Cytoplasmic dye transfer between metastatic tumor cells and vascular endothelium.

Metastatic colonization of a secondary organ site is initiated by the attachment of blood-borne tumor cells to organ-specific adhesion molecules expressed on the surface of microvascular endothelial cells. Using digital video imaging microscopy and fluorescence activated cell sorting techniques, we show here that highly metastatic cells (B16-F10 murine melanoma and R3230AC-MET rat mammary adenocarcinoma cells) previously labeled with the fluorescent dye BCECF begin to transfer dye to endothelial cell monolayers shortly after adhesion is established. The extent of BCECF transfer to endothelial cell monolayers is dependent upon the number of BCECF-labeled tumor cells seeded onto the endothelial cell monolayer and the time of coculture of the two cell types, as visualized by an increase in the number of BCECF-positive cells among cells stained with an endothelial cell-specific mAb. Dye transfer to BAEC monolayers proceeds with a progressive loss of fluorescence intensity in the BCECF-labeled tumor cell population with time of coculture. The transfer of dye is bidirectional and sensitive to inhibition by 1-heptanol. In contrast, poorly metastatic B16-F0 melanoma cells and non-metastatic R3230AC-LR mammary adenocarcinoma cells do not efficiently couple with vascular endothelial cells. It is inferred from these experiments and from the amounts of connexin43 mRNA expressed by tumor cells that tumor cell/endothelial cell communication is mediated by gap junctional channels and that this interaction may play a critical role in tumor cell extravasation at secondary sites.

Migrating endothelial cells are distinctly hyperglycosylated and express specific migration-associated cell surface glycoproteins.

Migration of endothelial cells is one of the first cellular responses in the cascade of events that leads to re-endothelialization of an injured vessel and neovascularization of growing tissues and tumors. To examine the hypothesis that endothelial cells express a specific migration-associated phenotype, we analyzed the cell surface glycoprotein expression of migrating bovine aortic endothelial cell (BAECs). Light microscopic analysis revealed an upregulation of binding sites for the lectins Concanavalin A (Con A), wheat germ agglutinin (WGA), and peanut agglutinin after neuraminidase treatment (N-PNA) on migrating endothelial cells relative to contact-inhibited cells. These findings were confirmed and quantitated with an enzyme-linked lectin assay (ELLA) of circularly scraped BAEC monolayers. The expression of migration-associated cell surface glycoproteins was also analyzed by SDS-PAGE. The overall expression of cell surface glycoproteins was upregulated on migrating BAECs. Migrating BAECs expressed Con A- and WGA-binding glycoproteins with apparent molecular masses of 25 and 48 kD that were not expressed by contact-inhibited BAEC monolayers and, accordingly, disappeared as circularly scraped monolayers reached confluence. Subconfluent BAEC monolayers expressed the same cell surface glycoconjugate pattern as migrating endothelial cells. FACS analysis of circularly scraped BAEC monolayers showed that the phenotypic changes of cell surface glycoprotein expression after release from growth arrest occurred before the recruitment of the cells into the cell cycle (3 vs. 12 h). Suramin, which inhibits endothelial cell migration, abrogated the expression of the migration-associated phenotype and induced the expression of a prominent 28-kD Con A- and WGA-binding cell surface glycoprotein. These results indicate that endothelial cells express a specific migration-associated phenotype, which is characterized by the upregulation of distinct cellular glycoconjugates and the expression of specific migration-associated cell surface glycoproteins.

Synthesis of cartilage matrix by mammalian chondrocytes in vitro. III. Effects of ascorbate.

