Identification of a tumor cell receptor for VGVAPG, an elastin-derived chemotactic peptide.

Extracellular matrix proteins and their proteolytic products have been shown to modulate cell motility. We have found that certain tumor cells display a chemotactic response to degradation products of the matrix protein elastin, and to an elastin-derived peptide, VGVAPG. The hexapeptide VGVAPG is a particularly potent chemotaxin for lung-colonizing Lewis lung carcinoma cells (line M27), with 5 nM VGVAPG eliciting maximal chemotactic response when assayed in 48-microwell chemotaxis chambers. Binding of the elastin-derived peptide to M27 cells was studied using a tyrosinated analog (Y-VGVAPG) to allow iodination. Scatchard analysis of [125I]Y-VGVAPG binding to viable M27 tumor cells at both 37 and 4 degrees C indicates the presence of a single class of high affinity binding sites. The dissociation constant obtained from these studies (2.7 X 10(-9) M) is equivalent to the concentration of VGVAPG required for chemotactic activity. The receptor molecule was identified as an Mr 59,000 species by covalent cross-linking of the radiolabeled ligand to the M27 tumor cell surface and subsequent analysis of the cross-linked material by electrophoresis and size-exclusion high performance liquid chromatography. These results suggest that M27 tumor cell chemotaxis to VGVAPG is initiated by high affinity binding of the peptide to a distinct cell surface receptor.

Coordinated expression of the vitronectin receptor and the urokinase-type plasminogen activator receptor in metastatic melanoma cells.

Integrin alpha v beta 3 is a marker of progression in malignant melanoma. Previously we reported that human melanoma cells derived from regional lymph node metastases had increased alpha v beta 3-mediated adhesion to lymph node vitronectin. In the present study, the expression and function of alpha v beta 3 were further investigated with emphasis on the functional relationship between alpha v beta 3 and the urokinase-type plasminogen activator system of proteolysis. We found that metastases-derived melanoma MeWo LNI 6I (6I) and MIM/8 LNI cells had a markedly increased expression of alpha v mRNA transcripts relative to the parent lines which was reflected in significantly elevated levels of the alpha v beta 3 heterodimers on the cell surface. These cells also expressed elevated levels of urokinase plasminogen activator receptor (uPAR) mRNA and had higher levels of surface bound urokinase plasminogen activator as detected by immunolabeling. To determine whether the expression of uPAR and alpha v were linked, alpha v synthesis in the metastatic melanoma cells was suppressed using alpha v antisense phosphorothioate oligonucleotides. This resulted in a marked decrease in detectable alpha v mRNA and protein and a corresponding substratum-specific reduction in cell adhesion to vitronectin. When uPAR expression in these cells was subsequently analyzed, we found a reduction of approximately 50% in uPAR mRNA levels. On the other hand, ligation of the alpha v beta 3 receptor on the melanoma cells by immobilized antibody resulted in a twofold increase in uPAR mRNA. The results suggest that the expression of uPAR in metastatic melanoma cells is linked to the expression and function of the vitronectin receptor.

Human melanoma cells derived from lymphatic metastases use integrin alpha v beta 3 to adhere to lymph node vitronectin.

Human melanoma is a highly metastatic cancer and the regional lymph nodes are generally the first site of metastasis. Adhesion to cryostat sections of human lymph nodes was therefore studied using two human melanoma models established from lymph node metastases, namely, MeWo cell lines of diverse metastatic potentials and a highly metastatic cell line of recent origin designated MIM/8. We found a good correlation between the metastatic potentials of the melanoma cells as measured in nude mice and their ability to adhere to cryostat sections of human lymph nodes. When adhesion to immobilized extracellular matrix proteins was measured, a significant increase in adhesion, which correlated with increased metastasis, was seen mainly on vitronectin and to a lesser extent on fibronectin. The adhesion to vitronectin and to the frozen sections were specifically blocked by an RGD-containing peptide, mAb 661 to vitronectin and mAb LM609 to integrin alpha v beta 3. FACS analysis revealed a significant and specific increase in cell surface expression of alpha v beta 3 on the metastatic cells as compared to the parent line. Together these results suggest that the adhesion of melanoma cells to lymph node vitronectin via the alpha v beta 3 receptor plays a role in the process of lymphatic dissemination.

Characterization of two highly metastatic variants of Lewis lung carcinoma with different organ specificities.

