Search

Bone is one of the most common sites of breast cancer metastasis. Metastases are often associated with bone destruction and are a major cause of morbidity. We examined structural bone changes induced by metastatic tumor in bone biopsies from 33 patients with metastatic breast carcinoma (20 from patients with pathological femoral fracture and 13 with no fracture) and 20 normal controls. In all metastatic biopsies bone remodeling was shown to be tumor volume-dependent. Bone resorption and bone formation were biphasic with both increasing at earlier stages of metastatic bone disease and decreasing later on. A comparison of patients with fracture and no fracture did not reveal statistically significant differences in the extent of bone destruction or trabecular thinning. Bone histomorphometry showed limited ability to explain the higher bone volume loss in fracture patients (decreases of 42% and 25%, respectively, in fracture and nonfracture patients compared with controls). However, changes in bone quality, including increased disconnectivity and decreased connectivity, as evaluated by node-strut analysis, suggested that there were more structural changes in the fracture compared with the nonfracture group. The nonfracture group included six patients with no radiological evidence of bone metastasis (occult metastasis). They showed a higher tumor volume and a twofold lower eroded surface compared with the rest of the group. The decrease in bone volume (14% lower than controls) was below the limit of X-ray detection. Because we observed no increase in osteoclast-related parameters and no correlation between osteoclast surface and eroded surface, we believe that, in occult metastasis, osteoclastic bone resorption is not an important factor in overall bone resorption. Quantitatively, the eroded surface in direct contact with tumor cells was threefold higher than the osteoclast surface in occult metastasis, whereas the rest of the metastatic group (27 of 33) showed predominantly osteoclast-mediated eroded surface. Node-strut analysis on occult metastasis revealed a significant increase in disconnectivity without a concomitant significant decrease in bone volume and trabecular thinning. We conclude that, in occult metastasis, bone resorption may be more osteoclast-independent and other mechanisms involving the tumor cells may be more prevalent., (Copyright 2002 Elsevier Science Inc.)

We have previously shown that circulating intravascular cells generally arrest by mechanical restriction in the hepatic sinusoids, causing rapid release of nitric oxide (NO) which is cytotoxic to these cells and inhibits their growth into metastatic tumours. Here, we present evidence that these NO-dependent cytotoxic mechanisms are susceptible to upregulation by lipopolysaccharide (LPS). Five x 10(5) fluorescently labelled melanoma cells were injected into the mesenteric vein of C57BL/6 mice to effect their localisation in the hepatic microvasculature. Test mice were then given 1 mg/kg LPS intraperitoneally (i.p.) to activate the microvascular cells. By electron paramagnetic resonance (EPR) spectroscopy, the expression of NO in the liver was significantly increased by 8 h in the LPS-treated mice. The non-selective NO synthase inhibitor L-NAME inhibited the induction of NO by LPS, while its inactive enantiomer D-NAME had no significant effect. Using immunohistochemistry (IHC), iNOS-positive microvascular cells were detected in the terminal portal venule (TPV) region of the liver 8 h after LPS stimulation. LPS treatment also increased the retention of melanoma cells in the liver between 8 and 24 h, especially in the TPV region. Eight hours after cell injection, local expression of VCAM-1 and ICAM-1 was detected by double-label immunohistochemistry at the sites of tumour cell arrest. Expression of these adhesion molecules was enhanced in mice treated with LPS. Using flow cytometry, 98% of the B16F1 melanoma cells expressed VLA-4, the counter receptor of VCAM-1, and approximately 1.5% expressed LFA-1, the counter receptor of ICAM-1. LPS did not significantly alter the expression of either counter receptor on melanoma cells in vitro or in vivo. By DNA end-labelling, the rates of melanoma cell apoptosis were significantly increased from 8 to 24 h in the TPV region (but not in the sinusoids) of LPS-treated mice. Fourteen days after tumour cell injection, the LPS-treated mice had a significantly smaller hepatic metastatic tumour burden than the control mice. These data suggest that LPS can inhibit the metastasis of melanoma cells in the liver by inducing the expression of NO and adhesion molecules by the hepatic endothelium. The induction of iNOS and the inducible cytotoxic effect of LPS appear to be primarily located within the TPV region of the liver acinus.

