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MDW40, a wheat germ agglutinin-resistant (WGAr) mutant of the highly metastatic tumor cell line called MDAY-D2, is restricted to local growth at the subcutaneous site of inoculation. The WGAr tumor cells acquire metastatic ability by fusing spontaneously with a normal host cell followed by chromosome segregation, a process accompanied by reversion of the WGAr phenotype (i.e., WGAs). Since lectin-resistant mutant cell lines often have oligosaccharide alterations that may affect membrane function and consequently metastatic capacity, we compared the major Asn-linked glycopeptides in WGAr and WGAs cell lines. [2-3H]mannose-labeled glycopeptides were separated into four fractions on a DEAE-cellulose column and then further fractionated on a concanavalin A-Sepharose column. Glycopeptide structures were determined by: (a) sequential exoglycosidase digestion followed by chromatography on lectin/agarose and Bio-Gel P-4 columns and (b) proton nuclear magnetic resonance analysis. The metastatic WGAs cells had a sialylated poly-N-acetyllactosamine-containing glycopeptide which was absent in the nonmetastatic mutant cell line. Unique to the mutant was a neutral triantennary class of glycopeptide lacking sialic acid and galactose; the WGAr lesion therefore appeared to be a premature truncation of the antennae of the poly-N-acetyllactosamine-containing glycopeptide found in the WGAs cells. High mannose glycopeptides containing five to nine mannose residues constituted a major class in both WGAr and WGAs cells. Lysates of both wild-type and mutant cells had similar levels of galactosyltransferase activity capable of adding galactose to the N-acetylglucosamine-terminated glycopeptide isolated from mutant cells; the basis of the WGAr lesion remains to be determined.

Several noninvasive techniques are currently utilized to safely assess cardiac function beyond the limits of the physical examination and EKG. These studies can accurately document rhythm and valvular abnormalities as well as the heart's ability to withstand stress. Results of these tests provide a solid base on which to decide if invasive diagnostic or therapeutic modalities may be required.

Neutral and acidic glycolipids in MDAY-D2, a highly metastatic murine tumor cell line, were examined and compared with glycolipids of MDW4 and D33W25-1, two lectin-resistant mutants of MDAY-D2 from distinct genetic complementation classes. D33W25-1 remained highly metastatic while MDW4 cells were found to be nonmetastatic (Dennis, J. W., Donaghue, T., Florian, M., and Kerbel, R. S., Nature (Lond.), 292: 242-245, 1981 and Dennis, J. W. et al., Cancer Res., 46: 4594-4600, 1986). Glycolipid structures were identified by fast-atom bombardment mass spectrometry, methylation analysis, exoglycosidase treatment, and immunostaining. The metastatic MDAY-D2 was found to contain GM3, GM2, IV3GalNAc-GM1b, and high levels of GM1a, GM1b, and GD1a. MDW4 showed a 3-fold decrease in total ganglioside content compared to MDAY-D2 and a corresponding increase in the precursor, glucosylceramide. MDW4 was deficient in GM1 and accumulated GM2 and NeuNG-GM2, indicating a lack of gangliosides having NeuNAc alpha 2-3Gal beta 1-3 terminal sequence. Neosynthesis of GD3 was also observed in MDW4. The metastatic mutant D33W25-1 had a similar pattern of gangliosides as that found in MDAY-D2 cells with N-glycolyl rather than N-acetyl neuraminic acid. These results suggest that the metastatic property of these cell lines may be related to the level of ganglioside, and that the substitution of N-glycolyl for N-acetyl neuraminic acid does not reduce metastatic capacity.

