Chemotactic responses of tumor cells to products of resorbing bone.

To explore possible mechanisms for the metastasis of malignant cells to bone, a model of tumor cell migration was developed, using Walker carcinosarcoma or malignant lymphoma cells. It was found that bone contains a factor that is strongly chemotactic for tumor cells. This factoor is released by a variety of agents that induce resorption of bone.

Phagocytosis of Candida albicans enhances malignant behavior of murine tumor cells.

Murine tumor cells were induced to phagocytize either Candida albicans or group A streptococcal cells. The presence of microbial particles within the tumor cell cytoplasm had no effect on in vitro tumor cell growth. However, when Candida albicans-infected tumor cells were injected into syngeneic mice, they formed tumors that grew faster, invaded the surrounding normal tissue more rapidly and metastasized more rapidly than control tumor cells. Tumor cells infected with group A streptococcal particles did not grow faster or show increased malignant behavior. These data indicate that the in vivo behavior of malignant tumor cells can be modulated by microbial particles, which are often present in the microenvironment of the growing tumor.

Vascular tube formation on matrix metalloproteinase-1-damaged collagen.

Connective tissue damage and angiogenesis are both important features of tumour growth and invasion. Here, we show that endothelial cells maintained on a three-dimensional lattice of intact polymerised collagen formed a monolayer of cells with a cobblestone morphology. When the collagen was exposed to organ culture fluid from human basal cell tumours of the skin (containing a high level of active matrix metalloproteinase-1 (MMP-1)), degradation of the collagen matrix occurred. The major degradation products were the $3 over 4$- and $1 over 4$-sized fragments known to result from the action of MMP-1 on type I collagen. When endothelial cells were maintained on the partially degraded collagen, the cells organised into a network of vascular tubes. Pretreatment of the organ culture fluid with either tissue inhibitor of metalloproteinase-1 (TIMP-1) or neutralising antibody to MMP-1 prevented degradation of the collagen lattice and concomitantly inhibited endothelial cell organisation into the vascular network. Purified (activated) MMP-1 duplicated the effects of skin organ culture fluid, but other enzymes including MMP-9 (gelatinase B), elastase or trypsin failed to produce measurable fragments from intact collagen and also failed to promote vascular tube formation. Together, these studies suggest that damage to the collagenous matrix is itself an important inducer of new vessel formation.

Rat lung fibroblast collagen metabolism in bleomycin-induced pulmonary fibrosis.

Endotracheal bleomycin administration in rats and other animal species causes rapid development of pulmonary fibrosis, characterized by increased lung collagen synthesis and deposition. To clarify the mechanism, lung fibroblasts from bleomycin-treated rats (BRF) were isolated and maintained in tissue culture. They were then compared with those from normal untreated control animals, with respect to several key parameters of collagen metabolism. BRF synthesized collagen at a rate 35-82% above normal rat lung fibroblasts (NRF). This difference did not appear to be due to the selection of a clone by the subculture process. Furthermore, analysis of newly synthesized collagen type composition, revealed a significantly lower ratio of type III to type I collagen. Noncollagenous protein synthesis, however, was not significantly different from normal. Collagenase production and growth rate were also unaffected. BRF, however, was morphologically indistinguishable from NRF, even at the ultrastructural level. Upon further bleomycin (1 microgram/ml) exposure in vitro, BRF could be further stimulated to synthesize collagen at 82% above the rate for untreated BRF. This is comparable to the 90% increase in NRF treated in vitro (compared with untreated NRF). These results would favor the conclusion that bleomycin induces pulmonary fibrosis, by causing directly and/or indirectly lung fibroblasts (or a certain line of lung fibroblasts) to synthesize collagen at a higher rate without any associated increase in growth rate. The data, however, do not rule out the possibility that the fibroblast isolation procedure has selected for a certain population of fibroblasts that may not be typical of the in vivo situation.

Antisense-mediated reduction in thrombospondin reverses the malignant phenotype of a human squamous carcinoma.

