Comparison of in vitro and in vivo approaches to studying brain colonization by breast cancer cells.

Brain metastases occur in 20 to 40% of patients with metastatic breast cancer. The process is complex and depends on successful cancer cell evasion from the primary tumor, distribution and survival within the blood stream and cerebral microvasculature, penetration of the blood brain barrier and proliferation within the brain microenvironment. The initial steps of brain colonization are difficult to study in vivo. Therefore, in vitro assays have been developed to mimic this process. Most commonly, in vitro studies of brain colonization focus on tumor cell adhesion to brain endothelial cells and transendothelial migration. We previously investigated breast cancer brain colonization from the blood stream in vivo and defined the time and process of brain entry for five different cancer cell lines in a mouse model. We now investigated if in vitro approaches can reliably emulate the initial steps that determine successful brain colonization in vivo. To this end, we optimized an in vitro model of the vascular blood brain barrier and compared the brain invasion properties of the in vivo characterized cell models with their ability to interact with and penetrate the blood brain barrier model in vitro. Our results show that the in vitro findings correlate only poorly with the vivo results. The limitations of the in vitro approaches are discussed in light of the in vivo processes. We conclude that investigation of mechanisms supporting the earliest steps of breast cancer brain metastasis from the blood stream will depend on in vivo analyses.

Integrin activation controls metastasis in human breast cancer.

Metastasis is the primary cause of death in human breast cancer. Metastasis to bone, lungs, liver, and brain involves dissemination of breast cancer cells via the bloodstream and requires adhesion within the vasculature. Blood cell adhesion within the vasculature depends on integrins, a family of transmembrane adhesion receptors, and is regulated by integrin activation. Here we show that integrin alpha v beta 3 supports breast cancer cell attachment under blood flow conditions in an activation-dependent manner. Integrin alpha v beta 3 was found in two distinct functional states in human breast cancer cells. The activated, but not the nonactivated, state supported tumor cell arrest during blood flow through interaction with platelets. Importantly, activated alpha v beta 3 was expressed by freshly isolated metastatic human breast cancer cells and variants of the MDA-MB 435 human breast cancer cell line, derived from mammary fat pad tumors or distant metastases in severe combined immunodeficient mice. Expression of constitutively activated mutant alpha v beta 3(D723R), but not alpha v beta 3(WT), in MDA-MB 435 cells strongly promoted metastasis in the mouse model. Thus breast cancer cells can exhibit a platelet-interactive and metastatic phenotype that is controlled by the activation of integrin alpha v beta 3. Consequently, alterations within tumors that lead to the aberrant control of integrin activation are expected to adversely affect the course of human breast cancer.

Kinetic analysis of integrin-dependent cell adhesion on vitronectin--the inhibitory potential of plasminogen activator inhibitor-1 and RGD peptides.

Distinct binding interactions between cell-surface receptors and extracellular matrix components are characteristic of multifunctional adhesion proteins such as vitronectin. The close proximity of binding sites for alpha(v)-integrins and plasminogen activator inhibitor-1 (PAI-1) on vitronectin may have consequences for cell adhesion and migration, or for the localized inhibition of plasminogen activators. In this study, the kinetics and reversibility of vitronectin-dependent cell adhesion via alpha(v)-integrins was investigated using RGD peptides and PAI-1 as competitors. Active, but not latent or cleaved PAI-1, and RGD peptides were effective in preventing cell adhesion to vitronectin provided the inhibitor was present at the time of cell seeding. In a concentration-dependent manner urokinase or thrombin abrogated the inhibitory effect of PAI-1. Following cell seeding onto a vitronectin substratum, delayed addition of RGD peptides or active PAI-1 (10-20 min post-seeding) resulted in the loss of their inhibitory potential. These data were supported by experiments in a purified system where delayed addition of active PAI-1 could no longer prevent vitronectin binding to immobilized alpha(v)beta3, while a cyclic RGD peptide gave some moderate inhibition. The apparent stabilization of vitronectin-integrin contacts was observed with immobilized native or multimeric vitronectin but not with the more rigid form of denatured, aggregated multimers. These results demonstrate that the cell adhesive properties of vitronectin depend on its conformational flexibility and can be tightly regulated in a spatio-temporal manner through direct competition of cellular integrins by soluble or matrix-bound factors such as PAI-1.

