site-specific experimental metastasis patterns of two human breast cancer cell lines in nude rats.

Animal models for breast cancer metastasis are valuable tools for studying mechanisms of metastasis and for preclinical testing of anti-metastatic therapy regimens. Using MA-11 and MT-1, two oestrogen and progesterone receptor-negative human breast cancer cell lines, we developed new models in nude rats with patterns of experimental metastasis resembling those frequently observed in humans. MA-11 cells showed a clear preference for growth in the CNS. Fourteen of 15 animals injected with MA-11 cells into the left ventricle of the heart developed hind-leg paralysis, and tumours were observed in the spinal cord. MT-1 cells consistently exhibited bone/bone marrow metastases after intracardial injection, in addition to tumours in the brain and spinal cord. When injected into the cisterna magna, both cell lines gave rise to leptomeningeal neoplastic disease. Injection of MA-11 cells into the tibial bone marrow resulted in tumours in only 2 of 13 rats, whereas all animals injected with MT-1 cells developed tumours. Only 2 of 6 rats injected i.v. with MA-11 cells developed lung colonies compared with all 9 animals injected with MT-1 cells. Cell-surface expression of the following was examined: EGP2; MUC1; EGFr; E- and N-cadherin; the alpha2, alpha3, alpha5, beta1 and beta4 integrins; c-erb-B2; and N-CAM. c-erb-B2 was expressed in a higher percentage of the bone-metastasizing MT-1 cells than the MA-11 cells, whereas E-cadherin was expressed in MA-11 but not MT-1 cells. In animals injected with MA-11 and MT-1 cells in the left cardiac ventricle, treatment with cisplatin and doxorubicin did not improve survival. In summary, these clinically relevant animal models may be used for studies related to site-specific growth and metastasis and for assessing effects of experimental therapy against human breast cancer.     

Vascular anatomy and organ-specific tumor growth as critical factors in the development of metastases and their distribution among organs

We have examined with 19 tumor cell lines the discrete roles that vascular anatomy and tumor-cell-organ-affinity play in the development of metastases and their distribution among organs. Spontaneous metastases of B16-G3.26 melanoma cells from a primary tumor growing in the foot pad of mice, or experimental metastases 21 days after intravenous tumor-cell injection resulted in tumor colonies only in the lungs. In contrast, when the lung microvasculature was bypassed, and the same cells given by systemic intra-arterial (s.i.a.) injection, large tumor colonies developed selectively in the ovaries, adrenal glands and bones, but rarely in the lungs. When animals injected i.v. were allowed to live with lung metastases for a long period of time, small tumor colonies began to develop in extra-pulmonary organs with a distribution identical to that seen after s.i.a. injection. Seven murine tumor cell lines (previously characterized by their ability to colonize primarily the lungs after i.v. injection) and 7 of the 8 studied human tumor cell lines colonized different specific extra-pulmonary organs after s.i.a. injection, frequently producing metastatic syndromes commonly described in patients with cancer, but rarely seen in animal models of metastasis. These results suggest that metastatic cells, even those capable of colonizing specific organs, do not freely circulate in the blood stream and lodge in specific tissues. In contrast, the cells must establish a vascular route of access to the target organ, e.g., through the systemic circulation from metastatic tumors in the lungs. Two cell lines considered to be tumorigenic but non-metastatic failed to colonize the lungs or extra-pulmonary organs after i.v. injection, but readily colonized specific organs after s.i.a. injection. Thus, tumor cells considered to be non-metastatic may be indeed metastatic if they are provided with vascular access to an organ more congenial to their growth requirements.     