Thrombospondin (TSP) is a trimeric glycoprotein which is synthesized and incorporated into the extracellular matrix by a wide variety of cells. TSP is involved in a number of cellular processes which govern tumor cell behavior including mitogenesis, attachment, migration, and differentiation. To directly assess the role of TSP in tumor cell growth and spread, a human squamous carcinoma cell line, with high TSP production and an invasive phenotype, was transfected with a TSP cDNA antisense expression vector. Five unique transfected clones were obtained with reduced TSP production. Expression of the transfected antisense sequence in these clones was verified by a ribonuclease protection assay. These clones demonstrated reduced growth rates in vitro when compared with a vector transfected control. After subcutaneous inoculation into athymic mice, the antisense clones formed either no tumors or tumors that were slow growing and highly differentiated. This contrasted with the vector-transfected clone which produced poorly differentiated, rapidly growing, invasive tumors. Our results argue in favor of a direct role for TSP in determining the malignant phenotype of certain human tumors.

All-trans retinoic acid (RA) stimulates events in organ-cultured human skin that underlie repair. Adult skin from sun-protected and sun-exposed sites responds in an identical manner to RA while neonatal foreskin responds differently.

Adult human skin from a sun-protected site (hip) and from a sun-exposed site (forearm) was maintained in organ culture for 12 d in the presence of a serum-free, growth factor-free basal medium. Cultures were incubated under conditions optimized for keratinocyte growth (i.e., in 0.15 mM extracellular Ca2+) or for fibroblast growth (i.e., in 1.4 mM extracellular Ca2+). Treatment with all-trans retinoic acid (RA) induced histological changes in the organ-cultured skin under both conditions which were similar to the changes seen in intact skin after topical application. These included expansion of the viable portion of the epidermis and activation of cells in the dermis. In sun-damaged skin samples, which were characterized by destruction of normal connective tissue elements and presence of thick, dark-staining elastotic fibers, a zone of healthy connective tissue could be seen immediately below the dermo-epidermal junction. This zone was more prominent in RA-treated organ cultures than in matched controls. Associated with these histological changes was an increase in overall protein and extracellular matrix synthesis. In concomitant studies, it was found that RA treatment enhanced survival and proliferation of adult keratinocytes and adult dermal fibroblasts under both low- and high-Ca2+ conditions. In all of these assays, responses of sun-protected and sun-exposed skin were identical. In contrast, responses of neonatal foreskin to RA were similar to those of adult skin in the presence of low-Ca2+ culture medium, but under conditions of high extracellular Ca2+ RA provided little or no additional stimulus. Together these studies suggest that the ability of RA to enhance repair of sun-damaged skin (documented in previous studies) may reflect its ability to influence the behavior of skin in a manner that is age dependent but independent of sun-exposure status.

Thrombospondin-induced adhesion of human keratinocytes.

Human epidermal keratinocytes obtained from normal skin attached and spread on thrombospondin (TSP)-coated plastic dishes but failed to attach and spread on untreated plastic culture dishes or dishes coated with fibronectin or laminin. These cells produced minimal amounts of immunoreactive TSP. Keratinocytes established in culture on MCDB 153 medium and maintained for one to three passages in an undifferentiated state by continued cultivation in this low Ca2+-containing medium attached and spread on plastic dishes as well as on TSP-coated dishes. These cells also secreted significant amounts of TSP into the culture medium. When the keratinocytes were incubated for one day in MCDB 153 medium supplemented with high Ca2+ or in MEM (which also contains high Ca2+), there was decreased secretion of TSP into the culture medium concomitant with a reduction in attachment and spreading on plastic culture dishes. Proteolytic fragments of TSP were examined for stimulation of keratinocyte attachment and spreading. A 140-kd fragment produced by removal of the 25-kd heparin-binding domain had similar activity to the intact molecule while the 25-kd fragment was without effect. Further proteolytic treatment of the 140-kd fragment gave rise to a fragment consisting of 120 kd and 18-D moieties held together in disulphide linkage. This fragment did not support attachment or spreading. This study reveals that normal epidermal keratinocytes grown under conditions that maintain the undifferentiated state are able to produce TSP and utilize it as an attachment factor. When keratinocytes are grown under conditions that promote differentiation, ability to produce and utilize TSP is diminished. Since TSP is present at the dermal-epidermal junction and because TSP promotes keratinocyte attachment and spreading, this molecule may play an important role in maintaining normal growth of the basal cell layer and may also participate in reepithelialization during wound repair.

