Lung Colony Formation Research Articles

Plasminogen activator activity in clonogenic cell populations separated from a murine fibrosarcoma.

Tumor cells from a murine fibrosarcoma (FSa) produce plasminogen activator (PA), a protease that converts the zymogen plasminogen into the trypsin-like enzyme plasmin. Several studies indicate that tumor cell invasion is accompanied by proteolysis and that PA, generated by highly malignant cells, is by far the most ubiquitous protease associated with malignant transformation. Subpopulations of FSa cells were isolated by using density gradient centrifugation and the ability of these populations to form lung colonies was compared with their associated levels of PA production. Five populations of cells from a murine fibrosarcoma were separated in continuous gradients of Renografin in the density range 1.05-1.18 g/cm2. The PA activities of an unseparated control cell lysate and cell lysates of the five separated populations were determined by using [125I]fibrin as a substrate in a reaction between cell lysate and plasminogen. The assay was based on the release of digested [125I]fibrin from the surface of Petri dishes into the supernatant solution, and the results were expressed as a percentage of the total radioactivity. The cell populations collected at densities of 1.05 and 1.09 (B1, B2) were the more clonogenic with relative clonogenic efficiencies of 2.6 and 3.3 times that of the unseparated tumor population, respectively. Analysis for PA demonstrated that enzyme formation was restricted mostly to these two populations. Cells from populations 4 and 5 did not secrete increased amounts of PA and had reduced clonogenic efficiencies compared with the unseparated FSa control population. These results are consistent with the hypothesis that PA activity is correlated with the clonogenicity of tumor subpopulations isolated from a heterogeneous and complex tumor system such as the FSa.

DMSO-induced changes in the procoagulant and fibrinolytic activity of B16 melanoma cells: influence on lung colony formation.

In this study DMSO (dimethylsulphoxide) was used as a tool to test the significance of in vitro modifications of procoagulant and fibrinolytic activity of tumor cells for their in vivo metastatic ability. B16 melanoma cells were chosen as the experimental model. After four days' treatment DMSO increased both the procoagulant and fibrinolytic (plasminogen activator) activity of B16 melanoma cells in a dose-related manner. DMSO treated cells showed significantly greater lung colonizing ability than untreated cells. Our results indicate that DMSO treatment in vitro can modulate procoagulant and fibrinolytic activity and the metastatic ability of B16 melanoma cells; however a direct causal relationship between these in vitro and in vivo effects remains to be established.

A new experimental metastasis model in athymic nude mice, the human malignant melanoma LOX.

The human tumor line LOX was established as an s.c. xenograft in nude mice from a lymph-node metastasis of a patient with malignant melanoma. I.v. injection into adult nude mice of single-cell suspensions prepared from xenografts resulted in progressively growing lung tumor colonies that killed the animals. No difference in colony formation was seen between cells taken from lung colonies and s.c. xenografts. An in vitro cell line, LOX-L, was established from lung colonies, and the monolayer cells, detached with EDTA, retained the same ability to form experimental lung metastases. In a total of 14 experiments, 82 of 89 mice receiving 1 X 10(6) viable tumor cells died with a mean survival time of 34.1 +/- 4.8 days. Long-term passaging in vivo and in vitro did not result in any alteration of the lung-colonizing potential of the LOX cells, whereas trypsinization of the cells before i.v. injection reduced lung colony formation. The life span was inversely related to the number of LOX cells injected, permitting estimation of the cell kill caused by chemotherapy. Mice injected i.v. with the LOX cells showed the same relative response to cis-diamminedichloroplatinum (CDDP) and mitozolomide (MZA) as did animals carrying s.c. xenografts. The LOX cells have shown a remarkable stability and similarity to the cells of the patient's tumor with respect to morphology, karyotype and chemosensitivity. The LOX model may be useful for testing effects of therapy on lung micro- and macrometastases, and the activity of antimetastatic agents, as well as for studying mechanisms involved in the metastatic process.

