Preferential organ attachment and invasion in vitro by B16 melanoma cells selected for differing metastatic colonization and invasive properties

Murine B16 melanoma sublines have been sequentially selected in vivo for low (B16-F1) or high (B16-F10) lung, high ovary (B16-O10) or high brain (B16-B15b) colonization, and in vitro for enhanced tissue invasion (B16-BL6). These B16 sublines were tested in vitro in a syngeneic organ adhesion/invasion assay to determine differences in tumor and/or host tissue properties that might account for preferential metastasis to certain sites. Tissues used were murine C57BL/6 lung, ovary and heart. In 8 independent experiments high lung-colonizing B16-F10 cells bound to and infiltrated into lung tissue better than ovary or heart tissue, while high ovary-colonizing B16-O10 cells attached to and invaded into ovary tissue at higher rates than lung or heart tissue. Only highly tissue-invasive B16-BL6 cells were able to invade heart tissue within 18 h in the experiments. The results suggest that organ metastatic colonization preferences by malignant cells may be determined, in part, by differences in the abilities of metastatic tumor cells to attach to and invade target tissue.     

Comparison of the in vitro and in vivo effects of retinoids either alone or in combination with cisplatin and 5-fluorouracil on tumor development and metastasis of melanoma

Purpose  Retinoids have previously been reported to inhibit proliferation of melanoma cell lines in vitro. However, the relative antimetastatic efficacy of various retinoids on melanoma in vivo is unknown. Therefore, we investigated the effects of different retinoids on the invasion and metastasis of murine melanoma B16-F10 cells in vitro and in vivo. Based on the findings, the antitumor effects of a selected retinoid either alone or in combination with cisplatin were also investigated in a preclinical mouse melanoma model. Methods  Cell proliferation and invasion analyses of murine melanoma B16-F10 cells were assessed in the presence of different retinoids, either alone or in combination with cisplatin (CDDP) or 5-fluorouracil (5-FU). Experimental lung metastasis assay was performed in this study to investigate the antimetastatic efficacy of retinoids. Additionally, a mouse melanoma model was used to assess the antitumor efficacy of a selected retinoid in combination with cisplatin. Results  Retinoids showed significant antiproliferation and anti-invasion effects on murine melanoma B16-F10 cells. Pretreatment with retinoids increased the sensitivity to CDDP but not to 5-FU in in-vitro. Moreover, the number of metastatic colonies formed in the lungs of mice injected intravenously with B16-F10 cells was significantly reduced by injecting the respective retinoid once a day for 10 days. Treatment with a combination of cisplatin and 13-cis-retinoic acid resulted in a significant reduction in primary tumor size and the number of lung metastatic nodules in melanoma-bearing mice. Conclusion  These results suggest that retinoids not only exhibit antimetastatic effect, but also enhance the antitumor activity of cisplatin in vivo.     

A variant actin (βm) reduces metastasis of mouse B16 melanoma

We recently reported an acidic actin co-expressed with β and γ actin in mouse B16 melanoma, whose expression was inversely correlated with the metastatic potential. The cDNA for this actin is slightly different from the hitherto recognized mouse β actin cDNA, and we designated it βm actin. In order to directly investigate the effects of βm actin on metastasis, we transfected the βm actin cDNA into a re-cloned B16-BL6 cell line which is more invasive than the highly metastatic cell line, B16-F10; we have already reported the suppressive effect of pm actin on the invasiveness of B16 F10. Here we report on the decline in the metastatic ability of βm-transfected cells. In the pm-transfected B16-BL6 cell line, we observed an increase in the organization of actin stress fibers, accompanied by a decrease in metastasis to the lung, in the invasion of collagen gels, in In vivo invasive-ness, and in cell migration on a glass plate covered with colloidal gold particles. We observed no correlation of pm actin expression either with cell attachment to Matrigel, or with type-IV collagenase expression. These results suggest that βm actin can play a role in reducing the invasiveness of mouse B16 melanoma, most probably through decreasing cell motility, which may thus result in suppression of the metastatic ability of cells.     