Role of endothelial-leukocyte adhesion molecule 1 (ELAM-1) in neutrophil-mediated lung injury in rats.

Two murine monoclonal antibodies (CL-3 and CL-37, both F(ab')2) to human endothelial-leukocyte adhesion molecule-1 (ELAM-1) were found to react immunohistochemically with rat pulmonary artery endothelial cells that had been pretreated with tumor necrosis factor (TNF alpha). CL-3, but not CL-37, blocked in vitro adherence of neutrophils to TNF alpha-treated endothelial cells and the killing of TNF alpha-treated rat endothelial cells by phorbol ester activated neutrophils. In rats treated systemically with CL-3, there was a 70% reduction in accumulation of neutrophils in glycogen-induced peritoneal exudates. Treatment of animals with CL-37 anti-ELAM-1 did not reduce neutrophil accumulation under the same conditions. When IgG immune complex deposition was induced in dermis and in lungs of rats, treatment with CL-3 anti-ELAM-1 markedly reduced vascular injury as measured by changes in vascular permeability (leakage of 125I-albumin) and hemorrhage (extravasation of 51Cr-red blood cells). The protective effects of CL-3 anti-ELAM-1 were related to greatly diminished recruitment of neutrophils (as assessed morphologically, by tissue extraction of myeloperoxidase, and by retrieval, via bronchoalveolar lavage, of neutrophils from lung). CL-37 had no protective effects in vivo after deposition of immune complexes in lung. Using either CL-3 or CL-37 anti-ELAM-1, immunohistochemical analysis of lungs undergoing IgG immune complex-induced injury revealed a striking upregulation of ELAM-1 in the lung vasculature (venules and interstitial capillaries), with a peak intensity developing between 3 and 4 h after deposition of immune complexes in lung. Vascular beds of spleen, liver, and kidney failed to show upregulation of ELAM-1 under these same conditions. The immunohistochemical reactivity of rat lung was abolished if the anti-ELAM-1 preparation was first absorbed with monolayers of human umbilical vein endothelial cells that had been pretreated with TNF alpha. Untreated human endothelial cells failed to cause loss of lung reactivity of the anti-ELAM-1 preparation. These data indicate that ELAM-1 is upregulated in the pulmonary vasculature of rats during deposition of immune complexes and that ELAM-1 appears to play an obligate role in the recruitment of neutrophils.

Response of Walker 256 carcinosarcoma cells to 12-O-tetradecanoylphorbol 13-acetate: possible regulation by endogenous cyclooxygenase metabolites.

Walker 256 carcinosarcoma cells (Walker cells) maintained in suspension culture responded to stimulation with 12-O-tetradecanoylphorbol 13-acetate [(TPA) CAS: 16561-29-8] by becoming temporarily adherent to the substratum. Both the control and treated cells produced very low levels of cyclooxygenase metabolites as detected by radioimmunoassay procedures. Levels of prostaglandin F2 alpha, 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha) (a prostacyclin metabolite), and thromboxane B2 were virtually the same as background, and prostaglandin E2 (PGE2) levels were only slightly higher. Studies employing high-performance liquid chromatography also failed to detect significant quantities of cyclooxygenase products in the supernatants from either the control or the stimulated Walker cells. Although the Walker cells maintained in culture failed to produce significant amounts of cyclooxygenase metabolites, they produced much greater amounts of these products, particularly PGE2 and 6-keto PGF1 alpha when they were maintained as an ascites tumor. Concomitant with the production of these metabolites was a loss in responsiveness to TPA in the adherence assay. Upon reestablishment in culture, the cells gradually reacquired the ability to respond to TPA. Over the same period, synthesis of cyclooxygenase products was curtailed. If the cells taken from ascites tumors were incubated with indomethacin so as to inhibit the production of cyclooxygenase metabolites, they rapidly regained responsiveness to TPA. These findings suggest that stimulus-coupled responses in the Walker cells may be regulated, at least in part, through the production of endogenous cyclooxygenase metabolites.