Augmentation of B16 melanoma lung colony formation in C57BL/6 mice having marked granulocytosis

The effect that polymorphonuclear leukocytes (PMN) may have on the metastatic colonization of tumor cells is controversial: some laboratories have reported that PMN can inhibit metastasis whereas others have shown an augmentation effect. We have exploited our finding that a particular fibrosarcoma (BMT-11) transplanted subcutaneously into syngeneic C57BL/6 mice induces a progressive increase in the number of circulating PMN, to re-examine this question. We used such "granulocytosis-positive" mice as recipients of B16 melanoma cells to examine the influence of significant granulocytosis on the level of lung tumor colonies. The number of B16 melanoma lung colonies detected after intravenous (i.v.) injection was significantly higher in BMT-11 tumor-bearing mice with granulocytosis than in control (non-tumor-bearing) mice. Retention of 125IUdR-labelled B16 cells 24 hr after the i.v. injection was 3 to 10 times greater in mice with granulocytosis than in controls. Either simultaneous injection, or preinjection of PMN with B16 cells, increased the lung-colonizing capacity of B16 melanoma cells. These results suggest that abnormally increased numbers of PMN in the peripheral blood, particularly in the lung circulation, can enhance the ability of tumor cells to colonize or metastasize.     

Inhibition of bone and muscle metastases of lung cancer cells by a decrease in the number of monocytes/macrophages

Attention has recently focused on the critical role of inflammatory responses in the tumor stroma that provide favorable conditions for cancer-cell growth and invasion/metastasis. In particular, macrophages recruited into the tumor stroma and activated, known as tumor-associated macrophages, are suggested to promote tumorigenesis. In this study, we examined the effect of a decrease in the number of monocytes/macrophages in peripheral blood and the tumor stroma on the development of bone and muscle metastases by lung cancer cells. Treatment with clodronate encapsulated by liposomes (Cl₂MDP-LIP) has been developed for the depletion of monocytes/macrophages in an animal model. Subcutaneous administration of Cl₂MDP-LIP markedly reduced the number of monocytes in peripheral blood, resulting in efficient suppression of both bone metastasis and muscle metastasis when lung cancer HARA-B cells were injected into the left cardiac ventricle of mice. Treatment with Cl₂MDP-LIP significantly reduced the number of macrophages in tumors and the number of osteoclasts in bone marrow, as well as peripheral monocytes in mice harboring lung cancer cells. In contrast, treatment with an osteoclast-targeting antibiotic, reveromycin A, inhibited bone metastasis by lung cancer cells, but not muscle metastasis. The survival of human macrophages in culture was found to be specifically blocked by Cl₂MDP-LIP, but not by reveromycin A. Cl₂MDP-LIP thus exerted antimetastatic effects in both bone and muscle whereas reveromycin A did so only in bone. Liposome-encapsulated bisphosphonate may modulate metastasis through decreasing the number of monocytes/macrophages in both peripheral blood and the tumor stroma, suggesting that tumor-associated macrophages might be suitable targets for antimetastatic therapy. ( Cancer Sci 2008; 99: 1595–1602)     

Severe combined immunodeficient-hu model of human prostate cancer metastasis to human bone