The biological properties and metastasis of two sublines of the Lewis lung carcinoma (3LL) which have maintained a stable pattern of organ-selective metastasis have been studied. Subline M-3LL, a lung-specific variant which originated from a lung metastasis of the parent line, metastasized only to the lung following injection of 10(4)-10(6) tumor cells i.v. or s.c. Lymphatic metastases of this tumor were rarely detected. Subline H-3LL which was developed from a rare, spontaneous hepatic metastasis of the parent line metastasized primarily to the liver, but pulmonary metastases have also been observed. While it grew at local s.c. sites, this tumor metastasized to the regional lymph nodes draining the tumor site, as determined by histology and by bioassay of the lymph nodes following their grafting into new recipient animals. Histologically, the two lines were indistinguishable with the exception of a higher incidence of giant cells detected in tissue sections and culture monolayers of the liver-colonizing variant H-3LL. Ten clones derived from each of the variant lines were expanded in vitro and inoculated i.v. While none of the ten clones derived from line M-3LL gave rise to extrapulmonary metastasis, nine of ten clones derived from Tumor H-3LL gave rise to hepatic metastasis. Highly metastatic clones selected from each tumor were subsequently used to study the patterns of distribution and arrest of radiolabeled tumor cells following their inoculation i.v. No correlation could be found between the initial distribution of the radiolabeled tumor cells and the organ selectivity eventually noted in the site of the metastases.

Identification of an Mr 64,000 plasma membrane glycoprotein mediating adhesion of tumor H-59 cells to hepatocytes.

Tumor H-59 is a variant of the Lewis lung carcinoma which is metastatic to the liver. In previous studies we have shown that liver metastasis in this tumor model correlates with adhesion in vitro to hepatocyte monolayers (Brodt, P., Clin. Exp. Metastasis, 7: 525-539, 1989). In an attempt to identify the adhesion molecule(s) involved, monoclonal antibodies were produced. One monoclonal antibody (MAb C-11) was highly specific to hepatocyte-adherent tumor cells. The antibody (an IgG1) and F(ab)2 fragments blocked tumor cell attachment to hepatocytes while having no effect on tumor cell adhesion to basement membrane proteins coated onto culture dishes. Western blot analysis of solubilized 11-59 plasma membranes or cell lysates showed that the antibody recognizes an Mr 64,000 protein. Treatment with N-glycosidase F prior to Western blot analysis revealed that N-linked carbohydrate residues constitute approximately 43% of the total weight of this molecule. This glycoprotein is only weakly expressed on tumor M-27, a lung-specific subline of the Lewis lung carcinoma (Brodt, P., Cancer Res., 46: 2442-2448, 1986), is undetectable in plasma membrane preparations obtained from spleen cells and thymocytes, but can be detected on cultured hepatocytes and in hepatocyte cell lysates. Pretreatment of the hepatocytes with MAb C-11 also resulted in inhibition of tumor cell adhesion. These results suggest that this glycoprotein mediates the attachment of H-59 cells to hepatocytes.

Changes in the host lymphocyte subsets during chemical carcinogenesis.

Changes in small lymphocyte subsets in the lymphoid organs of young C3H mice were studied following i.m. injection of a carcinogenic dose of 3-methylcholanthrene in trioctanoin oil. Using monoclonal anti-Lyt antibodies and a sandwich radiolabeling method with 125I-labeled rabbit anti-mouse Immunoglobulin, the lymphocyte subpopulations in the thymus, spleen, and draining lymph node were examined by radioautography. During the fifth week following the administration of the carcinogen and prior to the appearance of histologically evident tumor cells, a sharp decrease in the level of Ly-1,2+ small lymphocyte population in the thymus was noted which coincided with a considerable increase (10-fold) in the Ly-2+ and a small increase (1.7-fold) in the Ly-1+ population. During the same period, a similar increase in the Ly-2+ population was also observed in the draining but not in the contralateral lymph node. The high levels of Ly-2+ cells lasted for more than 4 weeks in the thymus while, in the draining node, they lasted for 2 weeks and dropped to normal levels (0 to 2%) simultaneously with the appearance of tumor cells identified in histological preparations. Smaller increases (3-fold) in the Ly-2+ subset were also noted in the spleen and contralateral node, but they were seen later (7 to 8 weeks) and were shorter in duration. These systemic increases coincided with the appearance of macroscopic tumor nodules. The relative incidence of immunoglobulin+ cells did not change significantly in any of the organs tested during the entire tumor induction phase, while the level of null cells underwent a slight increase (approximately 2-fold) in all organs tested immediately prior to and following the appearance of macroscopic tumors. None of these changes was observed in trioctanoin oil-injected control animals. The mixed lymphocyte reaction response of the draining node cells, but not of the spleen, was suppressed during the period of increased level of Ly-2+ cells. Furthermore, during this period, s.c. transplantation of a syngeneic mammary tumor in the same leg resulted in enhanced local growth as well as metastatic spread of the tumor to the lungs in 3-methylcholanthrene-treated mice. These findings suggest that a localized immunosuppression associated with the rise in the Ly-2+ cells may be of functional significance during carcinogen-induced tumor development.