Phenotypic stability of murine tumor cells in vitro and in vivo.

Murine fibrosarcoma cells that vary in their transplantability in syngeneic mice were isolated from a heterogeneous parent tumor (induced in a female C57BL/6 inbred mouse) and maintained in culture by serial passage. Several biologic properties that discriminate between the high- and low-transplantable lines (including adhesiveness, motility, and levels of chymotrypsin-like esterase activity), as well as properties that do not separate these lines (e.g., in vitro growth rates and levels of protease and glycosidase activities), were measured at periodic intervals over 2 years. Ability to induce primary tumors and to induce metastases from these tumors were evaluated at the same intervals. The high- and low-transplantable lines were also transplanted into syngeneic mice at tumorigenic doses. Isolates from primary and metastatic tumors induced in the animals were reestablished in culture and examined for the various characteristics of passages 5, 10, 20, and 30. The properties of the cells maintained continually in culture remained stable throughout the 2-year observation period. Tumor isolates showed some evidence of modulation immediately after reestablishment in culture, but by passage 10 they appeared to be identical to the prototype parent lines. These data show that the fibrosarcoma cells do not undergo continual phenotypic "drift" as has been suggested to occur with other tumor lines maintained by serial passage in animals or in culture.

Adhesive characteristics of tumor cell variants of high and low tumorigenic potential.

A variant subpopulation of C57BL/6 mouse fibrosarcoma cells that had very low tumorigenic potential was isolated from a highly tumorigenic parent fibrosarcoma cell culture. The adhesive characteristics of parent cells and variant cells were compared. The low-tumorigenic variant cells were released from the surfaces of plastic dishes, from protein-coated dishes, or from monolayers of fibroblasts or endothelial cells by protease treatment much more readily than were the parent cells. There was no difference between the variant cells and the parent cells in EDTA sensitivity or sensitivity to mechanical agitation under the conditions used. Also, no difference existed between the variant cells and the parent cells in rates of attachment to the surfaces of plastic dishes or to monolayers of endothelial cells. The variant cells were characterized by high levels of chymotrypsin-like esterase activity (two to three times increased over parent cell levels), but there was only a slight difference between the variant cells and the parent cells in caseinolytic or fibrinolytic activity.

Actin changes in normal human and rat leukocytes and in transformed human leukocytic cells.

The DNase I inhibition assay was used for the determination of the relative amounts of monomeric and total actin in normal human peripheral blood lymphocytes, normal Sprague-Dawley rat peritoneal leukocytes, and 6 transformed human cell lines of lymphoid and myeloid origins. In the normal lymphocytes and leukocytes, greater than 50% of the total actin was monomeric. In contrast, only 1 of the 6 transformed lines had greater than 50% of its actin in the monomeric form. In the other 5 lines, the percentage of actin in the monomeric form ranged from 23 to 39%. The normal lymphocytes, peritoneal leukocytes, and 2 of the transformed cell lines (CEM and K562) were examined in greater detail. The total actin (as a percentage of the total protein) was found to be much lower in the transformed cell lines than it was in the normal lymphocytes. The total amount of actin in the normal rat leukocytes was very similar to the amount in the normal human lymphocytes. In addition to these differences between the normal and transformed human cells, treatment of the normal lymphocytes with mitogenic doses of concanavalin A was found to significantly reduce the amount of monomeric actin in the cells. Similar treatment of the transformed cells produced no significant reduction in the monomeric actin.

Cell surface alpha-D-galactopyranosyl end groups: use as markers in the isolation of murine tumor cell lines with different cancer-causing potentials.