Selective interaction of a conformationally-constrained Arg-Gly-Asp (RGD) motif with the integrin receptor alphavbeta3 expressed on human tumor cells.

Two antigenized antibodies (AgAbs) were engineered to express peptidic Arg-Gly-Asp (RGD) motifs present in extracellular matrix molecules. The RGD tripeptide sequence was inserted in the third hypervariable loop of an immunoglobulin human/mouse chimeric heavy chain gene as a single or three repeat yielding two antibodies termed gamma1RGD and gamma1(RGD)3, respectively. The antibodies were used to target specific cell-surface receptors of the integrin type expressed by three human tumor cell lines, a melanoma (M21), and osteosarcoma (KRIB) and a fibroblastoma (WI-38). Based on in vitro adhesion assays and flow cytometric analysis, we found that all three cell lines interacted with gamma1(RGD)3 but not with gamma1RGD. Binding of tumor cells to surface-immobilized gamma1(RGD)3 was inhibited in a dose-dependent manner by the RGD-containing synthetic peptides GdRGDSP and RGDS. These synthetic peptides, but no a GDR-containing control peptide, interfered with the binding of tumor cells to surface-immobilized human fibronectin. In their soluble form, neither fibronectin nor gamma1(RGD)3 inhibited tumor cell adhesion to surface-immobilized fibronectin. Gamma1(RGD)3 specifically recognized integrin alphavbeta3 based on two criteria: reactivity with purified integrin receptors and binding to variants of M21 melanoma cells expressing alphavbeta3, alphaIIbbeta3 or no beta3 integrins, respectively. Collectively, our results indicate that the (RGD)3 loop in the antigenized antibody mimics the ligand function of natural extracellular matrix proteins and has a restricted receptor specificity for the alphavbeta3 integrin which is not inherent to short RGD containing peptides.

Clinical significance of alpha(v)beta3 integrin and intercellular adhesion molecule-1 expression in cutaneous malignant melanoma lesions.

Several lines of experimental evidence in in vitro and animal model systems suggest that the integrin alpha(v)beta3 plays a role in the tumorigenicity of human melanoma cells and that the blocking of alpha(v)beta3 ligand binding can inhibit tumor progression. However, there is only scanty information about the role of alpha(v)beta3 in malignant melanoma in a clinical setting. Therefore, in the present study, we have analyzed the distribution in lesions of melanocyte origin and in normal tissues of the alpha(v) integrin subunit and of the alpha(v)beta3 complex and their association with histopathological and clinical parameters of malignant melanoma. We have used as probes the monoclonal antibodies (mAbs) TP36.1 and VF27.263.15, which we have shown with a combination of serological and immunochemical assays to be specific for the alpha(v) subunit and for the alpha(v)beta3 complex, respectively. In immunohistochemical assays, mAb TP36.1 stained both benign and malignant lesions of melanocyte origin. In contrast, the reactivity of mAb VF27.263.15 was restricted to malignant lesions. Both mAbs displayed differential reactivity with primary melanoma lesions of different histotypes because they stained about 50% of acral lentiginous melanoma and superficial spreading melanoma lesions, at least 80% of nodular melanoma lesions, and none of the uveal melanoma lesions tested. Both mAbs TP36.1 and VF27.263.15 stained about 60% of lymph node metastases and 80% of cutaneous metastases. Expression of the alpha(v)beta3 complex in melanocytic lesions resembles that of intercellular adhesion molecule-1 (ICAM-1) in several respects: (a) both are expressed in a significantly (P < 0.004) larger proportion of malignant than of benign lesions; (b) expression of both molecules in primary melanoma lesions is significantly (P < 0.05) associated with lesion thickness; and (c) expression of both molecules in primary lesions from patients with stage I melanoma is significantly (P < 0.05) associated with an increased probability of disease recurrence following surgical excision. alpha(v)beta3 and ICAM-1 in primary melanoma lesions complement each other in predicting the outcome of the disease, because the association with prognosis was enhanced when primary lesions were stained by both anti-alpha(v)beta3 mAb VF27.263.15 and anti-ICAM-1 mAb CL203.4 or by neither mAb. Because alpha(v)beta3 has been suggested as a potential target of immunotherapy, its distribution in normal tissues was investigated. alpha(v)beta3 expression is restricted because it was only detected in ductal epithelium of parotid glands, thyrocytes, basal glands of the stomach, colonic and rectal epithelium glomeruli, Bowman's capsules and proximal and distal tubules of kidneys, and endometrial epithelium. These findings suggest that renal function will be a critical clinical parameter to monitor in therapies of malignant diseases relying on systemic administration of anti-alpha(v)beta3 mAb.