Chondrocytes isolated from bovine articular cartilage were plated at high density and grown in the presence or absence of ascorbate. Collagen and proteoglycans, the major matrix macromolecules synthesized by these cells, were isolated at times during the course of the culture period and characterized. In both control and ascorbate-treated cultures, type II collagen and cartilage proteoglycans accumulated in the cell-associated matrix. Control cells secreted proteoglycans and type II collagen into the medium, whereas with time in culture, ascorbate-treated cells secreted an increasing proportion of types I and III collagens into the medium. The ascorbate-treated cells did not incorporate type I collagen into the cell-associated matrix, but continued to accumulate type II collagen in this compartment. Upon removal of ascorbate, the cells ceased to synthesize type I collagen. Morphological examination of ascorbate-treated and control chondrocyte culture revealed that both collagen and proteoglycans were deposited into the extracellular matrix. The ascorbate-treated cells accumulated a more extensive matrix that was rich in collagen fibrils and ruthenium red-positive proteoglycans. This study demonstrated that although ascorbate facilitates the formation of an extracellular matrix in chondrocyte cultures, it can also cause a reversible alteration in the phenotypic expression of those cells in vitro.

Lung endothelial dipeptidyl peptidase IV is an adhesion molecule for lung-metastatic rat breast and prostate carcinoma cells.

Attachment of circulating tumor cells to endothelial cell adhesion molecules restricted to select vascular compartments is thought to be responsible for site-specific metastasis. Lung-metastatic rat R3230AC-MET breast and RPC-2 prostate carcinoma cells bound outside-out endothelial cell membrane vesicles, prepared by perfusion of the rat lung vasculature with a low-strength formaldehyde solution, in significantly higher numbers than their nonmetastatic counterparts R3230AC-LR and RPC-LR. In contrast, vesicles derived from the vasculature of a nonmetastasized organ (e.g., hind leg muscle) showed no binding preference for either of the four tumor cell lines. Lung-derived endothelial vesicles were used here to generate mAbs against lung endothelial cell adhesion molecules. The first group of mice were actively immunized against lung endothelial vesicles, whereas the second group was injected with syngeneic mouse antiserum against leg endothelial vesicles before active immunization with lung endothelial vesicles. 17 hybridoma supernatants obtained from the two fusions bound lung vesicles with at least a 10-fold higher affinity than leg vesicles. Seven (four obtained by a passive/active immunization protocol) stained rat capillary endothelia. One mAb, mAb 8.6A3, inhibited specific adhesion of lung-derived vesicles to lung-metastatic breast and prostate carcinoma cells. Purification of the antigen (endothelial cell adhesion molecule) from rat lung extracts revealed a protein with a 110-kD mol wt. NH2-terminal sequencing established identity with dipeptidyl peptidase IV which had been reported to serve as a fibronectin-binding protein. These results indicate that vesicles obtained from in situ perfused organs are a convenient immunogen for the production of antibodies to compartment-specific endothelial cell surface molecules, and reinforce the concept that endothelial cell surface components are selectively recognized by circulating cancer cells during metastasis formation.

Synthesis of cartilage matrix by mammalian chondrocytes in vitro. I. Isolation, culture characteristics, and morphology.

We describe the isolation and the ultrastructural characteristics of adult bovine articular chondrocytes in vitro. Slices of bovine articular cartilage undergo sequential digestions with pronase and collagenase in order to release cells. Chondrocytes are plated at high density (1 x 10(5) cells/cm2) in culture dishes or roller bottles with Ham's F-12 medium, supplemented with 10% fetal bovine serum. Before culture, chondrocytes are freed of surrounding territorial matrix. Within the first few days of culture they re-establish a territorial matrix. As time progresses, chondrocytes synthesize both territorial and extraterritorial matrices. The matrices are rich in collagen fibrils and ruthenium red-positive proteoglycans. These features are most apparent in mass roller cultures in which aggregates of cells and matrix appear as long streaks and nodules. This morphology reveals an organization of chondrocytes and their matrices that is similar to that of the parent articular cartilage in vivo.

Synthesis of cartilage matrix by mammalian chondrocytes in vitro. II. Maintenance of collagen and proteoglycan phenotype.