Synergism between N-acetylcysteine and doxorubicin in the prevention of tumorigenicity and metastasis in murine models

The thiol N-acetylcysteine (NAC) is a promising cancer chemopreventive agent which acts through a variety of mechanisms, including its nucleophilic and antioxidant properties. We have recently shown that NAC inhibits type-IV collagenase activity as well as invasion, tumor take and metastasis of malignant cells in mice. NAC is also known to attenuate the cardiotoxicity of the cytostatic drug doxorubicin (DOX, Adriamycin). The present study was designed to evaluate whether the combination of NAC and DOX treatments in mice injected with cancer cells could affect their tumorigenic and metastatic properties. Six separate experiments were carried out, using a total of 291 adult female mice. In experimental metastasis assays, in which B16-F10 melanoma cells were injected i.v. into (CD-1)BR nude mice, DOX significantly reduced the number of lung metastases when administered i.v. at a dose of 10 mg/kg body weight, 3 days after the i.v. injection of cancer cells. NAC inhibited lung metastases when added to the medium of cancer cells before their i.v. injection. The combined treatment with DOX and NAC, under various experimental conditions, was highly effective, showing a synergistic reduction in the number of mestastases. In tumorigenicity and spontaneous metastasis assays, in which B16-BL6 melanoma cells were injected s.c. into the footpad of C57BL/6 mice, DOX decreased the number of lung metastases when given i.p. at 2 mg/kg body weight. Oral NAC exerted significant protective effects, and considerably prolonged survival of mice. The combined treatment with DOX and NAC again showed synergistic effects on the frequency and weight of primary tumors and local recurrences, and completely prevented the formation of lung metastases in the experiment in which these end-points were evaluated at fixed times. While injection of DOX7 days after implantation of cancer cells failed to improve the cancer-protective effects of NAC, its injection after 1 day resulted in a striking inhibition of lung metastases. These findings demonstrate an evident synergism between DOX (given parenterally) and NAC (given with drinking water) in preventing tumorigenicity and metastases. The indications of these animal studies warrant further evaluation in clinical trials. © 1996 Wiley-Liss, Inc.     

Nerve growth factor effects on human and mouse melanoma cell invasion and heparanase production

The role of growth factor networks in regulating the progression of human melanocytes towards tumorigenicity and ultimately the malignant phenotype is poorly understood. In particular, the autocrine and paracrine influences that modulate cellular invasion and extracellular matrix degradative enzymes of melanoma cells remain undefined at the molecular level. We report here that nerve growth factor (NGF) can modify some metastasis-associated cellular properties of human and mouse melanoma cells. Treatment of early-passage human metastatic melanoma cells (MeWo) or their variants (3S5, 70W) with biologically active 2.5S NGF resulted in (a) delayed density-dependent inhibition of melanoma cell growth; (b) increased in vitro invasion through a reconstituted basement membrane; and (c) time- and dose-dependent induction of heparanase, a heparan-sulfate-specific endo-β-D-glucuronidase associated with human melanoma metastasis. These effects of NGF were most marked in the 70W brain-colonizing cells (70W > MeWo > 3S5). The NGF enhancement of heparanase secretion was not species-specific, since it was also observed in murine B16 melanoma cells; the highest NGF stimulation of heparanase was found in brain-colonizing murine B16-B15b variant (B16-B15b > B16-BL6, B16-F10, B16-F1). NGF also increased the invasive capacity of the human 70W and murine B16-B15b sublines in a chemoinvasion assay performed with filters coated with purified heparan sulfate proteoglycan (HSPG). The enhancement of chemotactic response and heparanase production was detected at NGF concentrations sufficient to fully saturate both low- and high-affinity NGF receptors (NGFR), the neurotrophin receptor (p75) and the trkA gene product, respectively. The results suggest that, in addition to the effects of NGF on cellular development and differentiation within the peripheral and central nervous systems, NGF can exert changes in the invasive properties of neuroectoderm-derived melanoma cells.     

Inhibition of spontaneous and experimental metastasis by a new derivative of camptothecin,CPT-11, in mice

Summary The antimetastatic effect of a new water-soluble derivative of camptothecin, 7-ethyl-10-(4-(1-piperidino)-1-piperidino) carbonyloxy-camptothecin (CPT-11), were examined in several metastatic murine tumor systems. Intravenous (i.v.) injection of CPT-11 into BALB/c mice inhibited lung metastasis by i.v. inoculated, metastatic colonic adenocarcinoma 26 (C26) cells, C26NL-17, in BALB/c mice. This treatment was also effective in C57BL/6 mice against lung metastasis by i.v. inoculated B16-F10 and B16-BL6 cells, highly metastatic variants of the B16 melanoma. Furthermore, intraperitoneal (i.p.) injection of CPT-11 significantly inhibited the growth of C26NL-22 cells, a highly metastatic variant of C26, inoculated subcutaneously (s.c.) into the left front footpads of BALB/c mice. Also, i.p. or i.v. injection of CPT-11 effectively inhibited the growth of 3LL tumors inoculated s.c. into the hind footpads of C57BL/6 mice. Moreover, following s.c. inoculation of either C26NL-22 or 3LL cells, combined surgical excision of the primary tumor and either i.p. or i. v. CPT-11 injections given before or after surgery markedly inhibited the formation of pulmonary metastases. These results show that a new derivative of camptothecin, CPT-11, has a potent inhibitory effect against both spontaneous and experimental lung metastasis.     