Molecular determinants of arg-gly-asp ligand specificity for beta3 integrins.

The Arg-Tyr-Asp (RYD) and Arg-Gly-Asp (RGD) sequences within the third complementarity-determining region of the heavy chain (H3) of murine recombinant Fab molecules OPG2 and AP7, respectively, are responsible for their specific binding to the platelet integrin alphaIIbbeta3. In this study, we evaluated the influence of divalent cation composition and single amino acid substitutions at key positions within H3 on the selectivity of these Fab molecules for integrin alphaIIbbeta3 versus the vitronectin receptor alphaVbeta3. The parent Fab molecule OPG2 (H3 sequence, HPFYRYDGGN) binds selectively to alphaIIbbeta3 and not at all to any other RGD-cognitive integrin, particularly alphaVbeta3, under any divalent cation conditions. The binding of the AP7 Fab molecule (HPFYRGDGGN) to alphaIIbbeta3 is not affected by the relative composition of calcium, magnesium or manganese. However, AP7 binding to alphaVbeta3, either expressed by M21 cells or as the purified integrin, is supported by manganese and inhibited by calcium. If the flanking asparagine 108 residue within the AP7 H3 loop is replaced by alanine (HPFYRGDGGA), the resulting Fab molecule AP7.4 binds selectively to alphaVbeta3 in a cation-dependent manner, but does not bind at all to alphaIIbbeta3 under any conditions. AP7.4 binding to alphaVbeta3 is supported by manganese, completely inhibited by calcium, and largely unaffected by magnesium. This behavior mimics that of the adhesive protein, osteopontin, another ligand that binds preferentially to alphaVbeta3. Despite these differences in specificity for alphaIIbbeta3 and alphaVbeta3, AP7 and AP7.4 remain selective for the beta3 integrins and do not bind to cell lines that express the RGD-cognitive integrins alphaVbeta5 or alpha5beta1. These results confirm that subtle changes in the amino acid composition immediately flanking the RGD or RYD motifs can have a profound effect on beta3 integrin specificity, most likely because they influence the juxtaposition of the arginine and aspartate side chains within the extended RGD loop sequence.

A single immunoglobulin-like domain of the human neural cell adhesion molecule L1 supports adhesion by multiple vascular and platelet integrins.

The neural cell adhesion molecule L1 has been shown to function as a homophilic ligand in a variety of dynamic neurological processes. Here we demonstrate that the sixth immunoglobulin-like domain of human L1 (L1-Ig6) can function as a heterophilic ligand for multiple members of the integrin superfamily including alphavbeta3, alphavbeta1, alpha5beta1, and alphaIIbbeta3. The interaction between L1-Ig6 and alphaIIbbeta3 was found to support the rapid attachment of activated human platelets, whereas a corresponding interaction with alphavbeta3 and alphavbeta1 supported the adhesion of umbilical vein endothelial cells. Mutation of the single Arg-Gly-Asp (RGD) motif in human L1-Ig6 effectively abrogated binding by the aforementioned integrins. A L1 peptide containing this RGD motif and corresponding flanking amino acids (PSITWRGDGRDLQEL) effectively blocked L1 integrin interactions and, as an immobilized ligand, supported adhesion via alphavbeta3, alphavbeta1, alpha5beta1, and alphaIIbbeta3. Whereas beta3 integrin binding to L1-Ig6 was evident in the presence of either Ca2+, Mg2+, or Mn2+, a corresponding interaction with the beta1 integrins was only observed in the presence of Mn2+. Furthermore, such Mn2+-dependent binding by alpha5beta1 and alphavbeta1 was significantly inhibited by exogenous Ca2+. Our findings suggest that physiological levels of calcium will impose a hierarchy of integrin binding to L1 such that alphavbeta3 or active alphaIIbbeta3 > alphavbeta1 > alpha5beta1. Given that L1 can interact with multiple vascular or platelet integrins it is significant that we also present evidence for de novo L1 expression on blood vessels associated with certain neoplastic or inflammatory diseases. Together these findings suggest an expanded and novel role for L1 in vascular and thrombogenic processes.