Commonly used in vivo models of prostate cancer metastasis include syngeneic rodent cancers and xenografts of human cancer in immunodeficient mice. However, the occurrence of osseous metastases in these models is rare, and in xenograft models, species-specific factors may limit the ability of human cells to metastasize to rodent bones. We have modified the severe combined immunodeficient (SCID)-human model to test the ability of circulating human prostate cancer cells to home to macroscopic fragments of human bone and other organs previously implanted into SCID mice. We have also compared the growth of human prostate cancer cells in various human and mouse tissue microenvironments in vivo. Macroscopic fragments of human fetal bone, lung, or intestine (16-22 weeks gestation) or mouse bone were implanted s.c. into male CB.17 SCID mice. Four weeks later, human prostate cancer cells were injected either i.v. via the tail vein (circulating cell colonization assay) or directly into the implanted tissue fragments transdermally (end organ growth assay). Tumor growth was followed for 6 weeks by palpation and magnetic resonance imaging. After 6 weeks, tumors were enumerated in implanted human and mouse organ fragments and native mouse tissue. Tumors were characterized by histology, immunohistochemistry, and chromosomal analysis. After i.v. injection, circulating PC3 cells successfully colonized implanted human bone fragments in 5 of 19 mice. Tumors were easily followed by palpation and imaging and had an average volume of 258 mm3 at autopsy. Histological examination revealed osteolysis and a strong desmoplastic stromal response, which indicated intense stromal-epithelial interaction. Bone tumors were subcultured, and chromosomal analysis demonstrated that the tumors were derived from the parental prostate cancer cell line. Microscopic tumor colonies were also found in a few mouse lungs after i.v. injection of PC3, DU145, and LNCaP cells, however the volume of the lung nodules was less than 1 mm3 in all of the cases. No colonization of human lung or intestine implants, the mouse skeleton, or other mouse organs was detected, demonstrating a species- and tissue-specific colonization of human bone by PC3 cells. Direct injection of 10(4) prostate cancer cells into human bone implants resulted in large tumors in 75-100% of mice. PC3 and DU145 bone tumors were primarily osteolytic, whereas LNCaP bone tumors were both osteoblastic and osteolytic. PC3 and LNCaP bone tumors showed a desmoplastic stromal response, which indicated intense stromal-epithelial interaction. All three of the cell lines formed tumors in implanted human lung tissue; however, the tumors were all < or = 10 mm3 in volume and showed minimal stromal involvement. No tumors formed after either s.c. injection or injection of cells into implanted mouse bone demonstrating both species- and tissue-specific enhancement of growth of human prostate cancer cells by human bone. The severe combined immunodeficient-human model provides a useful system to study species-specific mechanisms involved in the homing of human prostate cancer cells to human bone and the growth of human prostate cancer cells in human bone.     

Mortality of radiation chimeras in relation to the number of transplanted bone marrow and lymph node cells

Injection of lymph node cells along with therapeutic doses of bone marrow cells into irradiated animals can cause death if the lymphoid cells are capable of reacting against the host animal. The mortality and time of death are proportional to the number of lymphoid cells injected. Death can be as late as 70 days after irradiation. Injection of bone marrow cells alone can cause death but only when very large numbers of cells are given and only in certain donor-host combinations. Great numbers of bone marrow cells raise the death rate after injection of lymph node cells in certain donor-host combinations but counteract this killing effect in other combinations. The number of immunologically competent cells in bone marrow from both CBA and C57BL mice is small. In CBA mice an equivalent number of immunologically competent cells was estimated in 20 x 10(6) bone marrow and in 0.05 to 0.1 x 10(6) lymph node cells; for C57BL mice these figures were 50 x 10(6) bone marrow and 0.4 x 10(6) lymph node cells. To explain why bone marrow can apparently offset the killing effect of lymphoid cells, it is postulated that immunologically tolerant cells developing from pluripotent stem cells in bone marrow compete with immunologically competent lymphatic cells.     

埃本膦酸钠对肺癌骨转移动物模型的干预作用

目的 探讨埃本膦酸钠对肺癌细胞骨转移动物模型的干预作用。方法 通过贴骨接种人小细胞肺癌H446细胞制作骨转移动物模型,皮下注射埃本膦酸钠干预．利用放射学检查及病理分析等手段观察埃本膦酸钠对肺癌细胞骨转移的干预效果。结果 放射学检查显示裸鼠接种H446细胞后引起明显的肿瘤性骨损伤,骨病灶处的99Tcm-MDP放射性浓聚．肿瘤细胞大量侵袭至骨髓腔中。经埃本膦酸钠干预后,此骨损伤病程被明显抑制。结论 埃本膦酸钠可明显地干预肿瘤细胞对骨的侵犯,减轻肿瘤引起的骨损伤,可作为一个治疗骨转移的选择药物。     

A new experimental metastasis model in athymic nude mice, the human malignant melanoma LOX