Blood vessels facilitate the widespread dissemination of cancer cells in metastasis. Interactions between circulating intravascular cancer cells and the microvasculature involve mechanical contact and transient attachment, mediated by endothelial surface adhesion molecules and their ligands on the neoplastic cells. Initial interactions trigger a sequence of activation pathways that involve cytokines, growth factors, bioactive lipids, and reactive oxygen species produced by the cancer cell or the endothelium. These activation steps elicit the expression of integrin adhesion molecules in cancer cells and the endothelium, matrix metalloproteinases, and chemotactic factors that promote firm attachment of tumor cells to the vessel wall and transvascular penetration. On the other hand, induction of endothelial free radicals can be cytotoxic to cancer cells. Collectively, the sum of these interactions act as a rate-regulating step in the metastatic process.

The formation of liver metastases involves interactions between intravascular cancer cells and the hepatic microvasculature. Here we provide evidence that the arrest of intravascular B16F1 melanoma cells in the liver induces a rapid local release of nitric oxide (NO) that causes apoptosis of the melanoma cells and inhibits their subsequent development into hepatic metastases. B16F1 melanoma cells (5 x 10(5)) labeled with fluorescent microspheres were injected into the portal circulation of C57BL/6 mice. The production of NO in vivo was detected by electron paramagnetic resonance spectroscopy ex vivo using an exogenous NO-trapping agent. A burst of NO was observed in liver samples examined immediately after tumor cell injection. The relative electron paramagnetic resonance signal intensity was 667 +/- 143 units in mice injected with tumor cells versus 28 +/- 5 units after saline injection (P < 0.001). Two-thirds of cells arrested in the sinusoids compared with the terminal portal venules (TPVs). By double labeling of B16F1 cells with fluorescent microspheres and a TdT-mediated UTP end labeling assay, we determined that the melanoma cells underwent apoptosis from 4-24 h after arrest. The mean rate of apoptosis was 2-fold greater in the sinusoids than in the TPVs at 4, 8, and 24 h after injection (P < 0.05-0.01). Apoptotic cells accounted for 15.9 +/- 0.8% of tumor cells located in the sinusoids and 7.1 +/- 0.9% of tumor cells in the TPVs. The NO synthase inhibitor N(G)-nitro-L-arginine methyl ester completely blocked the NO burst (P < 0.001) and inhibited the apoptosis of B16F1 cells in the sinusoids by 77%. However, the rate of tumor cell apoptosis in the TPVs was not changed. There were 5-fold more metastatic nodules in the livers of N(G)-nitro-L-arginine methyl ester-treated mice (P < 0.05). The inactive enantiomer N(G)-nitro-D-arginine methyl ester had no effect on the initial NO burst or on apoptosis of tumor cells in vivo. Both annexin V phosphatidylserine plasma membrane labeling and DNA end labeling of apoptotic cells were demonstrated after a 5-min exposure (a time equivalent to the initial transient NO induction in vivo) of B16F1 cells to a NO donor in vitro. These results identify the existence of a natural defense mechanism against cancer metastasis whereby the arrest of tumor cells in the liver induces endogenous NO release, leading to sinusoidal tumor cell killing and reduced hepatic metastasis formation.

Background: This review summarizes evidence that the formation of bone metastases is the result of multiple synergistic cellular and molecular interactions between metastatic cells and the unique microenvironment in bone., Methods: Molecular technologies have been used to detect cancer cells in bone and to define their genotypic and phenotypic properties. Bone organ cultures have been employed to analyze the ability of tumor cells to modulate bone resorption and to study the effects of resorption products on the phenotypic properties of cancer cells. Experimental models of bone metastasis provide the ability to examine the effects of modulating specific host or tumor properties in vivo by quantifying their effects on the formation of bone tumors., Results: By means of the blood stream, cells from many common neoplasms seed bone marrow as an early clinical event. The subsequent growth of these cells into clinically significant metastatic lesions is associated with their ability to stimulate bone resorption through osteoclasts and macrophages or through a direct action on bone. In turn, the products of bone resorption, which include matrix-derived growth factors, act on the tumor cells to stimulate the expression of properties that promote their metastatic competence. These include the induction of integrin adhesion molecules, the stimulation of cell motility and chemotaxis, the enhanced expression of matrix metalloproteinases, and the stimulation of tumor cell growth., Conclusions: The interdependency of tumor cells and bone was recognized by Steven Paget over 100 years ago, and it provides a rational basis for the development of current therapeutic strategies against bone metastasis.