Cell lines from 2 3-methylcholanthrene-induced murine tumors were established in culture and examined for reactivity with Griffonia simplicifolia isolectin B4 (GS I-B4), a lectin that has strict specificity for terminal alpha-D-galactopyranosyl residues. Virtually all of the cells in both populations were strongly reactive, indicating the presence of this carbohydrate on the cell surface. Both tumor lines were exposed to human serum with antibodies to the blood group B antigen. More than 99.99% of the exposed cells were killed by this treatment. This is not surprising, because terminal alpha-D-galactopyranosyl residues comprise the blood group B antigen. From the few surviving cells, it was possible to establish cell lines resistant to the cytotoxic effects of the anti-blood group B antibodies. A total of 4 cell lines were independently obtained in this way. The human serum-resistant lines showed no detectable reactivity with GS I-B4, indicating that alpha-D-galactopyranosyl-deficient cell lines had been obtained. The parent and variant cells were compared for reactivity with anti-laminin antibodies. Both parent lines showed strong reactivity by immunofluorescence in the viable state, whereas the alpha-D-galactopyranosyl-deficient lines showed no reactivity. The parent and variant lines were also compared with regard to in vitro and in vivo growth. The alpha-D-galactopyranosyl-deficient lines had reduced in vitro growth capacity relative to the parent lines. More importantly, in contrast to the parent lines, these lines were significantly less tumorigenic than the parent lines when injected into syngeneic mice and did not metastasize spontaneously.

Characteristics of the chemotactic factor-mediated cell swelling response of tumor cells.

The interaction between Walker carcinosarcoma cells, maintained in ascites form in noninbred Sprague-Dawley rats and in cell culture, and appropriate factors induced directed migration (chemotaxis) of these cells as measured in the Boyden chamber assay. The response of the tumor cells to these factors was very similar to that of leukocytes to their chemotactic factors. In addition to inducing chemotactic responses, the interaction between Walker carcinosarcoma cells and appropriate chemotactic factors also led to a swelling of the cells, which could be measured with the use of Coulter Channelyzer C-1000. Associated with the cell-swelling response was a temporary drop in the cell count of a suspension of cells as measured with the use of a Coulter counter, model ZBI. These responses were also similar to what occurs when leukocytes interact with their chemotactic factors. When the suspension of tumor cells was pretreated with 2-deoxyglucose, the responses measured with the Coulter counters were almost completely abrogated. In contrast to this, treatment of the cells with various chemical agents, including cytochalasin B, colchicine, cycloheximide, chlorpromazine, and the Ca2+ ionophore A23187 failed to significantly reduce the cell-swelling response. Cytochalasin B slightly potentiated the response. These similarities to the responses of leukocytes suggested a common underlying mechanism.

Products of cells cultured from gliomas. V. Cytology and morphometry of two cell types cultured from glioma.

Explants of cells of a human glioma were evaluated with the nuclear fluorochrome 4',6-diamidino-2-phenylindole, by phase-contrast illumination, and by Giemsa staining correlated with double immunofluorescence for glial fibrillary acidic protein (GFAP) and fibronectin (FN). FN-positive (FN+) cells lacked GFAP detectable by immunofluorescence. Their mean nuclear-to-cytoplasmic ratio was large (0.192). Actual mean areas of nuclei (1,252 microns2) and cytoplasm (8,376 microns2) of FN+ cells compared with mean areas of fibroblasts suggested that the high nuclear-to-cytoplasmic ratio of FN+ cells was due to their microscopically evident reduced cytoplasmic spreading rather than to larger nuclei. Some FN+ cells showed marked variation in nuclear and nucleolar size and shape. Others had abnormal mitoses or hyperchromatic nuclei. GFAP-positive (GFAP+) cells lacked FN detectable by immunofluorescence. GFAP+ cells were smaller and less round than FN+ cells. Their usual location was growing on a layer of FN+ cells. The mean nuclear-to-cytoplasmic ratio (0.245) of GFAP+ cells was the highest in the study, surpassing the ratio of the continuous glioma line LM (0.176). Mean areas of nuclei (289 microns2) and of cytoplasm (1,350 microns2) of GFAP+ cells suggested that their high nuclear-to-cytoplasmic ratio was due to their microscopically evident reduced cytoplasmic spreading. Reduced spreading was associated with extension of long, thin cytoplasmic processes. The majority of GFAP+ cells showed marked cytoplasmic basophilia, nuclear hyperchromasia, and clumped chromatin. Features observed in both FN+ and GFAP+ cells from this high-grade astrocytoma are features associated with malignant transformation in more thoroughly studied tumor systems.

Phorbol myristate acetate-induced adherence of Walker 256 carcinosarcoma cells.