Separation of High and Low Metastatic Subpopulations from Solid Tumors by Centrifugal Elutriation

We have isolated from murine solid tumors (B16a) subpopulations of cells possessing high and low metastatic potential. Tumors were dispersed by collagenase treatment. The resulting heterogeneous population of cells (i.e., viable and non-viable tumor cells and host cells) were separated by centrifugal elutriation. Four of the fractions (100, 180, 260, 340) contained tumor cells of high viability (greater than 95%) and high purity (less than 1% host cell contamination). The four fractions were characterized by flow cytometry and found to differ in distribution of cells in G1, S and G2. The cell populations were also found to differ in metastatic potential as determined by their ability to form lung colonies following intravenous injection. The 340 fraction was approximately 5-fold more metastatic than the 100 fraction. We also observed that cells from the 100 fraction failed to induce platelet aggregation whereas cells from the 340 fraction induced significant platelet aggregation. These observations demonstrate that cells of B16a tumors are heterogeneous for phenotypic characteristics (i.e., metastatic potential; platelet aggregation, etc.) and that their ability to induce platelet aggregation is positively correlated with metastatic potential.

YIGSR, a synthetic laminin pentapeptide, inhibits experimental metastasis formation.

The invasion of tumor cells through basement membranes is a critical step in the formation of metastases. The binding of the malignant cells to laminin in the basement membranes allows their attachment and activates their invasiveness. Recently a synthetic nonapeptide from the B1 chain sequence of laminin was identified as a major site for cell binding. A pentapeptide within the nonapeptide sequence was found to reduce the formation of lung colonies in mice injected with melanoma cells and also to inhibit the invasiveness of the cells in vitro.

Interactions of Natural Effector Cells and Prostaglandins in the Control of Metastasis<xref ref-type="fn" rid="FN2">2</xref>

The roles of prostaglandins and nonspecific immune effector cells were examined in the growth and metastasis of a series of murine mammary adenocarcinomas growing in BALB/c mice. The spontaneous metastasis of the highly metastatic tumor designated 410.4 was inhibited by oral administration of the prostaglandin synthesis inhibitor indomethacin (INDO), whereas treatment of mice with the interferon inducer polyinosinic-polycytidylic acid (poly I:C) delayed tumor growth but did not affect the rate of spontaneous metastasis. Lung colony formation was dramatically reduced by poly I:C treatment following iv tumor cell inoculation (experimental metastasis), whereas oral INDO had no significant effect. Suppression of natural killer cell activity with anti-asialo GM1 (asGM1) antibody increased lung colony-forming ability of mammary tumor lines with either high, moderate, or low metastatic capacity. Although pretreatment of the recipient with INDO did not alter the incidence of experimental metastasis, culture of the tumor cells with INDO (1 micron) prior to iv inoculation inhibited the appearance of lung colonies by greater than 90%. This beneficial effect was dependent on an asGM1-positive population. These results suggest that in this murine mammary tumor system, prostaglandin synthesis contributes to metastatic capacity and that the beneficial effects of INDO may be attributable, in part, to the enhancement of tumor cell sensitivity to natural cytotoxic effector mechanisms.

Enhancement of lung colony formation by admixing irradiated with viable tumor cells: dependence on host status.

The study was designed to determine whether whole-body irradiation or stimulation of the reticuloendothelial system of mice influences the ability of heavily irradiated tumor cells to enhance formation of artificial metastases when given simultaneously with viable tumor cells. Experiments were performed with a nonimmunogenic sarcoma syngeneic to C3Hf/Kam mice. Whole-body irradiation augmented and stimulation of the reticuloendothelial system abolished the metastasis-enhancing effect of tumor cells. Another observation was that heavily irradiated tumor cells can enhance formation of metastases if given i.v. within several hours before or after i.v. injection of tumor cells.

A synthetic peptide from fibronectin inhibits experimental metastasis of murine melanoma cells.