Metastases arising from a subcutaneous injection of the DBA/2 tumor, MDAY-D2, as well as four drug-resistant variants (either wheat germ agglutinin-resistant, ouabain-resistant, or both, i.e., WGAR/OuaR) of MDAY-D2, were examined for the presence of a tumor-associated antigen (TAA). Of 15 mice examined, tumor antigen-loss variants were detected in only 1 animal. These antigen-loss metastases arose in a mouse injected with the WGAR variant called MDW4. The tumor at the site of inoculation retained the TAA, whereas all four of the metastases removed from liver, spleen and other tissues were antigen-loss variants. The antigen-loss variants were not killed by cytotoxic T cells (CTL) directed against the TAA of the parental tumor, did not competitively inhibit CTL lysis of MDW4 targets in a 'cold target' inhibition test, and were not able to elicit a CTL response. In vivo immunization-protection (challenge) experiments also showed that the metastases did not express the TAA of MDAY-D2. Unlike the WGAR phenotypes, which were lost in all spontaneous metastases recovered from MDW4-injected mice, loss of the TAA appeared to be an uncommon event. Antigen-loss tumor cell variants are discussed in terms of their relevance to metastasis, and in regard to their use in the study of T cell-mediated cytotoxicity of tumor cell populations.

The distribution of Fc receptors is not restricted to lymphoid cells - indeed, they were first discovered on macrophages and subsequently on a range of other leukocytes and granulocytes. It should not come as a surprise, therefore, to discover that solid, non-lymphoid tumors are generally filled to varying degrees with cells that have Fc receptor. After all, it has been known for over a century that such tumors can become infiltrated with all kinds of lymphoreticular host cells, an observation which has been claimed by some to be good morphologicalin-vivo evidence for the body's anti-cancer immune surveillance system. What did come as a surprise, then, was the claim that a considerable proportion - perhaps the vast majority - of such Fc-receptor-positive cells were tumorous, regardless of the nature or origin of the malignant tumor. R. S. Kerbel wrote in 1974 that the importance of lymphocyte-associated receptors could be 'eclipsed in terms of biological significance by the finding that an astonishing array of non-lymphoid and non-reticuliondothelial tumors appear to possess Fc types of receptors'. How does that statement hold up today? In this review, R. S. Kerbel and J. W. Dennis attempt to answer this question and summarize the current state of affairs., (Copyright © 1980. Published by Elsevier B.V.)

There is now considerable evidence that malignant transformation is accompanied by many changes in the oligosaccharide structures of glycoproteins and glycolipids. More recently, studies using tumour cell glycosylation mutants and inhibitors of Asn-linked oligosaccharide processing have suggested that the transformation-associated increase in GlcNAc beta 1-6Man alpha 1-6 Man beta- branching of complex-type oligosaccharides may enhance tumour cell metastasis. The synthesis of beta 1-6 branched oligosaccharides and related structures as well as their possible function in cell-cell interactions and tumour cell metastasis are discussed.

A novel model of tumor progression in metastatic cancer is described which grew out of attempts to derive stable non-metastatic variants from a highly metastatic mouse tumor called MDAY-D2. The variants were obtained by selection of so-called lectin-resistant (LecR) membrane mutants using toxic concentrations of wheat germ agglutinin (WGA) as the selective agent, after mutagenesis. Cloned WGAR variants almost all appeared to be highly tumorigenic and metastatic, but displayed altered growth properties which were highly suggestive of major cellular phenotypic alterations occurring prior to metastasis. This were confirmed with the discovery that spontaneous visceral metastases always consisted of WGA-sensitive (WGAS) 'revertant' tumor cells. Such revertants also arose at the site of the subcutaneous inoculation and, with time, comprised an increasing proportion of the tumor cells at that location. The WGAS/high metastatic phenotype was stable in vitro or in vivo, implying the WGAR leads to WGAS shift had an underlying genetic basis. Thus, it appeared that the WGAR tumor cells could not metastasize, because of either an intrinsic cellular defect or a host imposed barrier, but that this block could be circumvented through a genetic change in the WGAR tumor cells which was accompanied by reversion of the WGAR phenotype. Non-tumorigenic (tum-) WGAR variants were also obtained, but in these cases the mutagenesis treatment itself appeared responsible for development of the tum- phenotype. The reduced tumorigenicity had an underlying immunological basis, a finding which could be exploited to immunotherapeutically treat established visceral metastases of poorly immunogenic tumors. Throughout these studies, emphasis was placed on the considerable potential of using tumor cell populations having various stable drug-resistant genetic markers to monitor aspects of tumorigenesis, tumor progression, and metastasis.