Natural killer T cell ligand alpha-galactosylceramide inhibited lymph node metastasis of highly metastatic melanoma cells.

The role of natural killer T (NKT) cells in the prevention of multiple tumor metastasis was examined. The i.v. inoculation of a highly metastatic subline of B16-BL6 (B16-BL6-HM) melanoma cells resulted in the formation of metastatic nodules in lymph nodes in addition to lung, intrapleural cavity, and ovary. However, treatment of the mice with the NKT cell ligand alpha-galactosylceramide (alpha-GalCer) three times from 1 day after B16-BL6-HM melanoma inoculation caused a significant inhibition of multiple metastasis. Lymph node metastasis of B16-BL6-HM was almost completely blocked by alpha-GalCer treatment. This antimetastatic effect of alpha-GalCer was abolished in NKT cell-deficient mice. These results suggest that alpha-GalCer-activated NKT cells played a critical role in the prevention of lymph node metastasis of melanoma cells.     

Reduced Invasive and Metastatic Potentials of KAI1-transfected Melanoma Cells

KAI1 is a metastasis suppressor gene for human prostate cancer. To reveal the effect of KAI1 on the in vivo metastasis of tumors other than prostatic cancer, we transfected a human KAI1 cDNA into highly metastatic B16-BL6 murine melanoma cells and established stable transfectant clones with different expression levels of KAI1 message. The following results were obtained with the use of those transfectants. (1) Cell aggregation assay revealed a significantly enhanced Ca²⁺-independent aggregation of B16-BL6 cells by KAI1 cDNA transfection compared with mock transfectants ( P <0.01). (2) The in vivo phagokinetic activity and invasive ability of KAI1 transfectants were clearly decreased as compared with those of mock transfectants ( P <0.01). There was no significant effect of KAI1 expression on the in vitro or in vivo proliferation of B16-BL6 cells. (3) Lung colony formation of intravenously injected KAI1 transfectants in nude mice was significantly reduced as compared with mock transfectants or parental B16-BL6 cells ( P <0.01). These data suggest that KAI1 expression gives rise to the suppression of invasive and metastatic potentials of B16-BL6 cells.     

Natural Killer T Cell Ligand α-Galactosylceramide Inhibited Lymph Node Metastasis of Highly Metastatic Melanoma Cells

The role of natural killer T (NKT) cells in the prevention of multiple tumor metastasis was examined. The i.v. inoculation of a highly metastatic subline of B16-BL6 (B16-BL6-HM) melanoma cells resulted in the formation of metastatic nodules in lymph nodes in addition to lung, intrapleural cavity, and ovary. However, treatment of the mice with the NKT cell ligand α-galactosylceramide (α-GalCer) three times from 1 day after B16-BL6-HM melanoma inoculation caused a significant inhibition of multiple metastasis. Lymph node metastasis of B16-BL6-HM was almost completely blocked by α-GalCer treatment. This antimetastatic effect of α-GalCer was abolished in NKT celldeficient mice. These results suggest that α-GalCer-activated NKT cells played a critical role in the prevention of lymph node metastasis of melanoma cells.     

Genetically engineered mouse melanoma and mouse Lewis lung carcinoma models

Melanoma B16-F10 cells and Lewis lung carcinoma LL/2 cells were engineered with a bacterial gene -- chloramphenicol acetyl transferase (CAT) -- by establishing stable transductants. Expression of CAT in both cell types did not alter the ability of these cells to grow into tumors when injected subcutaneously into mice. In addition, the measurement of CAT levels in the lung using a simple ELISA assay revealed a close correlation with direct counting of metastatic nodules. Thus, the CAT-expressing cells will likely have wide ranging applications to quantify tumor metastasis especially in situations where visual counting is difficult. The availability of genetically labeled mouse B16-F10 melanoma and Lewis lung carcinoma cell lines will facilitate future studies of the mechanism and progression of cancer and the discovery of new therapies.     