The human tumor line LOX was established as an s.c. xenograft in nude mice from a lymph-node metastasis of a patient with malignant melanoma. I.v. injection into adult nude mice of single-cell suspensions prepared from xenografts resulted in progressively growing lung tumor colonies that killed the animals. No difference in colony formation was seen between cells taken from lung colonies and s.c. xenografts. An in vitro cell line, LOX-L, was established from lung colonies, and the monolayer cells, detached with EDTA, retained the same ability to form experimental lung metastases. In a total of 14 experiments, 82 of 89 mice receiving 1 X 10(6) viable tumor cells died with a mean survival time of 34.1 +/- 4.8 days. Long-term passaging in vivo and in vitro did not result in any alteration of the lung-colonizing potential of the LOX cells, whereas trypsinization of the cells before i.v. injection reduced lung colony formation. The life span was inversely related to the number of LOX cells injected, permitting estimation of the cell kill caused by chemotherapy. Mice injected i.v. with the LOX cells showed the same relative response to cis-diamminedichloroplatinum (CDDP) and mitozolomide (MZA) as did animals carrying s.c. xenografts. The LOX cells have shown a remarkable stability and similarity to the cells of the patient's tumor with respect to morphology, karyotype and chemosensitivity. The LOX model may be useful for testing effects of therapy on lung micro- and macrometastases, and the activity of antimetastatic agents, as well as for studying mechanisms involved in the metastatic process.     

Cilengitide inhibits metastatic bone colonization in a nude rat model

Integrins αvβ3 and αvβ5 are considered to play an important role in the pathogenesis of breast cancer bone metastases. This study investigates the effects of the αvβ3/αvβ5 integrin-specific inhibitor cilengitide during early metastatic bone colonization. The impact of cilengitide on the migration, invasion and proliferation of MDA-MB-231 human breast carcinoma cells as well as on bone resorption by osteoclasts was investigated in vitro. For in vivo experiments, nude rats were treated with cilengitide for 30 days starting one day after site-specific tumor cell inoculation in the hind leg, and the course of metastatic changes in bone was followed using flat-panel volumetric computed tomography (VCT) and magnetic resonance imaging (MRI). Vascular changes in bone metastases were investigated using dynamic contrast-enhanced (DCE-) MRI-derived parameters amplitude A and exchange rate coefficient kep. In vitro, cilengitide treatment resulted in a decrease in proliferation, migration and invasion of MDA-MB-231 cells, as well as of osteoclast activity. In vivo, the development of bone metastasis in the hind leg of rats was not prevented by adjuvant cilengitide treatment, but cilengitide reduced the volumes of osteolytic lesions and respective soft tissue tumors of developing bone metastases as assessed with VCT and MRI, respectively. DCE-MRI revealed significant changes in the A and kep parameters including decreased relative blood volume and increased vessel permeability after cilengitide treatment indicating vessel remodeling. In conclusion, during early pathogenic processes of bone colonization, cilengitide treatment exerted effects on tumor cells, osteoclasts and vasculature reducing the skeletal lesion size of experimental skeletal metastases.     

PDGFR signaling blockade in marrow stroma impairs lung cancer bone metastasis

Bone microenvironment and cell-cell interactions are crucial for the initiation and development of metastasis. By means of a pharmacologic approach, using the multitargeted tyrosine kinase inhibitor sunitinib, we tested the relevance of the platelet-derived growth factor receptor (PDGFR) axis in the bone marrow (BM) stromal compartment for the initiation and development of lung cancer metastasis to bone. PDGFRβ was found to be the main tyrosine kinase target of sunitinib expressed in BM stromal ST-2 and MC3T3-E1 preosteoblastic cells. In contrast, no expression of sunitinib-targeted receptors was found in A549M1 and low levels in H460M5 lung cancer metastatic cells. Incubation of ST-2 and human BM endothelial cells with sunitinib led to potent cell growth inhibition and induction of apoptosis in a dose-dependent manner. Similarly, sunitinib induced a robust proapoptotic effect in vivo on BM stromal PDGFRβ(+) cells and produced extensive disruption of tissue architecture and vessel leakage in the BM cavity. Pretreatment of ST-2 cells with sunitinib also hindered heterotypic adhesion to lung cancer cell lines. These effects were correlated with changes in cell-cell and cell-matrix molecules in both stromal and tumor cells. Pretreatment of mice with sunitinib before intracardiac inoculation of A549M1 or H460M5 cells caused marked inhibition of tumor cells homing to bone, whereas no effect was found when tumor cells were pretreated before inoculation. Treatment with sunitinib dramatically increased overall survival and prevented tumor colonization but not bone lesions, whereas combination with zoledronic acid resulted in marked reduction of osteolytic lesions and osseous tumor burden. Thus, disruption of the PDGFR axis in the BM stroma alters heterotypic tumor-stromal and tumor-matrix interactions, thereby preventing efficient engagement required for bone homing and osseous colonization. These results support the notion that concomitant targeting of the tumor and stromal compartment is a more effective approach for blocking bone metastasis.     