Two highly glycosylated membrane sialoglycoproteins designated P2A and P2B were isolated from the lymphoreticular tumor cell line called MDAY-D2 and shown to be structurally similar to the lymphocyte glycoprotein called leukosialin and to lysosome-associated membrane glycoprotein termed LAMP-1, respectively. The loss of sialic acid and polylactosamine sequences in glycosylation mutants of MDAY-D2 has been correlated previously with enhanced cell adhesion on extracellular matrix proteins. Since these two glycoproteins bear the majority of the sialylated oligosaccharides found in membrane fractions of MDAY-D2, they were tested for binding activity on extracellular matrix proteins. Both isolated glycoproteins bound to immobilized collagen type I with affinities that were dependent on their glycosylation. Enzymatic removal of sialic acid, polylactosamine, or complete asparagine-linked chains from purified P2B enhanced its binding to collagen, laminin, and fibronectin. In contrast, P2A bound specifically to collagen type I and the interaction required the presence of sialic acid residues which were sensitive to neuraminidase digestion but not to endoglycosidase F. The results suggest that oncodevelopmental regulation of oligosaccharide expression on P2A and P2B glycoproteins may modulate their binding to extracellular matrix glycoproteins.

Three independent isolates, one obtained spontaneously and the others obtained after in vitro mutagenesis, of a WGA-resistant mutation were compared to the highly metastatic parental murine cell line MDAY-D2 for alterations in plasma membrane glycoproteins and changes in metastatic behavior. The mutants were non-metastatic from an s.c. site of injection and poor organ-colonizers when administered i.v. Each of the mutant lines had the same lesion in N-linked oligosaccharide structure, which rendered the cells hypersensitive to the N-acetylglucosamine-binding lectin BSII in vitro. The phenotypic similarities between the 3 WGA-resistant isolates indicated that the mutation was directly related to the attenuated malignant phenotype. Direct confirmation was obtained by the isolation of a BSII-resistant clone of the mutant cells that co-reverted for lectin-sensitivity, lectin binding glycoproteins and malignant aggressiveness. The results indicate a direct relationship between malignant behavior and cell-surface oligosaccharide structure.

The rosetting of sheep erythrocytes (SRBC) coated with non-haemagglutinating monoclonal antibodies rather than conventional haemagglutinating antisera revealed readily detectable FcR on most splenic natural killer (NK) cells since 76% of splenic lymphocytes forming conjugates with YAC also rosetted with SRBC coated with high concentrations of monoclonal anti-SRBC antibody of the IgG2b subclass and since Ficoll depletion or enrichment of splenic lymphocytes rosetting with IgG2b-coated SRBC resulted in a corresponding 4-fold decrease or increase in conjugate-forming cells and a 10-fold decrease or increase in NK cytolytic activity. NK cells bound much less readily to monoclonal IgG2a and not at all to monoclonal IgG1 or IgM, but the degree of binding was directly proportional to the amount of antibody on the erythrocytes and was not isotope-restricted. In addition, immunofluorescent studies revealed that YAC-1-conjugated lymphocytes were Lyt-1-, Lyt-2-, partially Thy-1+ (60%), asialo(GM1+ (80%). Qa-4+ (77%), Qa-5+ (79%), and Ly-5+ (94%). In comparison, a proportion (39%) of alloimmune peritoneal exudate cells which conjugated with P815-2 also stained by immunofluorescence with anti-asialo GM1 antisera. Most (greater than 90%) P815-conjugated cells were Thy-1+, Lyt-2%, and a subpopulation of Lyt-1+2+ conjugates was observed (25%). Qa-5 and Ly-5 were also expressed on most (two-thirds) cytolytic T lymphocytes (CTL) conjugates, whereas Qa-4 and FcR for IgG2b were not detected. The best phenotype distinctions between NK cells and CTL were therefore based on the presence or absence of Lyt-2, Qa-4, and FcR for IgG2b on most effector cells. Anti-asialo-GM1 or monoclonal anti-Qa-4 and complement treatment greatly diminished both the frequency of NK conjugates and the percentage of conjugates with detectable IgG2b FcR or asialo-GM1. These results confirm that NK cells co-express asialo-GM1 and Fc receptors, at the single-cell level, and provide a simple method for greatly enriching NK populations at least 10-fold.