The haematogenous phase of cancer metastasis facilitates the transport of metastatic cells within the blood and incorporates a sequence of interactions between circulating intravascular cancer cells and the endothelium of blood vessels at the sites of tumour cell arrest. Initial interactions involve mechanical contact and transient adhesion, mediated by endothelial selectins and their ligands on the neoplastic cells. This contact initiates a sequence of activation pathways that involves cytokines, growth factors, bioactive lipids, and reactive oxygen species produced by either the cancer cell or the endothelium. These molecules elicit expression of integrin adhesion molecules in cancer cells and the endothelium, matrix metalloproteinases, and chemotactic factors that promote the attachment of tumour cells to the vessel wall and/or transvascular penetration. Induction of endothelial free radicals can be cytotoxic to cancer cells. Collectively, the sum of these interactions constitutes an interdependent relationship, the outcome of which determines the fate of the metastatic process., (Copyright 2000 John Wiley & Sons, Ltd.)

Bone metastases occur in approximately 80% of patients with advanced cancer. They are characterized by cancer cell growth and bone destruction that cause pain, fractures, anemia, and hypercalcemia. At diagnosis, bone metastases are usually incurable owing to their advanced development. However, the early stages in their formation are asymptomatic and begin as single micrometastatic cells from the blood stream. These cells can be detected by molecular analysis of bone marrow in approximately 30% of patients at the time of cancer diagnosis, but not all single micrometastatic cells develop into clinically significant bone metastases. A synergistic relationship exists between the micometastasis and the bone environment creating favorable conditions for the development and growth of disseminated tumor cells. Such bone metastases induce osteolysis or new bone formation, releasing growth factors and cytokines, which in turn amplify this pathological mechanism. The underling hypothesis, first proposed by Paget in 1889, is that the growth of disseminated tumor cells in bone is dependent on the fertility of the soil or bone itself. This article explores the most current opinions in this area of study and presents a comprehensive summary of the major factors involved.

The structural and functional heterogeneity of hepatocytes and non-parenchymal cells across the liver lobule or acinus has been well documented. The geographic distribution and potential for induced expression of adhesion molecules on murine hepatic microvascular cells has not been reported, although these molecules are able to influence the metastatic outcome of intravascular cancer cells. We have postulated that the expression of adhesion molecules on these cells is susceptible to regulation by environmental factors and that these molecules have a zonal distribution across the acinus. To test this hypothesis, we injected C57BL/6 mice with bacterial lipopolysaccharide, 1 microg/g body weight, i.p. At various time points (0-48 h) after stimulation, liver tissue sections were prepared for immunohistochemistry. Confocal microscopy was used to detect the expression of vascular cell adhesion molecule-1 (VCAM-1), E-selectin, intercellular adhesion molecule-1 (ICAM-1) and alpha v integrin. The expression patterns were quantitatively measured by histomorphometry. Under basal conditions, ICAM-1 was weakly expressed in terminal portal veins while minimal VCAM-1 and no E-selectin were detected. Following stimulation with lipopolysaccharide, VCAM-1 and E-selectin were expressed on the endothelium of terminal portal veins and on sinusoidal lining cells with significantly stronger expression in the periportal zone than midzone. VCAM-1 expression peaked at 4 h and decreased gradually by 48 h. E-selectin peaked at 2 h and disappeared by 12 h after stimulation. ICAM-1 expression showed a much stronger and more uniform expression across the acinus with the peak reached by 4 h and sustained for longer than 48 h after lipopolysaccharide administration. The alpha v integrin was not detected under basal conditions or after lipopolysaccharide stimulation. Expression of all these adhesion molecules (ICAM-1, VCAM-1, E-selectin and alpha v integrin) was induced by growth of B16F1 melanoma cells in the peritoneal cavity of the mouse. These results support the hypotheses that expression of microvascular adhesion molecules in the mouse liver is susceptible to regulation by environmental stimuli and has a zonal heterogeneity across the acinus.

Within the tumor-stromal microenvironment a disrupted balance between matrix metalloproteinases (MMPs) and their inhibitors compromises the integrity of the extracellular matrix and promotes malignancy. Tissue inhibitors of metalloproteinases (TIMPs) have been linked to tumor suppression in studies of genetically altered tissue culture cells and in analyses of clinical specimens in situ. We generated transgenic mice as a model system to test the relationship between TIMP-1 levels in a host organ and susceptibility to experimentally targeted metastasis. Ectopically overexpressed TIMP-1 in the brain resulted in a tissue microenvironment with elevated protein levels of this natural MMP inhibitor. Metastatic challenge provided by lacZ-tagged fibrosarcoma cells permitted high-resolution analysis of metastatic load and pattern. We found that elevated host TIMP-1 imposed resistance to experimental metastasis of fibrosarcoma: In TIMP-1 overexpressing mice, brain metastases were significantly reduced by 75% compared to wild-type littermates. Our findings demonstrate that ectopic TIMP-1 expression efficiently exerts a suppressive effect on metastasizing tumor cells.