Role of beta3 integrins in melanoma cell adhesion to activated platelets under flow.

Mechanisms mediating tumor cell attachment to the vessel wall under flow conditions are largely unknown. Therefore we analyzed the ability of human melanoma cells to adhere to an immobilized matrix during blood flow and determined the role of platelets in this process. In a parallel plate flow chamber, M21 melanoma cells were suspended in human blood and perfused over a collagen I matrix at a wall shear rate of 50 s-1 (2 dynes/cm2) to simulate venous flow over a thrombogenic surface. Melanoma cell interaction with the matrix or blood cells and platelets was monitored and quantified by fluorescence and confocal laser microscopy. Despite their ability to adhere to collagen I under static conditions, M21 cells failed to attach directly to this matrix during blood flow. However, they associated with adherent thrombi, and this resulted in stable melanoma cell arrest. Inhibition of platelet activation or platelet integrin alphaIIbbeta3 function abolished M21 cell attachment. Melanoma cell interaction with thrombi was specific and required beta3 integrin expression. M21-L cells which lack integrin alphavbeta3 failed to associate with thrombi and to arrest during blood flow. Transfection of these cells with the integrin subunits alphav or alphaIIb resulted in variants expressing alphavbeta3, as in the wild type, or alphaIIbbeta3. Both variants were able to associate with thrombi and to arrest during blood flow. Therefore, beta3 integrin-mediated binding to activated platelets represents an efficient mechanism for melanoma cell arrest under flow, and this may contribute to the role of platelets in hematogenous metastasis.

Vitronectin and its receptors.

The extracellular matrix protein vitronectin is recognized as an adhesive substrate by cells expressing at least one of four known vitronectin receptors: integrins alpha v beta 1, alpha v beta 3, alpha v beta 5 or alpha IIb beta 3. Cell interaction with vitronectin may induce spreading and migration and have an effect on cell growth and differentiation in specific processes, such as tumor growth and metastasis, wound healing, bone resorption and viral infection.

Involvement of integrin alpha V gene expression in human melanoma tumorigenicity.

Human melanoma originates in the skin and can lead to wide-spread metastatic disease. Analysis of melanoma biopsy material has shown that the vitronectin receptor, integrin alpha v beta 3, is a specific marker of the most malignant cells, i.e., vertically invasive primary lesions or distant metastases (Albelda, S. M., S. A. Mette, D. E. Elder, R. Stewart, L. Damjanovich, M. Herlyn, and C. A. Buck. 1990. Cancer Res. 50:6757-6764), suggesting a role for this adhesion receptor in the malignant growth of human melanoma tumors. A cell model was established to analyze the role of alpha v integrins on the tumorigenicity of human melanoma. From M21 human melanoma cells, stable variants were selected that lack alpha v gene expression and thus fail to express integrin alpha v beta 3 (M21-L cells). These cells not only lost the ability to attach to vitronectin but showed a dramatic reduction in tumorigenicity when transplanted into athymic nude mice, compared with M21 cells, even though both cell types showed identical beta 1 integrin expression and growth properties in vitro. M21-L cells were stably transfected with a cDNA-encoding alpha v. This resulted in the functional expression of integrin alpha v beta 3 on these cells and completely restored their tumorigenicity. Thus, integrin alpha v gene expression and the resulting adhesive phenotype are directly involved in the proliferation of human melanoma in vivo.

Glycolipid depletion using a ceramide analogue (PDMP) alters growth, adhesion, and membrane lipid organization in human A431 cells.