Treatment of nonadherent Walker 256 carcinosarcoma cells with phorbol myristate acetate (PMA) causes these cells to become adherent to noncellular foreign surfaces such as nylon fibers and plastic culture dishes and to monolayers of endothelial cells. Increased adherence is first observed after a short lag period (5 to 15 min) and is transient. Other tumor-promoting analogs of PMA also induce this response, while inactive analogs of PMA do not. Simultaneous treatment of the cells with 2-deoxyglucose, colchicine, cytochalasin B, and cycloheximide indicates that the adherence response of the cells is an energy-dependent process that requires an intact cytoskeleton but does not require protein synthesis. Inhibitors of phospholipids and arachidonic acid metabolism including indomethacin, nordihydroguaiaretic acid, and p-bromophenacyl bromide greatly inhibit PMA-induced adherence, but acetylsalicylic acid is much less effective. PMA also increases the rate of attachment to plastic dishes of cells which would normally attach, although slowly, and grow as substrate-attached cells. However, PMA treatment has no effect on the subsequent degree of susceptibility of these cells to release from plastic dishes mediated by proteolytic enzymes. These findings suggest (a) that PMA may be useful in delineating the initial events involved in the adherence of cells to cellular and noncellular surfaces and (b) that PMA may stimulate tumor cell adherence in a manner similar to that of chemotactic peptides may be useful in delineating the events associated with chemotactic factor stimulation of these cells.

Phorbol ester binding to chemotactically responding and nonresponding Walker 256 carcinosarcoma cells.

Phorbol esters induce, in the chemotactically responsive Walker 256 carcinosarcoma cells, functional responses that are similar to those induced by peptide chemotactic factors. These responses are presumed to result from ligand binding to cellular receptors, although this has not been formally demonstrated. In this study, it was shown that tritium-labeled phorbol dibutyrate [( 3H]PDB) bound to the Walker cells in a time- and concentration-dependent manner. Binding was inhibited by excess unlabeled PDB and was reversible. Half-maximal binding was achieved with a 31 nM concentration of [3H]PDB and occurred within 15 min after addition of the ligand. This is in accord with biological activity (i.e., cell-to-substrate adherence). Half-maximal cell adherence was observed with 25 nM PDB. Increased adhesiveness was detected as early as 15 min after exposure of the cells to the ligand. The response peaked at 30 to 45 min after exposure and fell off rapidly thereafter. A number of phorbol esters successfully competed with [3H]PDB binding to the cells. There was a direct relationship between the ability of these agents to compete for binding and their ability to induce biological activity. Using cell-to-substrate adherence as an indicator of biological activity allowed separation of responding and nonresponding populations. The biologically responsive cells and the nonresponsive cells both bound high levels of [3H]PDB. This suggests that receptor occupancy is, by itself, not sufficient for biological activity and that, in Walker cells, one or more points of control exist subsequent to ligand binding.

Induction of carcinoembryonic antigen secretion and modulation of protein secretion/expression and fibronectin/laminin expression in human colon carcinoma cells by transforming growth factor-beta.

We have recently reported that TGF-beta induces a response similar to that of planar polar differentiation promoters in human colon carcinoma MOSER cells. N,N-Dimethylformamide and TGF-beta had similar effects on MOSER cells with respect to reversible inhibition of growth (both in monolayer culture and semisolid medium), induction of fibronectin expression and the induction of morphological alterations (Cancer Res., 47:2950-2954, 1987). Since the expression of carcinoembryonic antigen (CEA) has been reported to be modulated by planar polar compounds that promote differentiation in colon carcinomas, we addressed the issue of whether the differentiation-like effects of TGF-beta on these cells would also encompass modulation of CEA expression in the MOSER cells. The biological modulating effects of TGF-beta on extracellular matrix glycoprotein expression and the expression and secretion of cellular proteins were also studied in view of the reported modulating effects of this growth factor on untransformed, noncolonic cells. In this communication we report that TGF-beta induced the synthesis of fibronectin and laminin but not collagen IV. TGF-beta also induced CEA secretion in a dose-dependent manner. Elevated CEA secretion was detected following 48 h of TGF-beta treatment and a 16-fold increase in CEA secretion was observed following 7 days of treatment. The cells were committed to secrete CEA following one dose of TGF-beta treatment. The enhanced expression of four cellular proteins (Mr 42,000, Mr 48,000, Mr 52,000, and Mr 55,000) and the enhanced secretion of three proteins (Mr 66,000, Mr 200,000, and Mr 400,000) were also induced. Some of these protein alterations were detected as early as 6-24 h following TGF-beta treatment. It is concluded that TGF-beta modulated the production and secretion of CEA, the synthesis of fibronectin and laminin, and the expression and secretion of several cellular proteins in the colon carcinoma MOSER cells. To our knowledge, this is the first report on the modulation of CEA and laminin by TGF-beta in tissue-cultured cells, and is the first report on the modulation of cellular proteins by this growth factor in human colon carcinoma cells.