Increased branching at the trimannosyl core of 'complex-type' Asn-linked oligosaccharides has been observed in both human and murine tumour cells, and appears to be associated with enhanced metastatic potential in several murine tumour models [Dennis, Laferte, Waghorne, Breitman & Kerbel (1987), Science 236, 582-585]. The lectin leucoagglutinin (L-PHA) requires the-GlcNAc beta 1-6Man alpha 1-6Man-linked lactosamine antenna in complex-type oligosaccharides for high-affinity binding and can be used to detect these structures in glycoproteins separated on SDS/polyacrylamide-gel electrophoresis. The major L-PHA-binding glycoproteins in the highly metastatic lymphoid tumour cell line called MDAY-D2 were purified and resolved into two major species, termed P2A (110 kDa) and P2B (130 kDa). P2A had L-PHA-reactive Asn-linked oligosaccharides with polylactosamine sequences as well as a large component of sialylated O-linked carbohydrates. The glycoprotein showed structural characteristics similar to those of leukosialin (i.e. CD43), a glycoprotein previously identified on the surface of leukocytes. Based on monosaccharide compositional analysis and glycosidase digestions, P2B was found to be 50-60% Asn-linked oligosaccharide containing polylactosamine sequences and sialic acid. The N-terminal peptide sequence of P2B was determined to be very similar to that of murine lysosomal membrane glycoprotein (LAMP-1), a ubiquitous glycoprotein found largely in the lysosomal membranes but also in the plasma membrane of several murine and human tumour cell lines.

MDW4, a wheat germ agglutinin (WGA)-resistant (WGAr) mutant of the metastatic murine tumor cell line MDAY-D2, was previously shown to be nonmetastatic in the syngeneic DBA/2 host. A substantial portion of the asparagine (Asn)-linked carbohydrate in MDW4 terminated in N-acetylglucosamine (GlcNAc) and appeared to be a premature truncation product of the sialylated poly-N-acetyllactosamine-containing complex found in MDAY-D2 cells. This lesion in carbohydrate structure has been shown to contribute to the more adhesive behavior of MDW4 cells on laminin, fibronectin, and type IV collagen and to the increased sensitivity of MDW4 to natural killer (NK) cell lysis in vitro. For further characterization of the relationship between Asn-linked carbohydrate structures, cell adhesion, NK cell sensitivity, and metastasis, mutants of MDW4 were selected for resistance to the GlcNAc-binding lectin from Bandeiraea simplicifolia seeds (BSII). Three independently selected BSII-resistant (BSIIr) mutants of MDW4 chosen for further study had a lectin resistance phenotype intermediate between that of MDAY-D2 and that of MDW4. Plasma membrane glycoproteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with iodinated WGA, BSII, and leukoagglutinin also indicated an intermediate phenotype, with the presence of both GlcNAc-terminating structures and sialylated complex. Compared to MDW4, the BSIIr mutants of MDW4 showed a return to the more malignant phenotype of MDAY-D2 when injected iv. The double mutants were less sensitive to NK cell lysis in vitro and to the in vivo effects of the NK cell boosting agent polyinosinic-polycytidylic acid. The double mutants retained the ability to attach to fibronectin, laminin, and collagen type IV in vitro, a property that may have contributed to their low malignancy when the cells were injected sc. MDW4 cells have been shown to fuse at low frequency with host-derived bone marrow cells at the sc site of injection, thereby acquiring the wild-type lectin resistance and metastatic phenotypes. The same process appears to occur in mice given an injection of the double mutants. The results suggest that the WGA-binding oligosaccharides found in MDAY-D2 and the BSIIr mutants of MDW4 enhance the malignant phenotype of the cells in the experimental metastasis assay.