Relationship between secreted urokinase plasminogen activator activity and metastatic potential in murine B16 cells transfected with human urokinase sense and antisense genes.

Murine melanoma B16-F1 cells of low metastatic potential were transfected with the human gene for the prepro form of urokinase in an SV40 expression vector (plasmid pSV2-uPA), and cells expressing high amounts of the human urokinase gene product were selected for by an enzyme-linked immunosorbent assay specific for human high molecular weight urokinase. Southern analysis showed one of the cell lines (clone 7) had incorporated 150 copies of the pSV2-uPA plasmid into its genomic DNA. The human urokinase synthesized by the pSV2-uPA-transfected murine B16 cells was found to be glycosylated and did not bind to the murine cell surface urokinase receptor sites. In an in vivo assay that measures metastasis from a primary tumor (spontaneous metastatic assay), clone 7 cells showed an increased ability to metastasize (12 of 12 mice showed metastatic tumors), while control cells showed a lower ability to metastasize (only 2 of 11 mice showed metastatic tumors). In a second in vivo assay, which measures only the steps of the metastatic migration process during which tumor cells extravasate from the blood and then grow into pulmonary tumors (lung colonization assay), a significant multifold increase in the ability to form lung tumors was shown by the high human urokinase-secreting B16-F1 cells. In B16-F10 cells incorporating an antisense sequence to preprourokinase (plasmid pSV1-ASuPA-265) and secreting significantly decreased amounts of murine urokinase, a corresponding significant decrease in lung colonization was observed. These results provide direct experimental support for a role of secreted (non-surface-bound) urokinase in the colonization steps of the metastatic process. Furthermore, the data indicate that the higher lung colonization ability of the B16-F10 line than of the B16-F1 line is primarily based on the quantitative differences in their abilities to produce urokinase.     

Immunochemical localization of heparanase in mouse and human melanomas

Heparanase, an endo-beta-D-glucuronidase, has been associated with melanoma metastasis. Polyclonal antibodies directed against the murine N-terminal heparanase peptide detected a Mr approximately 97,000 protein on SDS-PAGE of mouse melanoma and human melanoma cell lysates. In an indirect immunocytochemical study, human A375-SM and mouse B 16-BL6 melanoma cells were stained with the anti-heparanase antibodies. Heparanase antigen was localized in the cytoplasm of permeabilized melanoma cells as well as at the cell surface of unpermeabilized cells. Immunohistochemical staining of frozen sections from syngeneic mouse lungs containing micrometastases of B16-BL6 melanoma demonstrated heparanase localized in metastatic melanoma cells. Similar studies using frozen sections of malignant melanomas resected from patients indicated that heparanase is localized in invading melanoma cells. Our studies suggest that (a) the N-terminus of the heparanase molecule in mouse and human is antigenically related; (b) heparanase antigens are localized at the cell surface and in the cytoplasm of metastatic human and mouse melanoma cells; and (c) heparanase antigens are enriched in invasive and metastatic murine and human melanomas in vivo.     

DT-5461, a New Synthetic Lipid A Analogue, Inhibits Lung and Liver Metastasis of Tumor in Mice

We have investigated the antimetastatic effect of a new synthetic lipid A analogue, of low endo-toxicity, DT-5461, against two highly metastatic tumor cell lines, L5178Y-ML25 T-lymphoma and B16-BL6 melanoma cells in mice. Four intermittent i.v. administrations of DT-5461 at intervals of 4 days resulted in a significant inhibition of liver metastasis caused by i.v. injection of L5178Y-ML25 cells and lung metastasis of B16-BL6 cells in the experimental metastasis models. Intraperitoneal and intranasal administrations as well as i.v. administration of DT-5461 were also effective in preventing lung metastasis of the melanoma cells. Multiple administrations of DT-5461 before the surgical excision of primary tumors significantly reduced the number of lung colonies of melanoma cells and primary tumor size. Similarly, this treatment modality after the surgical excision of primary tumors showed a greater reduction of lung tumor colonies as compared with lipopolysaccharide, a synthetic lipid A (No. 506) and its analogue as well as untreated control in the spontaneous lung metastasis model. Furthermore, the group that received DT-5461 after the inoculation of lymphoma or melanoma cells showed significantly enhanced survival rate compared with the untreated control. These results suggested that DT-5461 may he therapeutically useful for the inhibition of tumor metastasis.     