Bone marrow macrophages support prostate cancer growth in bone

Resident macrophages in bone play important roles in bone remodeling, repair, and hematopoietic stem cell maintenance, yet their role in skeletal metastasis remains under investigated. The purpose of this study was to determine the role of macrophages in prostate cancer skeletal metastasis, using two in vivo mouse models of conditional macrophage depletion. RM-1 syngeneic tumor growth was analyzed in an inducible macrophage (CSF-1 receptor positive cells) ablation model (MAFIA mice). There was a significant reduction in tumor growth in the tibiae of macrophage-ablated mice, compared with control non-ablated mice. Similar results were observed when macrophage ablation was performed using liposome-encapsulated clodronate and human PC-3 prostate cancer cells where tumor-bearing long bones had increased numbers of tumor associated-macrophages. Although tumors were consistently smaller in macrophage-depleted mice, paradoxical results of macrophage depletion on bone were observed. Histomorphometric and micro-CT analyses demonstrated that clodronate-treated mice had increased bone volume, while MAFIA mice had reduced bone volume. These results suggest that the effect of macrophage depletion on tumor growth was independent of its effect on bone responses and that macrophages in bone may be more important to tumor growth than the bone itself. In conclusion, resident macrophages play a pivotal role in prostate cancer growth in bone.     

Increased experimental pulmonary metastasis in pregnant mice

The effect of pregnancy on experimental pulmonary metastasis was studied. Compared to the incidence of pulmonary metastasis induced by G6 cells in non-pregnant mice, the incidence of such metastasis was found to be greatly enhanced when the cells were injected i.v. in the latter half of pregnancy. The maximum enhancement was seen on the 15th day of pregnancy. The incidence of pulmonary metastasis returned to the level observed in non-pregnant mice when the cells were injected 4 days after parturition. Pregnancy also significantly increased the incidence of pulmonary metastasis of 2 other cell lines (3LL and Colon 26). Injection of G6 cells after hysterectomy performed on the 15th day of pregnancy resulted in decreased lung colonization, similar to that seen after parturition. Quantificative analysis of the arrest of G6 cells labeled with [¹²⁵1]-5-iodo-2′-deoxyuridine in the lungs showed that the tumor-cell clearance from the lungs during the 24–72 hr after tumor-cell injection was much slower in pregnant than in non-pregnant mice. The continuous administration of β-estradiol and/or progesterone, which maintained serum levels of the hormones equivalent to those prevailing on the 15th day of pregnancy, did not affect the lung colonization of G6 cells. Tumor-cell-platelet aggregation was more extensive with platelets obtained from mice at the 15th day of pregnancy than with those from non-pregnant mice. When platelets isolated from pregnant mice were injected into normal mice 5 min before G6 injection, lung metastasis was also enhanced. These findings suggest that a pregnant host is handicapped with regard to pulmonary metastasis, this being partly due to increased platelet-aggregating activity in response to tumor cells. © 1995 Wiley-Liss Inc.     

Functional role of platelets in experimental metastasis studied with cloned murine fibrosarcoma cell variants