Paracrine growth stimulation by hepatocyte-derived insulin-like growth factor-1: a regulatory mechanism for carcinoma cells metastatic to the liver.

Tumor H-59 is a subline of the Lewis lung carcinoma which is highly and preferentially metastatic to the liver. We used this carcinoma model to investigate the role of paracrine growth regulation by liver-derived factors in this organ-selective pattern of metastasis. We observed that serum-free medium conditioned by primary cultures of mouse hepatocytes was highly and specifically mitogenic for H-59 cells but had little effect on the proliferation of a second subline, i.e., carcinoma M-27, which is metastatic only to the lung. This mitogenic activity was hepatocyte-specific and could be blocked or depleted by a monoclonal antibody to insulin-like growth factor 1 (IGF-1). IGF-1 could in turn be detected in hepatocyte conditioned medium by the Western blot assay, and when added to serum-deprived cells, IGF-1 could stimulate the proliferation of H-59 but not M-27 cells. Furthermore, when expression of the IGF-1 receptor was analyzed by the Northern blot assay, we found that H-59 cells expressed significantly higher levels of mRNA transcripts encoding IGF-1 receptor. A ligand binding assay revealed that the number of IGF-1 binding sites on H-59 cells was 3.4-fold higher than that on M-27 cells. The results identify IGF-1 as the growth factor mediating the proliferative effect of hepatocyte conditioned medium and suggest that paracrine growth stimulation by hepatocyte-derived IGF-1 is a potential mechanism of selection in the process of liver colonization by these carcinoma cells.

Regulation of the Mr 72,000 type IV collagenase by the type I insulin-like growth factor receptor.

The Mr 72,000 type IV collagenase [matrix metalloproteinase 2 (MMP-2)] is known to play a central role in the process of invasion and metastasis, but its regulation is not clearly understood. We investigated the role of the type I insulin-like growth factor (IGF-I) in the regulation of tumor cell invasion and the synthesis of MMP-2. Highly invasive murine Lewis lung carcinoma subline H-59 cells, in which expression of the IGF-I receptor (IGF-IR) was blocked by antisense mRNA, had a significantly reduced invasion in reconstituted basement membrane (Matrigel) as compared with that of controls. These cells had a decrease of up to 6-fold in the level of MMP-2 mRNA transcripts, as assessed by reverse transcription-PCR, and a corresponding reduction in protein synthesis, as assessed by the Western blot assay and gelatin zymography. Conversely, overexpression of IGF-IR in a second, poorly invasive carcinoma subline (M-27) with low endogenous levels of the receptor increased MMP-2 mRNA and protein expression by up to 7.5- and 4-fold, respectively. Ligand-mediated activation of the IGF-IR induced MMP-2 synthesis in both cell types. The results identify IGF-I as a regulator of MMP-2 expression and cellular invasion.

A monoclonal antibody to Lewis lung carcinoma variant H-59 identifies a plasma membrane protein with apparent relevance to lymph node adhesion and metastasis.

Tumors H-59 and M-27, two stable metastatic variants of the Lewis lung carcinoma, differ in their ability to disseminate lymphatically. Tumor H-59 metastasizes to the regional lymph nodes regardless of the local site of growth and gives rise to widespread lymphatic dissemination, whereas tumor M-27 disseminates hematogenously without involvement of the regional nodes (P. Brodt, Cancer Res., 46: 2442-2448, 1986). In a previous paper we reported that this divergent potential to disseminate lymphatically correlated well with adhesion to frozen sections of syngeneic lymph nodes and spleens (P. Brodt, Clin. Exp. Metastasis, 7: 343-352, 1989). A monoclonal antibody (12/50) specific for tumor H-59 was subsequently generated. This antibody (an IgG1) but not three control antibodies, which reacted with tumor H-59, significantly reduced tumor cell binding to the frozen sections. Western blot analysis revealed that it recognized a plasma membrane protein of Mr 37,000 on tumor H-59 cells. No antibody binding was detected when solubilized plasma membrane preparations of tumor M-27 were used. Subsequent enzymatic assays indicated that the binding of monoclonal antibody 12/50 was insensitive to cell treatment with exoglycosidases but could be significantly reduced by pretreatment of the tumor cells with Pronase. Together these results suggest that monoclonal antibody 12/50 recognizes a cell surface adhesion protein relevant to lymphatic dissemination of this tumor.