Swainsonine, a plant alkaloid and potent inhibitor of Asn-linked oligosaccharide processing, has previously been shown to inhibit organ colonization by metastatic murine tumor cells and to inhibit the growth of transformed fibroblasts in soft agar. In this report, we show that swainsonine has antiproliferative activity against human tumor cells growing in tissue culture and as tumor xenografts in nude mice. The antiproliferative activity of swainsonine was additive with that of human interferon-alpha 2 (HuIFN-alpha 2) in cultures of HT29 colon carcinoma, SN12 renal carcinoma, and A375 melanoma cells. In vivo, the growth rate of HT29m human colon carcinoma tumors in athymic nude mice was reduced by supplementing their drinking water with swainsonine (49%) or by administering HuIFN-alpha 2 systemically (53%); combining these treatments reduced tumor growth by 78%. Combining swainsonine and HuIFN-alpha 2 treatments enhanced the activity of the interferon-inducible enzyme 2',5'-oligoadenylate [2',5'-oligo(A)] synthetase in HT29m tumors compared to that observed in tumors from mice treated with interferon alone. In vitro, swainsonine enhanced interferon-dependent induction of 2',5'-oligo(A) synthetase activity in low-density cultures of HT29m cells. However, swainsonine alone did not stimulate 2',5'-oligo(A) synthetase activity in vivo or in vitro, indicating that the antiproliferative effect of swainsonine is independent of interferon production. The results suggest that in addition to the previously reported antimetastatic activity of swainsonine, the plant alkaloid has antiproliferative activity that is independent from, but additive with, that of interferon in vivo and in vitro.

Previous studies demonstrated that growth in DBA/2 mice of MDW4, a wheat germ agglutinin-resistant (WGAr) mutant of the highly metastatic MDAY-D2 DBA/2 mouse tumor, led to the emergence of WGA-sensitive (WGAs) revertants having higher ploidy levels at the site of inoculation as well as at distant visceral metastases. The results implied that MDW4 was nonmetastatic but progressed to become metastatic in vivo only after a cellular change took place which was accompanied by extinction of the WGAr phenotype and acquisition of a higher number of chromosomes. Results presented here provide strong and direct evidence for the underlying mechanism being spontaneous cell fusion in vivo between the MDW4 (WGAr) tumor cells and normal host cells, at least some of which are of bone marrow origin. Thus, growth of the H-2d MDW4 tumor cells in (C3H X DBA/2)F1 (H-2k X H-2d) or (C57BL/6 X DBA/2)F1 (H-2b X H-2d) mice led to the appearance of WGAs revertants bearing the H-2k or H-2b major histocompatibility complex antigens associated with the C3H or C57BL/6 parental strains, respectively. Similarly, WGAs revertants of MDW4 were found to express H-2k antigens after growth in CBA/HT6T6 (H-2k) leads to DBA/2 bone marrow radiation chimeras. Attempts to mimic the in vivo hybridization process were successful in that in vitro somatic cell fusion between an ouabain-resistant (OuaR), 6-thioguanine-resistant (Thgr) derivative of the MDW4 mutant and either normal bone marrow or spleen cells resulted in loss of the WGAr phenotype in the hybrids (thus showing its recessive character) and increased malignant properties in vivo. An analysis of spontaneous frequencies of re-expression of various drug resistance genetic markers in several hybrid metastatic cells was also consistent with chromosome segregation of the sensitive alleles. The results show that tumor progression and the emergence of metastatic cell variants could arise as a consequence of tumor X host cell fusion followed by chromosome segregation. We also discuss the possibility that this type of event may normally be a very rare one during the growth of tumors, the frequency of which can be artificially amplified by the use of certain classes of lectin-resistant mutants carrying particular cell surface alterations.