The in vitro phenotype of bovine articular chondrocytes is described. Chondrocytes plated at high density in roller-bottle and dish cultures were maintained in vitro. The major matrix macromolecules, collagen and proteoglycan, synthesized by these cells were characterized during the course of the culture period. The chondrocytes synthesized mainly Type II collagen, which was found predominantly in the cell-associated matrix. The media contained a mixture of Type II and Type III collagens. Type I collagen was detectable in neither the medium nor the cell-associated matrix. The proteoglycan monomers found in media and cell-associated matrix had the same hydrodynamic sizes as monomers synthesized by cartilage slices or those extracted from adult articular cartilage. The majority of proteoglycans synthesized by the cells were found in high molecular weight aggregates which were readily recovered from the media and were extractable from cell-associated matrix with low ionic strength buffers. The results demonstrate the long-term in vitro phenotypic stability of the bovine articular chondrocytes. The advantages of the in vitro system as a model for studying the effects of external agents, such as drugs and vitamins, are discussed.

Blocking of lung endothelial cell adhesion molecule-1 (Lu-ECAM-1) inhibits murine melanoma lung metastasis.

The 90-kD lung endothelial cell adhesion molecule-1 (Lu-ECAM-1) selectively promotes Ca(2+)-dependent adhesion of lung-metastatic B16 melanoma cells. Corresponding with their metastatic performance, high lung-metastatic B16-F10 melanoma cells bind in significantly higher numbers to Lu-ECAM-1 than their intermediate and low lung-metastatic counterparts B16-L8-F10 and B16-F0, respectively. Maximum attachment is observed at a density of approximately 2.4 x 10(2) Lu-ECAM-1 sites/microns2 of plastic surface. B16 melanoma cell binding to Lu-ECAM-1 is blocked by mAb 6D3 and is competitively inhibited by soluble Lu-ECAM-1. C57B1/6 mice passively immunized with anti-Lu-ECAM-1 mAb 6D3 or actively immunized with purified Lu-ECAM-1 exhibit an anti-Lu-ECAM-1 antibody titer-dependent reduction in the number of B16 experimental metastases. Lu-ECAM-1 promotes neither binding nor metastasis of other lung-metastatic tumor cells (e.g., KLN205). Our data indicate that an "antiadhesion" therapy directed at interfering with the adherence of blood-borne tumor cells to organ-specific vascular endothelium is efficient in the control of metastasis formation in selective organ sites.

Regulation of tumor invasion by cartilage-derived anti-invasion factor in vitro.

The resistance of cartilage to tumor invasion was studied with the use of a novel in vitro culture system. Articular cartilage obtained from fresh metacarpophalangeal joints of preadolescent bovines was used as a growth surface for human TE-85 osteosarcoma cells and foreskin fibroblasts. Cartilage disks formed the bottoms of stainless-steel cylinders, providing closed growth chambers for these cells. Both invasive osteosarcoma cells and normal fibroblasts were unable to penetrate viable, unextracted cartilage during a 2-week culture period. When cartilage was devitalized by freezing and thawing, the tissue remained resistant to invasion. Cartilage, extracted with either 1 or 3 M guanidine hydrochloride, was invaded by osteosarcoma cells, but not by control fibroblasts. Invasion by osteosarcoma cells into salt-extracted cartilage was abolished when low concentrations of a cartilage-derived, anti-invasion factor were added to the culture medium. These data provided evidence that the resistance of cartilage to tumor invasion is regulated in part by tissue-derived proteinase inhibitors.

Modulation of the metastatic phenotype by 13-cis-retinoic acid.