Bone metastases are often associated with osteolysis and subsequent pathological fractures. To determine if metastatic human cancer cells can directly degrade non-mineralized and mineralized bone, we used prostate PC3 adenocarcinoma cell lines, which were originally established from skeletal metastases. We show that PC3 cells and their conditioned medium degraded non-mineralized, osteoid-like radiolabelled extracellular matrices from human Saos2 and U2OS osteoblast-like cells. These cells also directly degraded mineralized bone by inducing (45)Ca release from rat fetal calvariae and forming resorption pits on bone slices, an effect increased by transforming growth factor-beta(1). A role for matrix metalloproteinases in degradation was shown by: (1) stimulation by the phorbol ester TPA of PC3-induced matrix degradation and release of matrix metalloproteinase activity; (2) abrogation of matrix degradation by 1,10-phenanthroline, a metalloproteinase inhibitor, and (3) degradation of purified type I collagen by PC3 cells and their conditioned medium. We demonstrate that human prostate cancer cells can directly degrade bone-related matrices and that matrix metalloproteinases have a role in this process., (Copyright 2000 S. Karger AG, Basel.)

Basement membranes form a boundary between intravascular and extravascular compartments that is remodeled by matrix metalloproteinases (MMP) expressed by endothelial cells. These cells are at risk of exposure to reactive oxygen intermediates generated as a consequence of interactions with drugs, x-radiation, activated neutrophils, or cancer cells. Herein we have investigated the hypothesis that endothelial cells alter their expression of MMP after sublethel exposure to H2O2 and that this leads to degradation of adjacent basement membranes. Cultured human umbilical vein endothelial cells were treated with concentrations of H2O2 ranging from 1.5 to 32 microM or with 2 x 10(-6)M phorbol myristate acetate (PMA). After 24 hours, the cells were placed into serum-free medium for an additional 24 hours. This conditioned medium or cell lysates were studied by matrix degradation assays, gelatin zymography, immunoblots, and Northern analysis. H2O2-treated or PMA-treated cells, or their serum-free conditioned medium, caused a 2-fold increase in degradation of [3H]-proline-labeled endothelial basement membranes or purified type IV collagen compared to untreated cells. Endothelial cells constitutively expressed gelatinases at Mr 96,000 and 72,000, consistent with MMP-9 and inactive MMP-2. H2O2 exposure caused increased expression of these MMP and appearance of Mr 64,000 to 66,000 gelatinases corresponding to activated MMP-2. In cell lysates, H2O2 or PMA treatment led to increased expression of membrane-type MMP-1, an activator of latent MMP-2. The results suggest that oxidants such as H2O2 may stimulate MMP expression and influence the remodeling of vascular basement membranes by endothelial cells.

There have been few reported visual observations of metastatic cancer cell arrest in vivo. To seek evidence that inducible vascular adhesive properties can regulate hepatic metastasis, groups of 9-14 c57bl/6 mice were given 1.5 microg of interleukin-1alpha (IL-1alpha) 4 h before the injection of 3 x 10(5) B16F1 melanoma cells into a mesenteric vein. After 7 days, these mice had an 11-22-fold greater hepatic tumor burden than controls given i.p. saline. In both groups, small metastases were seen in the portal tract region. Twice as many 125I-labeled UdR-labeled B16F1 cells were detected in the livers of IL-1alpha-treated animals 5 min after injection, and 7 times as many were found after 24 h. Intravital videomicroscopy showed marked differences in the arrest pattern of the B16F1 cells between controls and IL-1alpha-treated mice. In controls, arrest occurred at a median distance of 32 microm beyond the sinusoidal inlet, where the median sinusoidal diameter was 16 microm. However, in IL-1alpha-treated mice, arrest occurred in the presinusoidal portal vein branches, which had a median diameter of 34 microm. Maximum observed tumor cell velocities were 2-fold less in the IL-1alpha-treated mice, although there was no significant difference in the flow rate of RBCs. To look for effects on the adhesive properties of the hepatic microvasculature, 5 x 10(4) B16F1 cells were incubated for 15 min on 5-microm sections of liver from control and IL-1alpha-treated mice. Three-fold more cells adhered to sections of liver from IL-1alpha-treated mice. This phenomenon was blocked by GRGDS peptides and by antibodies to E-selectin, ICAM-1, VCAM-1, and the alpha v integrin subunit. We postulate that pretreatment of mice with IL-1alpha alters a number of adhesive interactions between B16F1 cells and the hepatic microvasculature, contributing to the site of arrest and to the subsequent fate of the arrested cells.