In previous studies we have shown that the ability of murine tumor cells to metastasize in situ is directly linked to expression of -GlcNAc beta 1-6Man alpha 1-6Man beta 1-branched complex-type Asn-linked oligosaccharides in tumor-cell glycoproteins. Here we demonstrate that cell-surface expression of beta 1-6 branched oligosaccharides in metastatic tumor cells is specifically associated with increased invasion of human amnion basement membranes in vitro. Compared to nonmetastatic SP1 murine mammary carcinoma cells, 2 metastatic sublines expressed higher levels of beta 1-6 branched oligosaccharides and were found to be invasive but poorly adhesive on the amnion basement membrane. Swainsonine, a non-toxic inhibitor of Asn-linked oligosaccharide processing which blocks the pathway prior to initiation of the beta 1-6 linked antenna, blocked metastatic tumor-cell invasion and increased adhesiveness. Swainsonine and the metalloprotease inhibitor O-phenanthroline inhibited invasion, apparently via independent mechanisms. O-phenanthroline did not affect tumor-cell adhesion to the amnion basement membrane and swainsonine did not block secretion of metalloproteases, beta-hexosaminadase or tissue plasminogen activator activity by the tumor cells. These results suggest that tumor-cell invasion of basement membranes requires both secretion of hydrolase activities and expression of beta 1-6 branched complex-type oligosaccharides at the tumor cell surface, such oligosaccharides being associated with reduced tumor-cell adhesion to extracellular matrix.

Tetrafluoroethylene (TFE) was discharged onto woven polyethylene terephthalate (PET) prostheses, and the PET prostheses (50 mm long) with or without TFE were implanted into canine carotid arteries and aortas. Additional controls included polytetrafluoroethylene and Dacron. Specimens were explanted after one to 12 months, photographed, and sectioned for light and scanning and transmission electron microscopy, and thrombus-free surface areas calculated by computerized planimetry. Results showed no significant patency differences among carotid or aortic groups. However, both PET carotid groups had significantly greater thrombus-free surface areas. Histologically, both PET groups appeared identical. An endothelialized neointima covered PET carotid specimens by six months, compared with three months in the aortic position, with greater pannus ingrowth in both PET groups. Plasma polymerized TFE offered no additional advantage in these long-term experiments.

MDW4, a wheat germ agglutinin resistant mutant of the murine tumor line MDAY-D2, expresses abnormal asparagine-linked oligosaccharides, is less metastatic when injected intravenously, and is hypersensitive to natural killer (NK) lysis in vitro. To determine whether these phenotypes may be related, variants of the YAC-1 lymphoma and a YAC-1 X MDAY-D2 hybrid line were compared for sensitivity to four different lectins and to NK cell lysis in vitro. A relationship between sensitivity to concanavalin A (Con A) and NK cell lysis in vitro was observed. Although no single plasma membrane glycoprotein separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with 125I-labeled Con A correlated with NK and Con A sensitivities of the cell lines, a relationship between these phenotypes and the collective 125I-Con A staining intensity on the gels was apparent. In a more direct test of carbohydrate recognition by NK cells, specific glycopeptide structures isolated from tumor cells and added to the NK cell assay in microM quantities were found to inhibit tumor cell lysis. Thus, a subset of asparagine-linked oligosaccharides, including high mannose and some incomplete complex structures on a number of cell surface glycoproteins, appears to be recognized as part of the target structures for NK cell lysis. The administration of polyinosinic:polycytidylic acid stimulated splenic NK activity in vivo but had no effect on the growth of the NK-resistant MDAY-D2 cells. However, the low tumorigenicity of MDW4 cells injected intravenously was reduced further by pretreating the mice with polyinosinic:polycytidylic acid, which indicated a role for NK cells in the elimination of circulating tumor cells expressing high mannose and/or incomplete complex asparagine-linked oligosaccharides.

