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.

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 ®brosar-coma cells permitted high-resolution analysis of metastatic load and pattern. We found that elevated host TIMP-1 imposed resistance to experimental metastasis of ®brosarcoma: In TIMP-1 overexpressing mice, brain metastases were signi®cantly reduced by 75% compared to wild-type littermates. Our ®ndings demonstrate that ectopic TIMP-1 expression eciently exerts a suppressive eect on metastasizing tumor 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.

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.

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