Adhesive interactions between cells and the extracellular matrix occur at several stages of metastasis. Such interactions might be inhibited by synthetic peptide probes derived from the cell-binding regions of matrix molecules. Gly-Arg-Gly-Asp-Ser (GRGDS) is a pentapeptide sequence that appears to be critical for cell interaction with fibronectin. Coinjection of GRGDS with B16-F10 murine melanoma cells dramatically inhibited the formation of lung colonies in C57BL/6 mice. Two closely related control peptides, in which specific amino acids within the GRGDS sequence were transposed or substituted, displayed little or no activity. Inhibition by GRGDS was dose-dependent, noncytotoxic, and did not result from an impairment of cellular tumorigenicity. GRGDS may function by inhibiting tumor cell retention in the lung since radiolabeled B16-F10 tumor cells injected with the peptide were lost at a substantially greater rate than control cells.

Augmentation of B16 melanoma lung colony formation in C57BL/6 mice having marked granulocytosis.

The effect that polymorphonuclear leukocytes (PMN) may have on the metastatic colonization of tumor cells is controversial: some laboratories have reported that PMN can inhibit metastasis whereas others have shown an augmentation effect. We have exploited our finding that a particular fibrosarcoma (BMT-11) transplanted subcutaneously into syngeneic C57BL/6 mice induces a progressive increase in the number of circulating PMN, to re-examine this question. We used such "granulocytosis-positive" mice as recipients of B16 melanoma cells to examine the influence of significant granulocytosis on the level of lung tumor colonies. The number of B16 melanoma lung colonies detected after intravenous (i.v.) injection was significantly higher in BMT-11 tumor-bearing mice with granulocytosis than in control (non-tumor-bearing) mice. Retention of 125IUdR-labelled B16 cells 24 hr after the i.v. injection was 3 to 10 times greater in mice with granulocytosis than in controls. Either simultaneous injection, or preinjection of PMN with B16 cells, increased the lung-colonizing capacity of B16 melanoma cells. These results suggest that abnormally increased numbers of PMN in the peripheral blood, particularly in the lung circulation, can enhance the ability of tumor cells to colonize or metastasize.

Interference With Tumor Cell-Induced Degradation of Endothelial Matrix on the Antimetastatic Action of Nafazatrom2

The antithrombotic compound nafazatrom was evaluated in several in vivo and in vitro assays to elucidate the mechanism of its antimetastatic activity. C57BL/6 mice bearing B16 amelanotic subcutaneous tumors treated with 100 mg nafazatrom/kg/day exhibited a sixfold reduction in metastatic pulmonary lesions compared to lesion numbers in controls. The reduction in metastatic lesions was not accompanied by changes in primary tumor growth, and up to 1 microgram nafazatrom/ml did not inhibit tumor cell proliferation in vitro. Treatment of C57BL/6 mice with nafazatrom prior to iv inoculation of tumor cells failed to inhibit lung colony formation. In vitro exposure of exponentially growing B16 amelanotic cells to nafazatrom (1 microgram/ml for 72 hr) in culture did not change their ability to adhere to endothelial cell monolayers. B16 amelanotic cells degraded the matrix material of bovine endothelial cell monolayers; a heparin sulfate proteoglycan appeared to be the predominant matrix component released by these tumor cells, as judged by resistance to chondroitin ABC lyase and sensitivity to heparitinase and pronase degradation. Nafazatrom (1 microgram/ml for 72 hr) inhibited the solubilization of matrix components by approximately 60%. Tumor cell degradation of matrix components is an important event in the pathogenesis of metastasis. Thus the interference with this process appears to provide an explanation for the inhibition of malignant cell dissemination in vivo by nafazatrom.

Modulation of tumor growth by allogeneic blood transfusion.