Ultrasound has proven invaluable in detecting and evaluating pancreatic pseudocysts, and it is now a standard test to rule out complications of pancreatitis. In reviewing the authors' experience with 122 patients treated surgically for a pancreatic pseudocyst, five patients were identified in whom an ultrasound demonstrated a pseudocyst that was associated with an unexpected cancer at the time of operation. A sixth patient, with a pseudocyst documented by ultrasound, died prior to surgery and was found at autopsy to have metastatic common bile duct carcinoma. There was little difference in presenting symptoms, age, frequency of alcoholism, or physical findings compared with patients with pseudocysts secondary to pancreatitis. In two patients, pseudocysts were found in the tail of the pancreas at operation, in addition to carcinoma. In the other three patients, no pseudocyst was found; however, a subcapsular splenic hematoma was present in one. Five patients had metastatic disease, three from pancreatic adenocarcinoma, one from islet cell carcinoma, and one from a common bile duct carcinoma. One patient with a pancreatic adenocarcinoma confined to the head underwent a Whipple procedure and has no evidence of disease 6 months later. Malignancy may cause or coexist with pancreatic pseudocysts. Ultrasound is often not helpful in distinguishing pseudocysts associated with malignancy from those associated with pancreatitis. Biopsy should be performed to rule out malignancy when operating for pancreatic pseudocysts.

We have described a number of phenotypic and genotypic differences between defined tumor sublines from the Eb/ESb model system that differ greatly in metastatic capacity. Some of these differences are summarized in Figure 5. It can be seen that the differences are both structural and functional. They include differences in the glycosylation of membrane glycoproteins as well as differences in total membrane protein composition. The functional differences refer particularly to enzymatic activities, cell motility, and invasive capacity. In addition, a change in membrane fluidity and membrane dynamics was seen when we investigated membrane vesicles shed spontaneously from these tumor lines. ESb type cells were found to shed about three times as much membrane material as the low-metastatic, Eb cells. Since such vesicles carried degradative enzyme activity, they could play a role in the invasion process. The vesicles also carried TATA and thus could also play a role in preventing immunological attack against these tumor cells. Another immunological escape mechanism was found to be based on the ability of the high-metastatic cells to generate immunoresistant variants that no longer expressed the respective TATA. A cytogenetic comparison of such immunoresistant variants with the TATA+ line revealed several chromosomal changes. The contribution of chromosomal rearrangements to the ability of malignant cells to generate variants was discussed.

To follow the cellular progeny of the multiple-drug-marked benign murine tumor cell line MDW4 during its progression in vivo toward metastatic spread in DBA/2 mice, the following parameters were analyzed: retention of the drug-resistant markers ouabain resistance (OuaR) and thioguanine resistance (ThgR), lectin-resistance pattern (WGAR), and the karyotype of cell populations (and clones derived from these cells) removed at intervals from the solid tumor growing at the site of inoculation, as well as distant metastatic nodules. It was determined that the initially homogeneous inoculum composed of OuaR, ThgR, and WGAR hypotetraploid cells (mode: 68 +/- 2 chromosomes) was gradually overgrown and replaced by a new population of cells that were either OuaR or ouabain-sensitive but that became thioguanine-and lectin-sensitive and hyperploid (mode: 95 +/- 5). Regardless of the composition of the individual drug marker combinations, only cells with high chromosome contents were found to be able to disseminate to distant visceral organs and to rapidly produce metastases upon sc or iv reinjection. The presence of the same number of metacentric chromosomes in metastatic cells as in MDW4 and the coextinction of two recessive drug-resistant markers (WGAR and ThgR) suggested that cells endowed with invasive-metastatic potential represent the product of spontaneous somatic hybridization between the original nonmetastatic MDW4 cells and normal host cells of unknown origin. Such a fusion was followed by more or less extensive chromosome segregation that accounts for the karyotype mosaicism and the occasional drug marker heterogeneity identified in cell populations of metastatic nodules.