Prostate cancer frequently metastasizes to bone, and we propose that this process may be facilitated by the adhesion of metastatic cells to bone-derived type I collagen. We examined collagen receptor function and regulation in osteotropic PC-3 human prostatic carcinoma cells. PC-3 cell adhesion to immobilized human type I collagen was promoted by Mn2+ and Mg2+ ions and was RGD-independent. Antibodies directed against beta1 or alpha2 integrin subunits inhibited adhesion to collagen by 90% and 53%, respectively, suggesting involvement of the alpha2 beta1 receptor. Anti-alpha1 or anti-alpha3 antibodies had no effect on adhesion. Flow cytometry and immunoprecipitation of [35S]methionine-labeled cells demonstrated that alpha2 beta1 was the major collagen receptor expressed by PC-3 cells. The pretreatment of PC-3 cells with transforming growth factor-beta1 (TGF-beta1), a major bone-derived growth factor, caused a rapid (2 h) 2-fold increase in the de novo synthesis of alpha2 and beta1 integrin subunits, and also increased by 2- to 3-fold the adhesion and spreading of PC-3 cells on collagen. We conclude that alpha2 beta1 is the major collagen receptor employed by PC-3 cells, and that alpha2 beta1 upregulation by TGF-beta is associated with an increased adhesion and spreading on collagen. The data suggest that exposure of metastatic PC-3 cells to the high levels of TGF-beta in bone may promote their ability to adhere to bone-derived collagen, which may thereby facilitate the localization of metastatic cells in the skeleton.

The potential of tissue inhibitors of metalloproteinases (TIMPs) to inhibit neoplastic progression has been postulated from studies of genetically manipulated cells. To investigate whether the TIMP-1 expressed in a host tissue suppresses cancer in vivo and to identify the affected stages, we developed transgenic mice with constitutive overexpression or reduction of TIMP-1 in the liver. In double transgenic experiments, the TIMP-1 lines were crossed with a second transgenic line which expresses the Simian Virus 40t/T antigen (TAg). This viral oncogene leads to heritable development of hepatocellular carcinomas with a 100% incidence. Effects of TIMP-1 coexpression on the TAg-induced neoplasms were determined at the tissue and cellular level. Here, we report that overexpression of hepatic TIMP-1 blocked the development of TAg-induced hepatocellular carcinomas. High TIMP-1 levels inhibited not only the later stages in tumor development (growth and angiogenesis), but also events associated with tumor initiation (altered hepatocyte cytology and tissue architecture). We further show that an antisense-mediated reduction of TIMP-1 resulted in a more rapid tumor initiation and progression. These data demonstrate that intrinsic TIMP-1 levels contribute to a tissue's susceptibility to viral oncogene-induced tumorigenesis.

Prostatic carcinoma cells have a propensity to metastasize to bone, and we propose that this phenomenon may be promoted by the adhesion of metastatic cells to bone matrix. Bone matrix is produced by osteoblasts, and we have developed an in vitro model of bone matrix by isolating the substratum deposited by human osteoblast-like U2OS cells. The collagenous nature of this matrix was demonstrated by the incorporation of [3H]proline and its subsequent release by purified collagenase. Both U2OS matrix and purified type I collagen stimulated the adhesion of human PC-3 prostatic carcinoma cells. Human laminin supported adhesion to a much lesser extent, and PC-3 cells did not adhere to fibronectin. Adhesion of PC-3 cells to U2OS matrix closely resembled adhesion to purified type I collagen with respect to (a) inhibition by a collagen-derived peptide and by antibodies raised against alpha 2 or beta 1 integrin collagen receptor subunits; (b) lack of inhibition by RGD (Arg-Gly-Asp) peptides; (c) stimulation by Mn2+ and Mg2+ ions but not by Ca2+ ion; and (d) stimulation by the phorbol ester PMA (phorbol 12-myristate 13-acetate). This adhesion was also stimulated (2.3-fold) by transforming growth factor beta (TGF-beta), which is a major bone-derived growth factor. We conclude that human osteoblast-like matrix is an adhesive substrate for PC-3 prostate carcinoma cells. This adhesion appears to be mediated by the interaction of alpha 2 beta 1 integrin on PC-3 cells with matrix-derived collagen. The stimulation of this adhesion by TGF-beta suggests that the co-expression of TGF-beta and type I collagen in bone may synergistically facilitate the adhesion of metastatic cells to bone matrix proteins and thereby increase their localization in the skeleton.