The effect of a single blood transfusion on the formation and outgrowth of experimental lung metastases was assessed in two tumor models in rats. The transfusions were given either 1 week before (day -7) or 1 week after (day +7) tumor cell inoculation. The first approach was performed to investigate the effect of transfusions on the formation of lung colonies, the second approach to study the effect on the outgrowth of established metastases. The first tumor model used was a transplantable, nonimmunogenic sarcoma (LS175) in BN rats. Animals were injected i.v. with 10(5) tumor cells and the number of metastases developing in the lungs was counted after 18 days. Experimental animals received 1 ml of allogeneic WAG blood, controls were given 1 ml of syngeneic BN blood. A single allogeneic transfusion given on day -7 had no effect on the formation of LS175 lung colonies but, when given day +7, stimulated the outgrowth of established metastases. The second tumor model was a highly immunogenic transplantable basal cell carcinoma (BC1618) in inbred WAG rats. Rats were injected i.v. with 10(6) tumor cells and the numbers of lung colonies were counted after 21 days. Experimental animals were transfused with 1 ml of BN blood, controls received 1 ml of WAG blood. An allogeneic transfusion on day -7 led to a significant inhibition of lung metastases, whereas a transfusion on day +7 had no effect. The results clearly indicate that allogeneic blood transfusions can modulate tumor growth and metastasis. Although immunological factors seem to play a crucial role in this transfusion phenomenon, there was no clear-cut correlation between the observed effects (accelerated tumor growth vs inhibition of metastasis) and the type of immunomodulation evoked.

Antiplatelet pyrimido-pyrimidines and metastasis

The role of antiplatelet in relation to thieer potential antimetastatic activities has been reviewed and the effects of two pyimido-pyrimidine derivatives (RX-RA69 and RX-RA85 with strong antiplatelet activities investigated in metastasizing tumour models. The routes of administration and drug dosages were always chosen in such a way that good antiplatelet activities were obtained. RX-RA69 (20 mg/kg/day) given in the drinking water had no effect on spontaneous metastasis of Lewis lung carcinoma. RX-RA85 (20 mg/kg/day) did not influences spontaneous metastasis of B16 melanoma. On the other hand, giving RX-RA85 (8 mg/kg) daily i.p. to Lewis lung carcinoma bearing mice significantly increased th number of lung metastases but had no significant effect on primary tumour implant growth. Pretreating mice orally with 20 mg/kg RX-RA85 1 h before i.v. injection of B16 melanoma cells had no signi- ficant effect on lung colony number or distribution of extrapulmonary tumours while injecting the same dosage of RX-RA85 i.v. 1–2 h before tumour-cell injection decreased lung colony formation, but increased extrapulmonary tumour burden. The investigation like many others does not support the importance of platelets in metasis formation.

The relationship between lung colony and in situ assays

The relationship between three different assays: tumor control, tumor growth delay and lung colony formation, was examined after fast neutron and y ray irradiations. Fibrosarcomas (NFSa) in syngeneic C3Hf mice were irradiated locally with 60Co γ rays, fast neutrons or mixed beams (γ rays and fast neutrons). A comparison between the lung colony assay and the TRT 50 (50% tumor growth delay time) assay when cells were exposed to single doses of fast neutrons or γ rays, resulted in identical growth delay times. The fraction of cells surviving a single dose of fast neutrons, was 10 times higher than the surviving fraction of cells after a single dose of γ rays. Both doses resulted in the same tumor control probability (TCD 50 assay). Neither repair of potentially lethal damage nor tumor bed effect was sufficient to explain the difference between cell survival and tumor control probability. The surviving fraction of cells following fractionated irradiations of γ rays and fast neutrons were identical at 50% tumor control probabilities.

Characterisation of rat tumour cell hybrids: procoagulant and fibrinolytic activities.

The formation of lung colonies after i.v. injection of highly metastatic rat mammary adenocarcinoma cells (MAT 13762) was greatly reduced by concurrent treatment of rats with heparin. The procoagulant activity (PCA) of these cells, and of a non-metastatic adenocarcinoma (DMBA-8) has therefore been measured. These have been compared with PCA expressed by MAT 13762 cell derivatives including a non-metastatic hybrid clone (MAT 13762 X DMBA-8), its metastatic revertant, and clones selected in vivo from lung metastases. Potent PCA was expressed on intact MAT 13762 cells and in their spent culture media, the latter being sedimentable and associated with shed membrane vesicles. Cell-derived PCA, unlike thromboplastin, was equally effective in factor VII-deficient and normal bovine plasma. There were, however, no major differences in the expression of PCA (either cell-associated or shed) between the metastatic and non-metastatic cell types studied. Plasminogen activator (PA) production by these cells has also been measured. The results are discussed in the context of the possible role of fibrin formation and fibrinolysis in the metastatic process.