Loss of the metastatic phenotype in murine carcinoma cells expressing an antisense RNA to the insulin-like growth factor receptor.

The ability of malignant cells to form metastases in secondary sites remains a major obstacle to the curative treatment of cancer. Previously, we identified type 1 insulin-like growth factor (IGF-1) as a paracrine mitogen for highly metastatic murine carcinoma, H-59 cells. Here the role of IGF-1 and its receptor (IGF-1R) in metastasis was further investigated using H-59 cells transfected with a plasmid vector expressing IGF-1R cDNA in the antisense orientation. The transfectants had a markedly reduced expression of IGF-1R and lost the ability to respond to IGF-1 in vitro. When injected in vivo, either directly into the microvasculature of the liver or lung (experimental metastasis) or s.c. to allow the growth of primary local tumors (spontaneous metastasis), these cells did not give rise to any metastases under conditions which allowed wild-type or control transfectants to form multiple hepatic and pulmonary metastases. The results demonstrate that the IGF-1R can play a critical role in the regulation of carcinoma metastasis.

Rat prostate adenocarcinoma cells disseminate to bone and adhere preferentially to bone marrow-derived endothelial cells.

Approximately 70% of patients with prostatic cancer develop bone metastases. Metastatic prostate adenocarcinomas are associated with high mortality rates and represent a leading cause of cancer-related deaths among males. To study the host-tumor interactions underlying the predilection of prostate cancer cells for skeletal bone, an experimental model was developed using rat Dunning carcinoma Mat-LyLu cells. Inoculations of these cells into the left ventricle of the heart led to the development of spinal metastases in 100% of inoculated animals. A subline of Mat-LyLu (Mat-LyLu-B5) was subsequently selected through the sequential inoculation of bone marrow-derived carcinoma cells into the left ventricle and was found to have an increased metastatic potential compared to the parental line. The possible role of tumor cell adhesion to host cells in the process of bone marrow colonization was then investigated in vitro using the metastatic line and primary cultures of rat bone marrow-derived stromal cells. It was found that the adhesion of the metastatic Mat-LyLu cells to a bone marrow stromal cell culture highly enriched for endothelial cells was significantly higher than the adhesion to other bone-derived cells, including nonendothelial bone marrow stromal cells (3.5x) and osteoblasts (1.7x). It was also significantly higher than the adhesion to rat fibroblasts (7x) and to hepatic endothelial cells (7.5x). The results suggest that the adhesion of prostate carcinoma cells to the bone marrow endothelium may play a role in their metastasis to bone.

Eradication of hepatic metastases of carcinoma H-59 by combination chemoimmunotherapy with liposomal muramyl tripeptide, 5-fluorouracil, and leucovorin.

We have investigated the effect of a combined chemoimmunotherapy protocol with liposomal muramyl tripeptide phosphatidylethanolamine (MTP-PE), 5-fluorouracil (5-FU), and 5-formyltetrahydrofolate (leucovorin) on the growth of hepatic metastases using carcinoma H-59, a liver-homing subline of the Lewis lung carcinoma (P. Brodt, Cancer Res., 46: 2442-2448, 1986). C57BL/6 mice inoculated with the tumor cells via the intrasplenic route received three i.v. injections of liposomal MTP-PE, the first of which was administered 3 days prior to tumor cell inoculation. Chemotherapy with 5-FU and leucovorin at the maximal tolerated doses (30 mg/kg per injection) was initiated immediately after tumor inoculation and continued on alternate days for a total of 4 injections. The incidence of liver metastases in animals which received the combined therapy was compared to that in animals treated with chemotherapy or immunotherapy alone. We found that while the number of liver metastases was reduced in all of the treatment groups as compared to control untreated or placebo-treated animals, the combined effect of 5-FU leucovorin and liposomal MTP-PE was significantly better than that of chemotherapy or immunotherapy alone. This was reflected in a reduced incidence (70% as compared to 100% in all other groups) and in a significant reduction in the number and size of the liver nodules. Our results suggest that the efficacy of 5-FU and leucovorin in the treatment of hepatic metastases could be significantly augmented by the addition of the liposome-encapsulated immunoadjuvant MTP-PE.

Rapid induction of cytokine and E-selectin expression in the liver in response to metastatic tumor cells.