Bone metastases are a frequent clinical problem in patients with breast, prostate, and other cancers. Formation of these lesions is a site-specific process determined by multiple cellular and molecular interactions between the cancer cells and the bone microenvironment. Clinical studies, and in vivo and in vitro experimental approaches, have been useful to dissect different stages of this process. Mechanisms identified as relevant to cancer spreading and tumoral growth in the bones include (a) early vascular spread of cancer cells to bones; (b) adhesion of cancer cells to the bone microvasculature and matrix components; (c) presence of growth factors and chemo-attractants in bone; (d) osteolysis by osteoclasts, tumor associated macrophages, and cancer cells; and (e) tumor-induced local osteoblastic proliferation. Although none of these mechanisms alone are responsible for the development of bone metastases, their investigation may lead to novel therapeutic approaches that specifically block these stages and, thus, may hinder development of bone metastasis. The use of bisphosphonates and other experimental strategies already is being tested in clinical trials.

Background: Pulmonary endothelial damage can be caused by agents that generate oxidants, e.g., bleomycin, hyperoxia, neutrophils or x-irradiation. In animals with intravascular cancer cells, there is increased tumor cell arrest and the subsequent formation of metastatic tumors at the sites of such endothelial injury. We have previously shown that Walker 256 (W256) tumor cells, stimulated with phorbol esters (phorbol 12-myristate 13 acetate) or the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine, generate chemiluminescence that is inhibitable by catalase. Such activated cells can injure cultured endothelial monolayers. The purpose of the present study was to quantify and obtain morphological confirmation of the generation of reactive oxygen species by W256 cells in vitro, and to determine if this phenomenon could be morphologically detected in vivo during the metastatic process., Experimental Design: The production of oxidants from W256 cells was quantitated in vitro by the scopoletin fluorescence assay, by a ferrithyiocyanate colorimetric assay (Thurman reaction), and confirmed morphologically, in vitro and in vivo, by the formation of cerium perhydroxide (Ce[OH]2OOH) deposits from cerium chloride (CeCl3). To demonstrate generation of reactive oxygen species in vivo, we examined W256 cells collected from the pulmonary circulation and at sites of spontaneous metastasis in the lung after intramuscular tumor transplantation, or cells arrested in the lungs after intravenous injection. The specificity of the CeCl3 reaction was confirmed by blocking in the presence of catalase., Results: As measured by the loss in scopoletin fluorescence and by generation of ferrithiocyanate 5 x 10(6) activated W256 cells produced an equivalent of 18 nM of H2O2 per hour A. Ce-[OH]2OOH deposits were identified in vitro on the surface of W256 cells, and at points of attachment between W256 cells and cultured endothelial cell monolayers. In vivo, CeCl3-derived deposits were seen on circulating W256 cells and on W256 cells that had arrested in the lungs following the intravenous injection of activated or non-activated W256 cells, or in spontaneous pulmonary metastases which formed after intramuscular tumor inoculation. Pretreatment of tumor-bearing animals with phorbol 12-myristate 13 acetate increased the number of CeCl3-derived deposits more than 2 fold. Catalase inhibited the formation of the electron-dense deposits in vitro and in vivo., Conclusions: These data provide morphologic evidence that cancer cells can produce reactive oxygen species in vivo and suggest that free radicals might contribute to endothelial damage during the metastatic process.