KLN 205 murine squamous carcinoma cells were grown in medium supplemented with the retinoid 13-cis-retinoic acid (RA) to study the relationship between RA-induced cell surface changes and alterations of the metastatic phenotype. Modulation of the cell surface glycoconjugate expression was measured by flow cytometric analysis of the RA-treated tumor cells stained with fluoresceinated lectins. RA treatment (5 X 10(-6) and 5 X 10(-7) M) altered the glycoconjugate expression of KLN 205 cells in a selective, dose-dependent fashion. Tumor cells grown in RA-supplemented medium for more than 4 days demonstrated greatly increased binding of fluoresceinated Griffonia simplicifolia I lectin, peanut lectin, wheat-germ lectin, concanavalin A, and soybean lectin (P less than .001), but the increased binding of Ulex europaeus lectin was of a much smaller magnitude (P = .02). After 15 days of growth in these noncytotoxic or cytostatic concentrations of RA, malignant KLN 205 cells had a greatly decreased proclivity to metastasize, as measured by the lung colony assay (P = .0003). The RA-induced cell surface glycoconjugate changes preceded the decrease in experimental metastatic potential. Since enzymatic (neuraminidase) alteration of the tumor cell surface to produce glycoconjugate expression similar to that seen in RA-treated cells also reduced the ability of the KLN 205 cells to form lung colonies (P = .0022), it is suggested that RA-induced alteration of the cell surface carbohydrate antigens is related to the decreased experimental metastatic potential seen in tumor cells treated with RA.

Tumorigenicity of human breast cancer is associated with loss of the Ca2+-activated chloride channel CLCA2.

The human Ca2+-activated chloride channel-2 (CLCA2) is expressed in normal breast epithelium but not in breast tumors of different stages of progression. Northern analysis of nontransformed and transformed breast epithelial cell lines revealed CLCA2 expression in the nontransformed cell line MCF10A and the nontumorigenic cell line MDA-MB-453, whereas all tumorigenic cell lines were negative (MDA-MB-231, MDA-MB-435, MDA-MB-468, and MCF7). When stably reintroduced into CLCA2-negative MDA-MB-231 and MDA-MB-435 cells, CLCA2 expression reduced Matrigel invasion in vitro and inducibility of s.c. and metastatic tumors of MDA-MB-231 cells in nude mice. Our results suggest that CLCA2 may act as a tumor suppressor in breast cancer.

Correlations between cell surface protease activities and abnormalities of occludens junctions in rat bladder carcinoma in vitro.

Microenvironmental alterations, i.e., proteolytic enzymes, may play a causative role in abnormalities of zonulae occludentes. To test this hypothesis, we compared in vitro the ultrastructure of three carcinoma cell lines which were derived from N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide-induced tumors of the rat urinary bladder. One of these lines had a high cell surface protease activity; the other two lines exhibited relatively low activities. Quantitative electron microscopy data revealed differences in configuration and distribution of zonula occludens-intramembrane fibrils among these cell lines, as indicated by means and standard deviations of zonulae occludens widths, and numbers of intramembrane fibrils. Although the total length of the intramembranous fibrils per square micrometer of occludens junction area was not statistically different in the three lines, junctional morphology varied greatly. Thus, carcinoma cells with high surface protease activities are able to synthesize near-normal amounts of intramembrane fibrils but are unable to assemble normal zonulae occludentes. This indicates that alterations in zonula occludens morphology, which have been induced by exogenous proteolytic enzymes, are identical to those observed in a cell line with high cell surface protease activity.

In vitro determination of tumor invasiveness using extracted hyaline cartilage.

Our previous observation that salt-extracted, devitalized cartilage could be penetrated by malignant tumor cells but was nonpermissive to fibroblastic ingrowth led us to postulate that this matrix might be used as a test connective tissue to discriminate in vitro between noninvasive and invasive tumor cell lines. In a novel in vitro system, salt-extracted, bovine articular cartilage was therefore used as a growth surface for defined noninvasive, invasive, and metastatic carcinoma cell lines, derived from chemical carcinogen-induced tumors of the rat urinary bladder. As monitored by thin-section electron microscopy, salt-extracted cartilage was readily penetrated by the invasive and metastatic rat bladder carcinoma cell lines. The metastatic cell line could be differentiated from the invasive, nonmetastatic cell line by its greater depth of invasion. In contrast, noninvasive carcinoma cells as well as normal bladder epithelial cells lacked the capacity to erode and penetrate the extracted matrix of the articular cartilage. Using these defined cell lines, salt-extracted cartilage can be used to reproducibly discriminate between carcinomas having different invasive potentials. This assay system may have diagnostic application for the in vitro staging of tumors.