The involvement of platelets in experimental metastasis was studied with cloned cell lines derived from PAK 17, a recently induced methylcholanthrene-induced C57BL/6 mouse fibrosarcoma. Tumor cell-induced platelet aggregation and lung colonization assays were used to distinguish three major stable phenotypes among the clones: a low metastatic-low platelet aggregating type, e.g., clone PAK 17.12; a low metastatic-high platelet aggregating type, e.g., clone PAK 17.14; and a high metastatic-high platelet aggregating phenotype, e.g., clone PAK 17.15. Clones with high metastatic but low platelet aggregating potential were not observed in the study. Intravenously injected PAK 17.14 and PAK 17.15 cells, but not PAK 17.12 cells, induced greater than 50% reductions in circulating platelet levels in C57BL/6 mice. Since highly metastatic clone PAK 17.15 cells consistently induced high levels of tumor cell-induced platelet aggregation regardless of the platelet donor, it was selected to study the relationship between its tumor cell-induced platelet aggregation and lung colonizing abilities. (a) A 93% decrease in lung colony number resulted in mice injected with 100 micrograms of prostacyclin immediately before injection of clone PAK 17.15 cells. Prostacyclin was also able to inhibit, in a dose dependent fashion (0-5 ng), platelet aggregation induced by clone PAK 17.15 cells in vitro. (b) A 92% reduction in lung colony number occurred in mice showing marked thrombocytopenia following injection of 100 micrograms of rabbit anti-mouse platelet antibody 24 h before tumor cell injection. (c) A greater than 80% reduction in clone PAK 17.15 lung colony number was observed in mice rendered thrombocytopenic by i.v. injection of 0.038 units of neuraminidase 24 h before i.v. injection of 10(5) tumor cells. These results suggest that platelets are required for successful lung colonization by clone PAK 17.15 cells. However, the presence in this fibrosarcoma of high platelet aggregating-poorly metastatic cells, such as clone PAK 17.14, demonstrates that while the ability to aggregate platelets is necessary for successful metastasis by some tumor cells, it is insufficient if tumor cells lack other critical properties required for completion of the metastatic cascade.     

C-C chemokine receptor 5 on stromal cells promotes pulmonary metastasis

We have shown that mice that express the C-C chemokine receptor 5 (CCR5) have enhanced local tumor growth and an impaired response to vaccine therapy compared with CCR5 knockout (CCR5(-/-)) mice. Here, we extend these observations to evaluate the function of CCR5 in pulmonary metastasis and the mechanism underlying the diminished tumor growth in CCR5(-/-) mice. Lung metastases were counted in wild-type (WT) and CCR5(-/-) mice following the injection of 1 x 10(6) B16-F10 melanoma cells. These results were compared with those from syngeneic bone marrow chimeric mice formed by the transfer of WT bone marrow into irradiated CCR5(-/-) and CCR5(-/-) marrow into irradiated WT mice. Intact CCR5(-/-) mice developed fewer metastases than WT mice (40.2 versus 70.6; P < 0.05). Bone marrow chimeras formed by the transfer of WT bone marrow into CCR5(-/-) hosts had fewer metastases than WT hosts injected with knockout marrow (46.6 versus 98.6; P < 0.01). Adoptive transfer of CCR5-expressing leukocytes also failed to promote metastasis in CCR5(-/-) mice. However, the i.v. transfer of WT pulmonary stromal cells into CCR5(-/-) mice increased the number of metastases compared with transfer of CCR5(-/-) stromal cells (102.8 versus 26.0; P < 0.05). These results show for the first time that CCR5 expression on stromal and not hematopoietic cells contributes to tumor metastasis. Therefore, recently developed CCR5 inhibitors may have a novel benefit in cancer therapy.     

DT-5461, a new synthetic lipid A analogue, inhibits lung and liver metastasis of tumor in mice.

We have investigated the antimetastatic effect of a new synthetic lipid A analogue, of low endotoxicity, DT-5461, against two highly metastatic tumor cell lines, L5178Y-ML25 T-lymphoma and B16-BL6 melanoma cells in mice. Four intermittent i.v. administrations of DT-5461 at intervals of 4 days resulted in a significant inhibition of liver metastasis caused by i.v. injection of L5178Y-ML25 cells and lung metastasis of B16-BL6 cells in the experimental metastasis models. Intraperitoneal and intranasal administrations as well as i.v. administration of DT-5461 were also effective in preventing lung metastasis of the melanoma cells. Multiple administrations of DT-5461 before the surgical excision of primary tumors significantly reduced the number of lung colonies of melanoma cells and primary tumor size. Similarly, this treatment modality after the surgical excision of primary tumors showed a greater reduction of lung tumor colonies as compared with lipopolysaccharide, a synthetic lipid A (No. 506) and its analogue as well as untreated control in the spontaneous lung metastasis model. Furthermore, the group that received DT-5461 after the inoculation of lymphoma or melanoma cells showed significantly enhanced survival rate compared with the untreated control. These results suggested that DT-5461 may be therapeutically useful for the inhibition of tumor metastasis.     