Adhesion of cancer cells to endothelium is thought to be a prerequisite to extravasation during the haematogenous phase of metastasis, and is enhanced after perturbation of the endothelium by interleukin-1 (IL-1). The inducible endothelial adhesion molecules, E-selectin, VCAM-1/alpha 4 beta 1 and vitronectin receptor have been reported to mediate attachment of cancer cells to IL-1-treated endothelial cells. We have examined the relative contribution of these molecules by quantifying the adhesion of a panel of 22 human, 125I-labelled cancer cells and the rat W256 tumour to untreated and IL-1-treated endothelial monolayers in the presence of relevant neutralising antibodies. Antibodies against E-selectin inhibited the adhesion of HL-60 leukaemia cells and two colon carcinomas. Anti-alpha 4 beta 1 antibodies blocked adhesion of four melanomas, five sarcomas and one lung carcinoma. Anti-vitronectin receptor antibodies inhibited adhesion of 14 of the 22 human cell lines to IL-1-treated endothelial cells. Adhesion of seven cell lines was inhibited by more than a single antibody. In contrast, adhesion of one of the cancer cell lines was unaffected by any of the antibodies, suggesting involvement of other IL-1-inducible endothelial adhesion molecules. Moreover, none of the antibodies altered the attachment of cancer cells to unstimulated endothelial monolayers. We conclude that the mechanisms of cancer cell adhesion to the endothelium are influenced by endothelial activation and by the adhesive repertoire of the cancer cell.

We have studied the effect of 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (APD) on the morphology of rat bone and the metastatic behavior of Walker 256 (W256) cancer cells in the rat skeleton. Male Fischer rats (150-175 g) received s.c. injections for 7 days with APD (0.5 mg/kg body weight/day) (+ APD; n = 20) or with vehicle (-APD; n = 20). Subsequently, 10 + PD and 10 -APD rats received i.m. injections with W256 cells (+ W256), and the remaining rats received injections of vehicle (-W256). All rats were killed 14 days later. Trabecular bone volume was increased by 46 +/- 3% by APD treatment alone and was decreased by 56 +/- 7% (SEM) by W256 tumor burden alone. After 14 days of tumor burden, + APD/+ W256 rats had 3-fold more trabecular bone than did -APD/+W256 rats. Despite this bone-sparing effect, APD treatment of +W256 rats was associated with a 2.6-fold increase in skeletal tumor burden, while metastatic tumor burden in the liver, lungs, and kidneys was unaffected. The increased skeletal tumor burden in + APD/+ W256 rats was accompanied by an increase in the growth rate of W256 cells located in bone. Independent of APD treatment, W256 cells located adjacent to trabecular bone surfaces had greater growth rates than did W256 cells in the marrow, located > 50 microns from trabecular bone. In summary, the APD-induced increase in trabecular bone volume in rats is associated with a selective increase in skeletal tumor burden and an increased growth rate of W256 cells in the skeleton.

This article describes various adhesion molecules and reviews evidence to support a mechanistic role for adhesion molecules in the process of cancer metastasis. A variety of evidence supports the involvement of specific adhesion molecules in metastasis. 1. For example, some cancer cells metastasize to specific organs, irrespective of the first organ encountered by the circulating cancer cells. This ability to colonize a specific organ has been correlated with the preferential adhesion of the cancer cells to endothelial cells derived from the target organ. This suggests that cancer cell/endothelial cell adhesion is involved in cancer cell metastasis and that adhesion molecules are expressed on the endothelium in an organ-specific manner. 2. Further, inclusion of peptides that inhibit cell adhesion, such as the YIGSR- or RGD-containing peptides, is capable of inhibiting experimental metastasis. 3. Metastasis can be enhanced by acute or chronic inflammation of target vessels, or by treatment of animals with inflammatory cytokines, such as interleukin-1. In vitro, cancer cell/endothelial cell adhesion can be enhanced by pretreating the endothelial cell monolayer with cytokines, such as interleukin-1 or tumor necrosis factor-alpha. This suggests that, in addition to organ-specific adhesion molecules, a population of inducible endothelial adhesion molecules is involved and is relevant to metastasis. 4. Further support for this model is found in the comparison to leukocyte/endothelial adhesion during leukocyte trafficking. Convincing evidence exists, both in vivo and in vitro, to demonstrate an absolute requirement for leukocyte/endothelial adhesion before leukocyte extravasation can occur. The relevance of this comparison to metastasis is reinforced by the observation that some of the adhesion molecules involved in leukocyte/endothelial adhesion are also implicated in cancer cell/endothelial adhesion. The involvement of adhesion molecules suggests a potential therapy for metastasis based on interrupting adhesive interactions that would augment other treatments for primary tumors.