Flow cytometric analysis of heterogeneity in blood group-related antigen expression in a human urinary bladder carcinoma cell line, 647V.

Previous immunohistological studies showed a relationship between expression of blood group-related antigens (BG-Ag) and invasive potential in human urinary bladder carcinoma, but the marked variability of antigen staining within many individual tumors has obscured the biological basis of this finding. We studied the expression of the A, H, and T (Thomsen-Friedenreich) BG-Ag by flow cytometry in a human bladder carcinoma cell line (647V) using fluoresceinated BG-Ag-specific lectins (Dolichos bifloris, Ulex europaeus, and Arachis hypogaea). Cell cycle compartments were quantitated by flow cytometry using propidium iodide staining. Expression of all three antigens was highly variable, but staining for each antigen produced a distinct profile. T antigen expression appeared independent of A or H antigen expression. Cell populations sorted by T antigen expression showed heritable antigenic differences persistent over many weeks in culture. However, much of the T antigen variability was nonheritable, since the stable staining profiles of the sorted cells were intermediate between the parental and the profiles obtained immediately after sorting. The nonheritable antigenic variation did not appear entirely explainable by cell size or cell cycle fluctuation. These results were confirmed by isolating 64 clones from the dim part of the T antigen staining profile, 19 of which had a persistently dim phenotype. The variability of BG-Ag expression by human bladder carcinoma cells in vitro may explain the staining patterns observed in the study of antigen expression in resected human bladder carcinomas.

Development of an in vitro and in vivo epithelial tumor model for the study of invasion.

Three continuous cell lines were isolated from N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT)-induced carcinoma of the Fischer rat urinary bladder by standard explant techniques. RBTCC-2 carcinoma cells were derived from a noninvasive FANFT tumor of Stage 0, RBTCC-5 carcinoma cells were from an invasive FANFT tumor of Stage B2, and RBTCC-8 carcinoma cells were from a s.c. metastasis of a FANFT tumor of Stage D2. Invasive and metastatic carcinoma cells were differentiated from their noninvasive counterparts by cellular and nuclear pleomorphism, cell size, nuclear:cytoplasmic ratio, number of nucleoli, and abnormalities of occludens junctions. Using low (less than 10) and high (greater than 80) passages of these cell strains, tumorigenicity experiments in syngeneic rats showed that the normal in vivo progression of FANFT tumors was interrupted by the isolation of carcinoma cells to cell culture. Histological appearance and biological behavior of tumor isografts closely resembled those of the original FANFT tumors. This was best demonstrated when tumor cells were inoculated adjacent to rat femurs. The destruction of bone, monitored radiographically and histologically, served as a measure of the invasive potential of the tumor cells. Destruction and deep invasion were observed only with isografts of invasive and metastatic carcinoma cells, presumably due to collagenolytic activity. Despite rapid degradation of bone by these isografts, the natural resistance of cartilage to tumor invasion could not be overcome. These carcinoma cell lines, together with their normal epithelial counterparts and the major supporting cells of connective tissue characterized previously by our laboratory, provide a unique system to study tumor invasion.

Desmosome ultrastructure and biological behavior of chemical carcinogen-induced urinary bladder carcinomas.