ECGF, a polypeptide of bovine hypothalamic derivation, is the most potent endothelial cell mitogen known, with mitogenic and chemotactic effects well demonstrated in vitro on human endothelial cells. These effects are synergized by heparin. In vivo re-endothelialization of blood-contacting biomaterials may be enhanced by bonding ECGF and heparin to prosthetic surfaces. Long woven Dacron (24 mm) and woven PDS vascular prostheses were treated first with human plasma fibronectin (10 micrograms/cm2). Porcine sodium heparin (20 micrograms/cm2) was added by means of fibronectin's heparin affinity. Pure 125I-ECGF (95% alpha, 5% beta; 1 ng/cm2) was next fixed by the heparin affinity of ECGF and followed by a second heparin layer (20 micrograms/cm2) to synergize with and stabilize ECGF. 125I-ECGF adherences were determined by scintillation counts. Attachment efficiency averaged 25%. Prostheses were interposed into rabbit aortas and harvested in triplicate from 0 to 30 days to establish in vivo washout curves. After explantation, residual 125I-ECGF was eluted from prostheses, and intact ECGF was identified by SDS gel electrophoresis. Similarly prepared but nonradioiodinated Dacron and PDS prostheses were explanted after 7 days and their ECGF eluted off for in vitro activity documentation. This ECGF retained its mitogenic properties, causing a 1000% to 1200% increase in 3H-thymidine incorporation into newly synthesized DNA in test murine LE-II cells. Fibronectin-heparin-ECGF fixation to blood-contacting biomaterials may enhance spontaneous re-endothelialization and/or hasten the confluence of transplanted endothelial cells.

Increased--GlcNAc beta 1-6Man alpha 1-6Man beta--branching in asparagine-linked oligosaccharides has been observed in murine and human tumor cells and has recently been linked to enhanced metastatic potential in experimental tumor models. Leukoagglutinin (L-PHA) requires the beta 1-6-linked lactosamine antenna (beta 1-6 branch) for high affinity binding and was used in this study to quantitate these structures on glycoproteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Normal rodent tissues and cell lines were used to standardize the experimental conditions required to quantitate beta 1-6-branched oligosaccharide structures and the glycosyltransferase activity which initiates the synthesis of the antenna, beta 1-6 N-acetylglucosamine (GlcNAc)-transferase V (EC 2.4.1.155). Secondly, the levels of L-PHA-reactive oligosaccharide were compared in a series of benign and malignant human breast biopsies. Normal human breast tissue and benign lesions showed low expression but 50% of the primary malignancies examined showed significantly elevated L-PHA reactivity. GlcNAc transferase V activities in the human breast carcinomas and in normal murine tissues correlated with the levels of L-PHA reactive oligosaccharide in the tissues. GlcNAc transferase V showed similar ranges of activities, differing by approximately 5-fold between high and low expressing mouse tissues; fibroblasts with and without an activated H-ras oncogene; and low and high expressing human breast carcinomas. The results show that beta 1-6 branching in asparagine-linked oligosaccharides is dependent on tissue-specific regulation of GlcNAc transferase V activity. Secondly, a subset of human breast malignancies showed elevated levels of beta 1-6-branched oligosaccharides compared to benign samples, suggesting that further studies are warranted to determine whether the presence of these oligosaccharides is associated with metastatic disease and reduced patient survival time.

Neoplastic transformation has been associated with a variety of structural changes in cell surface carbohydrates, most notably increased sialylation and beta 1-6-linked branching of complex-type asparagine (Asn)-linked oligosaccharides (that is, -GlcNAc beta 1-6Man alpha 1-6Man beta 1-). However, little is known about the relevant glycoproteins or how these transformation-related changes in oligosaccharide biosynthesis may affect the malignant phenotype. Here it is reported that a cell surface glycoprotein, gp 130, is a major target of increased beta 1-6-linked branching and that the expression of these oligosaccharide structures is directly related to the metastatic potential of the cells. Glycosylation mutants of a metastatic tumor cell line were selected that are deficient in both beta 1-6 GlcNAc transferase V activity and metastatic potential in situ. Moreover, induction of increased beta 1-6 branching in clones of a nonmetastatic murine mammary carcinoma correlated strongly with acquisition of metastatic potential. The results indicate that increased beta 1-6-linked branching of complex-type oligosaccharides on gp 130 may be an important feature of tumor progression related to increased metastatic potential.