Swainsonine inhibition of spontaneous metastasis

We have previously shown that swainsonine, administered systemically to C57BL/6 mice, inhibited the pulmonary metastasis of iv injected B16-F10 melanoma cells by a mechanism involving interleukin-2 production and augmentation of natural killer cell activity. From this finding, which uses an "experimental metastasis" model system, we considered: (a) whether swainsonine would be effective in the inhibition of authentic or spontaneous metastasis; (b) whether the drug would also inhibit metastasis formation in organs other than the lungs; and (c) whether the drug would block the metastasis of tumor cells of different histological type or origin. Our data indicated that swainsonine effectively inhibited the spontaneous metastasis of B16-BL6 melanoma (by 88%) and M5076 reticulum sarcoma (by 95%) murine tumor cells to the lung and liver, respectively. In both cases, the antimetastatic activity of the drug increased as a function of the concentration in drinking water up to 3 micrograms/mL. These findings indicate that the antimetastatic activity of swainsonine is not limited to artificial or experimentally induced metastasis nor to a single tumor type or specific organ. The inhibition of metastasis is likely due to a combination of events, which are currently under investigation.     

Inhibition of Tumor Cell Invasion by Ubenimex (Bestatin) in vitro

We have investigated the effect of the immunomodulator ubenimex (bestatin) on tumor cell invasion of reconstituted basement membrane (Matrigel). The invasion of B16-BL6 melanoma cells and Lewis lung carcinoma (3LL) cells into Matrigel-coated filters was inhibited by the presence of bestatin in a concentration-dependent manner. The prctrcatment of either tumor cells or Matrigel with bestatin, however, had little effect on the invasion of tumor cells. Since bestatin was found to inhibit aminopeptidase in addition to its immunomodulating activities, the inhibition of tumor invasion by bestatin is likely to be associated with the action as an enzyme inhibitor. Other aminopeptidase inhibitors, arphamcnine B and amastatin A, could also inhibit tumor cell invasion into Matrigel. Bestatin inhibited hydrolyzing activities towards substrates of aminopeptidases in B16-BL6 melanoma cells. However, bestatin did not have any effect on the haptotactic migration and adhesion of tumor cells to the substrates. These results indicated that bestatin may inhibit tumor cell invasion through a mechanism involving its inhibitory action on aminopeptidases in tumor cells.     

DT-5461, a new synthetic lipid A analogue, inhibits lung and liver metastasis of tumor in mice.

We have investigated the antimetastatic effect of a new synthetic lipid A analogue, of low endotoxicity, DT-5461, against two highly metastatic tumor cell lines, L5178Y-ML25 T-lymphoma and B16-BL6 melanoma cells in mice. Four intermittent i.v. administrations of DT-5461 at intervals of 4 days resulted in a significant inhibition of liver metastasis caused by i.v. injection of L5178Y-ML25 cells and lung metastasis of B16-BL6 cells in the experimental metastasis models. Intraperitoneal and intranasal administrations as well as i.v. administration of DT-5461 were also effective in preventing lung metastasis of the melanoma cells. Multiple administrations of DT-5461 before the surgical excision of primary tumors significantly reduced the number of lung colonies of melanoma cells and primary tumor size. Similarly, this treatment modality after the surgical excision of primary tumors showed a greater reduction of lung tumor colonies as compared with lipopolysaccharide, a synthetic lipid A (No. 506) and its analogue as well as untreated control in the spontaneous lung metastasis model. Furthermore, the group that received DT-5461 after the inoculation of lymphoma or melanoma cells showed significantly enhanced survival rate compared with the untreated control. These results suggested that DT-5461 may be therapeutically useful for the inhibition of tumor metastasis.     

In vitro assay demonstrates similar invasion profiles for B16F1 and B16F10 murine melanoma cells

The variants of the B16 murine melanoma cell line were assayed for their invasive characteristics in the membrane invasion culture system (MICS) and concomitantly tested for their ability to form lung metastases in vivo. Specifically, the B16F1 (low metastatic variant) and the B16F10 (high metastatic variant) murine melanoma cell lines were examined for their ability to invade human amniotic basement membranes (BMs) in vitro and simultaneously examined for lung colony formation in vivo. The B16F1 and B16F10 cell lines both demonstrated similar invasion profiles over 72 h with the total percent invasion through the BMs for both cell lines not exceeding 5.0%. In vivo observations reconfirmed the significant difference in the metastatic capabilities of the 2 variants. These data suggest that tumor cells with differing metastatic propensities can invade an amniotic BM at similar rates, but their survival and metastatic lesion forming capabilities in vivo may vary considerably.     