Inhibition by dihydropyridine class calcium channel blockers of tumor cell-platelet-endothelial cell interactions in vitro and metastasis in vivo

Three calcium channel blockers of the dihydropyridine class were tested in vitro for their effects on tumor cell-platelet-endothelial cell interactions and in vivo for antimetastatic properties. Felodipine, nimodipine and nifedipine inhibited tumor cell-induced platelet aggregation in vitro in a dose-dependent manner. These compounds also inhibited platelet-enhanced tumor cell adhesion to endothelial cells in vitro. Lung colony formation (“experimental” metastasis) and spontaneous pulmonary metastasis were inhibited by felodipine, nimodipine and nifedipine. From the present studies on three calcium channel blockers of the dihydropyridine class we hypothesize that calcium channel blockers may represent a new generic class of antimetastatic agents.

Influence of migratory blood cells on the attachment of tumor cells to vascular endothelium.

The purpose of this study was to determine whether the presence of migratory blood cells in association with tumor emboli had the capacity to alter the attachment of tumor cells to vascular endothelium. Highly metastatic RT7-4bs rat hepatocarcinoma cells were labelled with [125I]UdR before being allowed to form mixed cellular spheroids incorporating resident peritoneal macrophages, activated peritoneal macrophages, polymorphonuclear leukocytes, splenic T lymphocytes, or splenic B lymphocytes derived from both normal and tumor-bearing animals. The presence of polymorphonuclear leukocytes or activated macrophages led to a considerable increase in the number of tumor cells attaching to endothelial cell monolayers in vitro. The presence of T or B lymphocytes from either normal or tumor-bearing rats was without effect on tumor attachment to endothelium. Increased tumor cell retention in the lungs was evident for mixed spheroids containing tumor cells and polymorphonuclear leukocytes compared to homotypic tumor spheroids composed of tumor cells alone. Furthermore, preinjection of polymorphonuclear leukocytes intravascularly or inoculation of tumor cells as heterotypic spheroids containing polymorphonuclear leukocytes increased lung colony formation over that obtained after inoculation of tumor cells alone. Several simple sugars were tested for their ability to block tumor cell, polymorphonuclear leukocyte or activated macrophage binding to endothelium in vitro. The results indicate that the glycosylated cell surface components mediating tumor cell attachment to endothelium are not identical with those mediating attachment of either polymorphonuclear leukocytes or activated macrophages. Medium conditioned during mixed spheroid formation was without effect on tumor cell attachment to endothelium. We conclude that the presence of some, but not all classes of leukocytes can modulate tumor cell attachment to vascular endothelium, an effect most likely mediated by a mechanism involving direct contact between the leukocytes and the endothelial cell monolayer.

Modulation of natural and acquired immunity of rats to tumour isografts.

Syngeneic tumour cell suspensions were injected into the foot or intravenously in normal rats or rats bearing subcutaneous tumours. Resistance was assessed by measuring the swelling of the foot due to the growth of the tumours or by counting lung colonies or metastases. The tumours included two methyl-cholanthrene-induced fibrosarcomas (D7, D8), a metastatic variant of D8 (D8M) and a spontaneous adenocarcinoma (ST-2). D8, D8M and ST-2 induced concomitant immunity to challenge in the foot. D8 and ST-2, but not D8M, induced concomitant immunity in the lung. Concomitant immunity was, in each case tested, non-specific and could be expressed against a tumour which itself failed to induce concomitant immunity. Irradiation significantly enhanced the growth of D7, D8 and ST-2 in the feet of non-immune rats. Irradiation, carrageenan and silica significantly reduced the growth of D8M in the feet of non-immune rats. Irradiation increased the growth of D8 and D8M and carrageenan increased the growth of D8 in the feet of concomitantly immune rats (i.e., depressed resistance). Carrageenan decreased lung colony formation by D8 in normal and concomitantly immune rats and silica decreased colony formation by D8 in concomitantly immune rats (i.e., increased resistance). Thymus-deprived rats allowed greater growth of D8 in the feet but still expressed concomitant immunity. It is concluded that: (1) both natural and acquired tumour immunity may be modulated non-specifically in rats; (2) concomitant immunity, which has a large non-specific component, may contribute to the suppression of metastases but may also have site specificity; (3) the exact nature of the rat host-tumour relationship differs from tumour to tumour.