In the quantitative electron microscopic study, we examined the relationship of desmosomes to tumor invasiveness in chemical carcinogen (N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide)-induced urinary bladder transitional cell carcinomas in the Fischer rat. The number of a desmosomes per unit area of plasma membrane was significantly reduced in carcinomas. However, the percentage of cell surface area occupied by desmosomes was greater in carcinomas than in controls. This was accounted for on the basis of increases in desmosomal size, which result from squamous differentiation within the tumors. Areas of transitional cell differentiation and squamous differentiation demonstrated an equal capacity for invasiveness. Desmosomes were abundant in invading nests of tumor cells. These findings cast doubt on the validity of the concept of decreased intercellular adhesion as a prerequisite for tumor invasion, since strong interadhesion is probably a function of the area occupied by the intercellular junctions.

Connective tissue degradation by invasive rat bladder carcinomas: action of nonspecific proteinases on collagenous matrices.

The invasive RBTCC-8 rat bladder carcinoma cell line (passage number, greater than 100) and its derivates, the RBTCC-8 tumor isografts and the 1-RBTCC-8 daughter cell line (fourth passage), express proteolytic activities of broad substrate specificity, which allow them to efficiently degrade extracellular (collagenous) matrices. Cell-associated, collagenolytic activity is evidenced by the release of hydroxyproline from collagen substrates of types I and IV, by visualizing the low-molecular-weight collagen breakdown products on sodium dodecyl sulfate-polyacrylamide gels, and by the depth of invasion into extracellular matrices in our bone invasion assays. Fractionated by diethylaminoethyl column chromatography, the major collagenolytic activities against collagens of types I and IV coelute in a relatively narrow peak within a NaCl gradient. The pooled collagenolytic diethylaminoethyl fractions contain: (a) two chymotrypsin-like, catheptic activities; (b) activity against a synthetic elastase substrate; (c) gelatinase activity; and (d) caseinolytic activity. Despite efficient collagenolysis, a vertebrate-type collagenase cannot be detected in any of our tumor samples, even after trypsin activation of the tumor cell extracts. The mechanism of action of these nonspecific proteinases is thought to be that of collagen "crosslinkases." The neutral proteinase activities are highest in RBTCC-8 tumor isografts, intermediate in the fourth passage 1-RBTCC-8 carcinoma cell line, and lowest in the RBTCC-8 carcinoma cell line of high passage number. The levels of these nonspecific enzyme activities are well correlated with the depth of invasion into bony matrices, as shown by our invasion assays.

Endothelial cell membrane vesicles in the study of organ preference of metastasis.

Many malignancies exhibit distinct patterns of metastasis that appear to be mediated by receptor/ligand-like interactions between tumor cells and organ-specific vascular endothelium. In order to study endothelial cell surface molecules involved in the binding of metastatic cells, we developed a perfusion method to isolate outside-out membrane vesicles from the lumenal surface of rat lung microvascular endothelium. Lungs were perfused in situ for 4 h at 37 degrees C with a solution of 100 mM formaldehyde, 2 mM dithiothreitol in phosphate-buffered saline to induce endothelial cell vesiculation. Radioiodinated rat lung endothelial cell membrane vesicles bound lung-metastatic tumor cells (B16F10, R323OAC-MET) in significantly higher numbers than their low or nonmetastatic counterparts (B16F0, R323OAC-LR). In contrast, leg endothelial membrane vesicle showed no binding preference for either cell line. Neuraminidase treatment of vesicles abolished specificity of adhesion of lung-derived vesicles to lung metastatic tumor cells. These results demonstrate that in situ perfusion is an appropriate technique to obtain pure endothelial cell membrane vesicles containing functionally active adhesion molecules. The preferential binding of lung-derived endothelial cell membrane vesicles by lung metastatic tumor cells is evidence of the importance of endothelial cell adhesion molecules in the formation of metastases.

Molecular cloning and biochemical characterization of a truncated, secreted member of the human family of Ca2+-activated Cl- channels.