Inhibition by human recombinant tissue inhibitor of metalloproteinases of human amnion invasion and lung colonization by murine B16-F10 melanoma cells

The human tissue inhibitor of metalloproteinases (TIMP) is a glycoprotein with a molecular weight of 28,000. It appears to be ubiquitous in human mesoderm tissues and has previously been shown to be identical to the collagenase inhibitor isolated from human skin fibroblasts. TIMP inhibits type I- and IV-specific collagenases and other neutral metalloendoproteinases that may be responsible for the degradation of extracellular matrix in tumor cell metastasis. In this work we have utilized recombinant human TIMP (rTIMP) obtained by expression of its cDNA gene (Carmichael et al., Proc. Natl. Acad. Sci. USA, 83:2407, 1986). The rTIMP is shown to have similar inhibition properties as natural TIMP against human skin fibroblast collagenase. In an in vitro amnion invasion assay system, rTIMP inhibited the invasion of B16-F10 murine melanoma cells through the human amniotic membrane at an identical concentration to that reported previously for natural TIMP. The mechanism by which rTIMP inhibits amniotic membrane invasion was compared to the mechanism by which the fibronectin receptor binding peptide RGDS and the aminin receptor binding peptide YIGSR inhibit amnion invasion. RGDS and YIGSR inhibited strong binding of the tumor cells to the amniotic membrane. In contrast rTIMP did not inhibit the cell adhesion step in amnion invasion, but actually increased the number of tumor cells that were tightly bound to the amnion. Thus rTIMP appears to inhibit a later step in the amnion invasion process, following B16-F10 cell adhesion. C57BL/6 mice treated with i.p. injections of rTIMP every 12 h for 6.5 days showed a significant inhibition of metastatic lung colonization by B16-F10 murine melanoma cells. While the rTIMP inhibited the number of metastatic lung tumors formed, it had no significant effect on the size of the lung tumors. Furthermore, tumors grown s.c. in mice receiving 12-h i.p. injections of rTIMP for 6.5 days, as in the in vivo colonization assay, showed no difference in size from controls. Thus the anticolonization effect of rTIMP appears not be due to an effect on tumor growth, but on the invasion step itself. The inhibition of lung colonization in C57BL/6 mice by rTIMP is one of the first examples showing an antimetastatic effect of a selective metalloproteinase inhibitor in a mammalian animal model, and supports an essential role for metalloproteinase(s) in the extravasation and invasion of tumor cells during lung colonization by blood-borne tumor cells.     

Experimental studies with the use of metastatic clones isolated from mouse tumors

Low and high metastatic clones were isolated from mouse colon 26 by repeated cloning of tumor cells in the lung. These clone cells showed an interesting metastatic capacities, low and high lung metastasis. The results indicate that original tumor consisted of various and heterogeneous cell clones having different metastatic capacities. Using highly metastatic clones, NL-17 and B16-F10 and B16-BL6, adriamycin and 5-fluorouracil were found similarly effective to both parental and cloned tumor lines. Although there are several reports to indicate heterogeneous drug sensitivity of metastatic clones from the parental line, present study indicated the similar drug sensitivity. Therefore, heterogeneity of drug sensitivity is scheduled to check with different antitumor drugs.     

Inhibition of tumor cell invasion by ubenimex (bestatin) in vitro

We have investigated the effect of the immunomodulator ubenimex (bestatin) on tumor cell invasion of reconstituted basement membrane (Matrigel). The invasion of B16-BL6 melanoma cells and Lewis lung carcinoma (3LL) cells into Matrigel-coated filters was inhibited by the presence of bestatin in a concentration-dependent manner. The pretreatment of either tumor cells or Matrigel with bestatin, however, had little effect on the invasion of tumor cells. Since bestatin was found to inhibit amino-peptidase in addition to its immunomodulating activities, the inhibition of tumor invasion by bestatin is likely to be associated with the action as an enzyme inhibitor. Other aminopeptidase inhibitors, arphamenine B and amastatin A, could also inhibit tumor cell invasion into Matrigel. Bestatin inhibited hydrolyzing activities towards substrates of aminopeptidases in B16-BL6 melanoma cells. However, bestatin did not have any effect on the haptotactic migration and adhesion of tumor cells to the substrates. These results indicated that bestatin may inhibit tumor cell invasion through a mechanism involving its inhibitory action on aminopeptidases in tumor cells.