Inhibition of tumor cell-platelet interactions and tumor metastasis by the calcium channel blocker, nimodipine.

Nimodipine, a dihydropyridine calcium channel blocker, was evaluated in vitro for its ability to inhibit platelet aggregation induced by B16 amelanotic melanoma (B16a) and Walker 256 carcinosarcoma (W256) cells, and for its ability to inhibit platelet-enhanced B16a and W256 adhesion to rat microvascular endothelial cells. Nimodipine produced a dose-dependent inhibition of tumor-cell-induced platelet aggregation (TCIPA). Platelets enhanced tumor cell adhesion to endothelium both in the presence and absence of overt platelet aggregation. However, the greatest enhancement of adhesion occurred under aggregatory conditions. Nimodipine at a dose of 40 micrograms/ml inhibited platelet-enhanced adhesion to endothelium under aggregatory and nonaggregatory conditions. Nimodipine was tested in vivo for its ability to inhibit both "experimental' and spontaneous metastasis. Nimodipine produced a 46 per cent inhibition of lung colony formation at a dose of 5 mg/kg body-weight. Over a dose range of 0.1-80 mg/kg, nimodipine produced a significant dose-dependent inhibition in the formation of lung metastases from a subcutaneous tumor. The in vitro results demonstrate that a dihydropyridine calcium channel blocker can inhibit tumor cell-platelet-endothelial cell interactions. The in vivo results suggest that these compounds may be a new class of antimetastatic agent.

The use of cell fusion to analyse factors involved in tumour cell metastasis.

Cell fusion has been used to study some of the factors involved in the process of metastasis. Highly metastatic rat mammary adenocarcinoma cells were fused with various non-metastatic cells and the hybrid clones isolated. These were then tested for their metastatic potential either by injecting the cells intravenously and measuring lung colony formation or by injecting the cells subcutaneously and measuring their ability to form lymphatic metastases. With most hybrid clones tested, the metastatic potential was either inhibited or greatly suppressed; thus this phenotype is a recessive characteristic. We also monitored the hybrid cells' ability to produce plasminogen activator (PA) a serine proteinase thought to be involved in the formation of metastatic lung foci. Whilst the highly metastatic parent cells produced large quantities of PA, none could be detected in the non-metastatic lines. Although the hybrid clones produced little PA activity this could not be correlated with their decreased metastatic potential in that one clone, after extensive in vitro culture, reverted to a more metastatic line without a concomitant increase in PA activity. The suppressed PA activity may be due to the presence of an inhibitor that is spontaneously produced by the hybrid cells.

Tumor-associated macrophages of mouse mammary tumors. I. Differential cytotoxicity of macrophages from metastatic and nonmetastatic tumors.

Macrophages were isolated from a series of transplanted mouse mammary tumors, originally derived from a single spontaneously arising tumor, to determine whether macrophage content or function correlated with any of a wide range of tumor properties. None of the tumor cell lines differed significantly in susceptibility to killing by MVE-2-activated peritoneal macrophages (cytotoxicity ranged from 40 to 63%). No significant differences were observed in macrophage content among the five tumor lines, nor was there any correlation between macrophage content and tumor weight, time since transplantation, or ability to metastasize. A significant association was observed, however, between a tumor's ability to metastasize to lung spontaneously and the tumoricidal activity of that tumor's infiltrating macrophages. Significant tumoricidal activity was seen when macrophages isolated from every metastatic tumor studied were used, whereas macrophage-mediated tumoricidal activity was observed in only 35% (6 of 17) of nonmetastatic tumors. Macrophage cytotoxicity was associated with spontaneous metastasis and not with lung colony formation per se.