DT-5461, a New Synthetic Lipid A Analogue, Inhibits Lung and Liver Metastasis of Tumor in Mice

We have investigated the antimetastatic effect of a new synthetic lipid A analogue, of low endo-toxicity, DT-5461, against two highly metastatic tumor cell lines, L5178Y-ML25 T-lymphoma and B16-BL6 melanoma cells in mice. Four intermittent i.v. administrations of DT-5461 at intervals of 4 days resulted in a significant inhibition of liver metastasis caused by i.v. injection of L5178Y-ML25 cells and lung metastasis of B16-BL6 cells in the experimental metastasis models. Intraperitoneal and intranasal administrations as well as i.v. administration of DT-5461 were also effective in preventing lung metastasis of the melanoma cells. Multiple administrations of DT-5461 before the surgical excision of primary tumors significantly reduced the number of lung colonies of melanoma cells and primary tumor size. Similarly, this treatment modality after the surgical excision of primary tumors showed a greater reduction of lung tumor colonies as compared with lipopolysaccharide, a synthetic lipid A (No. 506) and its analogue as well as untreated control in the spontaneous lung metastasis model. Furthermore, the group that received DT-5461 after the inoculation of lymphoma or melanoma cells showed significantly enhanced survival rate compared with the untreated control. These results suggested that DT-5461 may he therapeutically useful for the inhibition of tumor metastasis.     

Mechanisms of invasion and metastasis in human neuroblastoma

Neuroblastoma is the second most common solid tumor in children that is metastatic in 70% of patients at the time of diagnosis. The ability of neuroblastoma cells to colonize distant organs like the bone marrow and the bone is the result of close interactions between tumor cells and the microenvironment. Significant progress has been recently made in our understanding of the mechanisms that promote the colonization and invasion of the bone by neuroblastoma cells and these mechanisms are reviewed in this article. How this understanding is now allowing us to test new therapeutic agents specifically targeted at interfering with neuroblastoma metastasis is then discussed.     

The serum glycoprotein fetuin-A promotes Lewis lung carcinoma tumorigenesis via adhesive-dependent and adhesive-independent mechanisms

Fetuin-A is a serum glycoprotein in the cystatin family associated with the regulation of soft tissue calcification. We tested the role of systemic fetuin in tumor cell growth and metastasis by injecting Lewis lung carcinoma (LLC) cells into fetuin-A null and their wild-type (WT) littermate control C57BL/6 mice via the tail vein, s.c., and intrasplenic routes. In the experimental metastasis assay, the lungs of the WT mice were filled with metastatic nodules, whereas the lungs of the fetuin-A null mutant mice were virtually free of colonies at the end of 2 weeks. Lung colonization responded to the levels of serum fetuin-A in a dose-dependent manner, as observed by the formation of half as many colonies in mice heterozygous for the fetuin-A locus compared with homozygous WT mice and restoration of lung colonization by the administration of purified fetuin-A to fetuin-A-null mice. Serum fetuin-A also influenced the growth of LLC cells injected s.c.: fetuin-A-null mice developed small s.c. tumors only after a substantial delay. Similarly, intrasplenic injection of LLC cells resulted in rapid colonization of the liver with metastasis to the lungs within 2 weeks in the WT but not fetuin-A null mice. To examine the mechanism by which fetuin-A influences LLC colonization and growth, we showed that LLC tumor cells adhere to fetuin-A in a Ca(2+)-dependent fashion, resulting in growth of the tumor cells. These studies support the role of fetuin-A as a major growth promoter in serum that can influence tumor establishment and growth.