A novel family of chloride channel proteins has recently been discovered including two bovine (Lu-ECAM-1, bCLCA1), one murine (mCLCA1), and two human (hCLCA1 and hCLCA2) members. Here, we describe the cloning, expression, and molecular characterization of a truncated human homolog, tentatively named hCLCA3. It was cloned from a human spleen cDNA library and is expressed in numerous tissues including lung, trachea, spleen, thymus, and mammary gland as determined by reverse transcriptase-polymerase chain reaction. Unlike all previously known CLCA family members which consistently encode an approximately 125-kDa transmembrane protein that is cleaved to form a heterodimer of two proteins of approximately 90 and 35 kDa, the 3.6-kb hCLCA3 mRNA encodes a 37-kDa glycoprotein that corresponds to the N-terminal extracellular domain of its homologs. Moreover, when expressed in human embryonic kidney 293 or Chinese hamster ovary cells, this 37-kDa glycoprotein is secreted into the culture supernatant. These observations suggest that hCLCA3 is a structurally divergent member of the CLCA family of proteins and that it does not act as a channel protein but has distinct, yet unidentified functions.

Is the Fischer 344/CRJ rat a protein-knock-out model for dipeptidyl peptidase IV-mediated lung metastasis of breast cancer?

Fischer 344/CRJ rats harbor a G633R substitution in dipeptidyl peptidase IV (DPP IV) that leads to retention and degradation of the mutant protein in the endoplasmic reticulum (Tsuji E, Misumi Y, Fujiwara T et al. Biochemistry 1992; 31 (47): 11921-7). However, when these rats were used as a 'protein knock-out' model in further evaluating the previously established role of DPP IV in metastasis, lung colonization of the highly metastatic MTF7 rat breast cancer cell line was reduced by only 33% relative to normal Fischer 344 rats. To examine whether lung endothelia leak expression of mutant DPP IV and whether mutant DPP IV exhibits the same adhesion qualities as wild type DPP IV, detailed immunohistochemical, biochemical, transfection, and FACS analyses were performed to assess the surface expression of mutant DPP IV on lung endothelia and transfected HEK293 cells and adhesion assay to compare the adhesion qualities of wild-type and mutant DPP IV. Both endothelial and transfected HEK293 cells expressed mutant, enzymatically inactive DPP IV on their surfaces, albeit at greatly reduced levels when compared to expression of wild type DPP IV. Purified mutant DPP IV had identical adhesion qualities for lung-metastatic MTF7 cells as wild type DPP IV, and competitive inhibition of MTF7 lung colonization by truncated DPP IV confirmed involvement of mutant DPP IV in lung metastasis of Fischer 344/CRJ rats. Although metastasis appears to be mediated by several, often parallel mechanisms involving multiple tumor and host factors, these data indicate that altered expression of a single component can drastically change the outcome of metastatic disease.

A new adhesion assay using buoyancy to remove non-adherent cells.

A new adhesion assay was developed that utilizes buoyancy, rather than washing or centrifugation, to remove non-adherent cells. Biotinylated cells were added to wells containing cell monolayers or purified protein substrates. Non-adherent cells were then removed by floatation on a dense Percoll solution. The adherent cells were fixed tightly to the plate with a Percoll/glutaraldehyde fixative and quantitated by streptavidin: horseradish peroxidase chemistry. In a side-by-side comparison of buoyancy and washing assays, the buoyancy method detected B16F10 binding to purified fibronectin at a 4-fold lower fibronectin concentration and human umbilical vein endothelia cell (HUVEC) binding to laminin at a 10-fold lower laminin concentration than did washing assays. In cell to cell adhesion assays, the buoyancy method was able to detect significantly greater binding of mononuclear leukocytes and KM12-L4 colon carcinoma cells to IL-1 beta treated human umbilical vein endothelial cells (HUVEC). The binding of human promyelocytic leukemia HL60 cells to control and IL-1 beta treated HUVEC was the same (approximately 60%) with the buoyancy method, while a washing assay demonstrated 8-fold higher binding (51% vs. 6%) of HL60 on IL-1 beta treated cells. The buoyancy assay is useful for detecting weak cell to protein adhesion and may be useful for detecting cell to cell adhesion when background binding is sufficiently low.