The invasion of blood vessel walls is a critical step in cancer metastasis, in which endothelial cells and their vascular basement membranes act as barriers to tumor cell passage. Here we report that Walker 256 carcinosarcoma (W256) cells degrade subendothelial matrices by a process involving both the generation of hydrogen peroxide and the secretion of a matrix metalloproteinase. As an assay of basement membrane degradation, [3H]proline-labeled subendothelial matrices were exposed to W256 cells in the presence or absence of the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). The release of [3H]proline, in the presence of 5 x 10(6) W256 cells, was increased from 49 +/- 2.5 to 64 +/- 2.2% by the addition of 10(-6) M fMLP. In the presence of fMLP-activated W256 cells, [3H]proline release was completely inhibited by the addition of 2000 units/ml catalase or by the metalloproteinase inhibitors 1,10-phenanthroline and EDTA at concentrations > or = 10 micrograms/ml. alpha 1-Antitrypsin or alpha 2-macroglobulin were without effect. Cell-free supernatants obtained from activated W256 cells were also able to promote basement membrane degradation. Electrophoresis of the cell-free supernatants from fMLP or PMA-activated W256 cells in gelatin-containing sodium dodecyl sulfate-polyacrylamide gels revealed a major band of gelatinolytic activity at 94 kDa. The 94-kDa band represented the activity of a latent gelatinase since incubation with 1 mM 4-aminophenylmercuric acetate (APMA; a known activator of latent metalloproteinases) resulted in the loss of gelatinolytic activity at 94 kDa and the appearance of five new bands of lower molecular weight (M(r) 86, 79, 74, 70, and 66 kDa). Two of these lower molecular weight bands (M(r) 86 and 66 kDa) were also detected in the absence of APMA, following 10-fold concentration of the cell-free supernatants. When the cell-free supernatants of phorbol myristate acetate-activated W256 cells (concentrated 10-fold) were incubated with increasing concentrations of hydrogen peroxide (35 to 70 mM), the band at 66 kDa demonstrated enhanced gelatinolytic activity. We suggest that W256 cells can secrete a latent metalloproteinase of molecular weight 94 kDa which, when activated by hydrogen peroxide, can degrade subendothelial matrices.

Blood/vessel wall cell interactions depend, in part, on the expression of adhesion receptors on cell surfaces, such as expression of the vitronectin receptor (VnR) on the apical surface of endothelial cells (ECs) for platelet/EC adhesion. However, it is unclear how receptor expression is regulated from within cells. In previous studies, we found that ECs metabolize linoleic acid into the lipoxygenase monohydroxide, 13-hydroxyoctadecadienoic acid (13-HODE), and that the intracellular level of 13-HODE correlates inversely with VnR expression and platelet adhesion to the EC apical surface. In this study, we determined the physical associations of 13-HODE and VnR in unstimulated and stimulated ECs, ie, at times when ECs were and were not adhesive for specific ligands and platelets, using double antibody immunofluorescent staining techniques and binding assays. 13-HODE and the VnR were colocalized within unstimulated ECs. When ECs were stimulated, 13-HODE was no longer detectable, either in or outside the ECs, and the VnR was detected on the apical surface of the ECs. These changes were paralleled by increased vitronectin binding and increased platelet adhesion to the ECs. We suggest that colocalization of 13-HODE with VnR reflects a 13-HODE/VnR interaction, confining the VnR in a nonadhesive form inside unstimulated ECs, and, as a result, the ECs are nonadhesive. When the ECs are stimulated, 13-HODE and VnR dissociate, allowing the VnR to relocate on the EC surface, where the VnR undergoes a conformational change resulting in increased EC adhesivity.

Previously, we have demonstrated that stimulation of endothelial cells (ECs) with interleukin-1 alpha (IL-1 alpha) enhances the synthesis and expression of the vitronectin receptor (VnR), promotes VnR-dependent adhesion of human A549 adenocarcinoma cells to ECs, and is associated with decreased EC 13-hydroxyoctadecadienoic acid (13-HODE) synthesis in vitro. To determine whether these observations are relevant in vivo, we examined the acute retention and subsequent metastasis of intravenously-injected B16F10 melanoma cells in murine lungs, in relation to vessel wall 13-HODE. In C57BL/6 mice pretreated with IL-1 alpha, vessel wall 13-HODE was decreased and B16F10 lung entrapment and metastasis were increased. The latter two events were blocked by pretreating the animals with the GRGDS peptide. These data suggest a relationship between vessel wall 13-HODE synthesis, adhesion molecule expression, and adhesion of B16F10 cells to the endothelium.