Glycolipids were depleted from the membranes of human A431 cells using 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an inhibitor of glucosylceramide synthetase. After 6 days of culture in the presence of 5 microM D-threo-PDMP, glycolipid content was reduced to approximately 5% of control levels. By contrast, synthesis per cell of phosphatidylcholine, sphingomyelin, triglycerides, and glycoprotein was relatively unchanged in PDMP-treated cells. In parallel with glycolipid depletion, PDMP-treated cells exhibited a rapid loss of epithelial cell morphology, a reduced rate of cell growth, and inhibition of cell-substrate adhesion. The effects of D-threo-PDMP on cell morphology and substrate adhesion were blocked by exogenous GM3 addition and were not observed with L-threo-PDMP (a relatively inactive enantiomer). Fluorescence photobleaching and recovery (FPR) was used to investigate the hypothesis that glycolipids influence cell behavior, in part, by changing the diffusion characteristics of membrane proteins and lipids. Diffusion coefficients and mobile fractions of two integral membrane proteins, the EGF receptor and a class I MHC antigen, did not differ significantly between control and PDMP-treated cells. Diffusion coefficients of lipid probes, NBD-PC and fluorescent GM1 ganglioside, were similarly unaffected by glycolipid depletion. However, lipid probes did show a significant increase in mobile fraction (the fraction of lipids that are free to diffuse) in PDMP-treated cells. This increase was blocked by culturing cells in the presence of exogenous GM3 ganglioside. The results suggest that glycolipids play a role in the formation of lipid domains in A431 cell membranes. Glycolipid-mediated changes in membrane lipid organization may influence receptor activation and transmembrane signaling, leading to changes in cell growth, morphology, and adhesion.

Distinct biological consequences of integrin alpha v beta 3-mediated melanoma cell adhesion to fibrinogen and its plasmic fragments.

Fibrinogen/fibrin and its proteolytic fragments serve as potential adhesive substrates during thrombosis, wound healing, and cancer. In this report we examined the biological response of human melanoma cells exposed to fibrinogen and its naturally occurring plasmic breakdown products that are known constituents of the tumor stroma. Plasmin treatment of fibrinogen first results in fragment X, which is characterized by removal of the COOH-terminal portion of the alpha chain including an RGD sequence (A alpha 572-575). Further digestion leads to fragment D comprising primarily an intact COOH-terminal stretch of the gamma chain containing the platelet adhesion sequence HHLGGAKQAGDV. In a sensitive adhesion assay M21 human melanoma cells utilized integrin alpha v beta 3 to attach to all three of these ligands. However, only intact fibrinogen promoted significant cell spreading, while fragment X produced minimal spreading and fragment D promoted only adhesion. These results indicate that fibrinogen contains at least two alpha v beta 3-dependent adhesive sites and these promote distinct biological responses of human melanoma cells. The differential functional properties of these ligands directly correlate to their relative binding affinity for purified alpha v beta 3 as measured in a solid-phase receptor binding assay. These results provide evidence that a single integrin can promote distinct biological signals depending on the molecular nature of the ligand binding event.

A ceramide analogue inhibits T cell proliferative response through inhibition of glycosphingolipid synthesis and enhancement of N,N-dimethylsphingosine synthesis.

The ceramide analogue 1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol (PDMP) (particularly the D-threo isomer, D-PDMP) caused inhibition of cell growth in some types of cells, and this growth-inhibitory effect has been attributed to inhibition of UDP-Glc:Cer beta-Glc transferase, resulting in reduced glycolipid synthesis and increased free ceramide [Inokuch, J., & Radin, N. S. (1987) J. Lipid Res. 28, 565-571; Okada, Y., et al. (1988) FEBS Lett. 235, 25-29]. In view of increasing evidence that the T cell proliferative immune response is modulated by glycosphingolipids (GSLs), the reagent D-PDMP was used to evaluate the role of GSLs in this respect. Con A induced or PHA-induced mitogenesis of C3H/HeJ mouse splenocytes, as well as IL2-dependent CTLL cell growth, were strongly inhibited in a dose-dependent manner when cells were preincubated in the presence of 5-10 microM D-PDMP, but not with its stereoisomer L-PDMP. Closely associated with this growth-inhibitory effect in the presence of D-PDMP, levels of essentially all GSLs, including GM3 and other gangliosides, were greatly reduced, whereas ceramide accumulated. Importantly, metabolically labeled radioactive bands, corresponding to free sphingosine and N-monomethylsphingosine, were found to be present in very small quantities (5-12%) relative to the band corresponding to N,N-dimethylsphingosine (DMS), which showed significant accumulation in D-PDMP-treated lymphocytes. The quantity of IL2 receptors and their affinity to IL2 on T cells did not change, but IL2-dependent tyrosine phosphorylation was greatly stimulated, following D-PDMP treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Melanoma-associated gangliosides in the fish genus Xiphophorus.