In vitro and in vivo interaction between murine fibrosarcoma cells and natural killer cells.

Murine fibrosarcoma cells were examined for sensitivity to killing by natural killer (NK) and natural cytotoxic lymphocytes from mouse spleens. These tumor cell lines were sensitive to killing by effector cells which were nonadherent to plastic or nylon wool, Thy-1 negative, asialo-GM1 negative, and present in the spleens of beige mice, nude mice, and A/J mice, as well as in the spleens of normal syngeneic and allogeneic control mice. This indicates that the cytotoxic effects were due to natural cytotoxic lymphocytes rather than to NK lymphocytes, T-cells, or macrophages. Although the fibrosarcoma cells were not killed in vitro by endogenous NK cells, these tumor cells were able to "cold target" compete for Yac-1 (an NK-sensitive target) killing and to bind to asialo-GM1-positive, nonadherent spleen lymphocytes in a target cell binding assay. This suggests that the fibrosarcoma cells were recognized by NK cells. In addition, these cell lines were killed in a 4-h NK cytotoxicity assay by polyinosinic-polycytidylic acid-activated effector lymphocytes. The interaction between NK cells and the murine fibrosarcoma cells may have in vivo significance. When syngeneic mice were treated with anti-asialo-GM1 serum to eliminate NK activity and then given i.v. injections of the fibrosarcoma cells, many more lung tumors developed than in control animals. The structural basis for the recognition of the murine fibrosarcoma cells by the NK effector cells is not known. However, laminin may be involved. When the fibrosarcoma cells, which have receptors for the laminin molecule, were preincubated with laminin, they were reduced in their ability to compete for the killing of Yac-1 cells by the NK effectors and had reduced capacity to bind to NK cells in a target cell binding assay.

Monocyte killing of human squamous epithelial cells: role for thrombospondin.

Human peripheral blood monocytes maintained in culture for 18 h were examined for killing of normal human keratinocytes and squamous carcinoma cells. Keratinocytes grown under conditions which maintain the undifferentiated state were highly sensitive to killing but these cells became resistant to killing after induction of differentiation. A line of squamous carcinoma cells obtained from an undifferentiated tumor (designated as UM-SCC-11B) was sensitive to killing while a second line obtained from a more well-differentiated tumor (designated as UM-SCC-22B) was resistant. Several observations suggested that interaction of monocytes with the squamous epithelial cells was mediated, in part, through thrombospondin (TSP). Monocytes synthesized TSP and were positive by immunofluorescence for surface TSP. The normal and malignant squamous epithelial cells also expressed surface TSP as well as unoccupied TSP receptors and our previous studies have shown that both TSP and its receptor are much more prominently displayed on the undifferentiated cells than on the differentiated cells. A series of anti-TSP monoclonal antibodies inhibited killing. These included an antibody directed against the Mr 25,000 NH2-terminal region of the molecule which has heparin-binding activity and three antibodies the epitopes of which lie within the Mr 140,000 non-heparin-binding fragment of TSP. High concentrations of exogenously added TSP as well as the recombinant form of the heparin-binding domain from the TSP molecule also partially inhibited killing while laminin and fibronectin were ineffective. Taken together, these data suggest that TSP and TSP receptors on monocytes and squamous epithelial cells play a role in monocyte-mediated killing of the squamous epithelial cells.