Gangliosides from benign and malignant melanomas and from normal skin of the fish genus Xiphophorus were isolated and analyzed by thin-layer chromatography. Individual ganglioside components were characterized by mapping according to their sialic acid content and by cleavage with neuraminidases. In all three tissues examined, sulfatide and the gangliosides NeuAc-GalCer (GM4), II3NeuAc-LacCer (GM3), II3NeuAc-GgOse3Cer (GM2), and II3(NeuAc)2-LacCer (GD3) were found. Ganglioside GD3 yielded a positive reaction, following immunoadsorption with mouse monoclonal antibody R24 on thin-layer plates. Two alkali-labile disialoganglioside species were specifically recognized by mouse monoclonal antibody D1.1, thus indicating the presence of O-acetyl-neuraminic acid residues. One of them, a major ganglioside component of the malignant melanoma, was identified as O-acetyl-GD3, since it could be converted to the R24-positive GD3 ganglioside after alkaline saponification. The other one appears to be restricted to the malignant tumor and represents a novel melanoma-associated ganglioside derivative. It was characterized as O-acetyl(NeuAc)2-nLc4Cer by exoglycosidase cleavage, by proving its neutral carbohydrate backbone as type II-chain lacto-series oligosaccharide using mouse monoclonal antibody 1B2, and by its cross-reaction with antibody R24 following alkaline treatment. Using antibody R24 and cryopreserved tissue sections of both benign and malignant amelanotic melanomas from albino fishes, it was demonstrated that one of the main melanoma-associated gangliosides, GD3, was exposed predominantly in the malignant tumor. Thus, the chemical nature and even the immunohistochemical localization of the gangliosides in fish melanomas proved to be very similar to those of the known gangliosides in the phylogenetically distant human melanomas.

Carbohydrate analysis of chicken heart glycolipids.

Neutral and acidic glycolipids were extracted from chicken hearts. The neutral and acidic compounds were separated by preparative thin-layer chromatography into eight and two fractions, respectively. Total hydrolysis by mineral acid, permethylation analysis, and sequential cleavage with exoglycosidases showed the presence of glycolipids that belong to the globo- and gala-oligosaccharide series, i.e., the monohexosylceramides Glc-Cer and Gal-Cer, the dihexosylceramides Gal beta 1-4Glc-Cer and Gal alpha 1-4Gal-Cer, the tetrahexosylceramides GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-Cer and GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal-Cer (III3GalNAc alpha-Ga3Cer) and four subfractions of the Forssman glycolipid GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc-Cer. With the notable exception of III3GalNAc alpha 1-Ga3Cer, all glycolipids with terminal GalNAc alpha 1-3GalNAc1 reacted on thin-layer chromatograms with a monoclonal anti-Forssman antibody. The major components of the acidic fraction glycolipids were characterized as the lactose-based gangliosides Glac1 (GM3) and Glac2 (GD3).

Glycosphingolipid biosynthesis in early chick embryos.

Early chick embryos at the age of 22 h (neurula stage) and 48 h (20-25 somite stage) were explanted from eggs and cultured in vitro in the presence of radioactive sugar precursors. Metabolically labelled glycosphingolipids were isolated. Amongst these, neutral and acidic components, the latter including sulfatide and gangliosides, were identified. Cleavage by exoglycosidases, as well as immunostaining with antibodies on thin-layer plates, showed that at both embryonic stages glycosphingolipids were synthesized that belong to the globo series (globoside, Forssman glycolipid), the lacto series (lactoneotetraosylceramide, nLc4Cer, and two nLc4Cer-based gangliosides, a monosialo and a disialo species), and the ganglio series (ganglioside Gtet1a and higher sialylated derivatives).