The cytokine-inducible endothelial cell adhesion receptor E-selectin has been implicated in cancer metastasis. Previously, we reported that experimental liver metastasis of Lewis lung carcinoma subline H-59 cells could be abrogated in animals treated with an anti-E-selectin antibody. To gain further insight into the functional relevance of E-selectin expression to liver colonization, we investigated here the time course of cytokine and hepatic E-selectin expression after the intrasplenic/portal inoculation of H-59 cells by using a combination of reverse transcription-PCR, Northern blot analysis, immunohistochemistry, and in situ hybridization. In parallel, we analyzed cytokine induction in response to the injection of Lewis lung carcinoma subline M-27 and murine melanoma B16-F1 cells, which do not spontaneously metastasize to the liver. In livers derived from normal or saline-injected mice, only minimal basal levels of TNF-alpha and IL-1 mRNA were detectable by RT-PCR. Rapid cytokine mRNA induction was noted within 30-60 min of H-59 injection, reaching maximal levels at 4-6 h. This was followed by the appearance of E-selectin mRNA, which was detectable at 2 h after injection and reached maximal levels at 6-8 h, declining to basal levels by 24 h. In situ hybridization analysis and immunohistochemistry localized E-selectin mRNA and protein, respectively, to the sinusoidal endothelium. M-27 cells failed to induce cytokine or E-selectin expression, whereas B-16 cells elicited a delayed and more short-lived response. The results demonstrate that upon entry into the hepatic circulation, tumor cells can rapidly trigger a molecular cascade leading to the induction of E-selectin expression on the sinusoidal endothelium and suggest that E-selectin induction may contribute to the liver-colonizing potential of tumor cells.

Urokinase overproduction results in increased skeletal metastasis by prostate cancer cells in vivo.

We previously reported that urokinase (uPA) is produced by the human prostate cancer cell line, PC-3, and could function as a growth factor for cells of the osteoblast phenotype. To examine the role of uPA in metastasis to the skeleton and to extraskeletal sites, we have developed a homologous model of uPA overexpression in a rat prostate cancer cell line. Full length cDNA encoding rat (r) uPA was isolated and subcloned as a 1.4-kilobase XbaI-BspHI fragment in the sense and antisense orientation into the Moloney murine leukemia retroviral vector pYN. The control (pYN) and experimental (pYN-ruPA, pYN-ruPA-AS) plasmids were transfected into Dunning R 3227, Mat LyLu rat prostate carcinoma cells. Experimental clones expressing at least 5-fold higher (pYN-ruPA) or 3-fold lower (pYN-ruPA-AS) than controls were selected, and control and experimental cells were inoculated into the left ventricles of inbred male Copenhagen rats. Animals were sacrificed at timed intervals to examine the evolution of metastatic lesions. Control animals developed metastases to the lumbar vertebrae resulting in spinal cord compression and hind limb paralysis at 20-21 days postinoculation. Animals inoculated with cells overexpressing uPA developed hind limb paralysis significantly earlier (by day 14-15 postinoculation). Additionally, more widespread skeletal (ribs, scapula, and femora) metastases were seen. Serum from experimental animals showed a progressive elevation in alkaline phosphatase levels, and histological examination of lumbar metastases revealed markedly increased osteoblastic activity over that observed in control animals. In contrast to this, animals inoculated with cells underexpressing uPA developed hind limb paralysis significantly later (days 25-29 postinoculation) and displayed decreased tumor metastasis. These studies support a role for the catalytic domain of uPA in enhancing both skeletal and nonskeletal prostate cancer invasiveness and are consistent with a role for the growth factor domain of uPA in mediating an osteoblastic skeletal response.

Differences in the repertoires of basement membrane degrading enzymes in two carcinoma sublines with distinct patterns of site-selective metastasis.

Basement membrane-degrading enzymes of two clonal sublines of the murine Lewis lung carcinoma with distinct patterns of organ-selective metastasis were analyzed. Subline M-27 is highly metastatic to the lung and does not form liver metastases, while subline H-59 is highly metastatic to lymph nodes and liver, but not to lung. Qualitative and quantitative differences in the enzymatic profiles were found. H-59 cells which were significantly more invasive in vitro in the Matrigel invasion assay were found by zymogram analysis to secrete high levels of a 72 kDa gelatinase, while M-27 cells produced low levels of this gelatinase and of a higher molecular weight species which migrated in the 107 kDa region. On the other hand, M-27 cells produced significantly higher levels of urokinase type plasminogen activator (uPA) as indicated by a fibrinolysis assay and by Western blot analysis. Northern blot assays revealed an increase of approx. 3-fold in mRNA for cathepsin B in tumor M-27 which was reflected in a quantitative difference in plasma membrane cathepsin B levels as detected by Western blot analysis. H-59 cells on the other hand expressed approx. 8.5-fold more mRNA for cathepsin L. The quantitative differences in the levels of basement membrane degrading proteinases released by these tumor cells suggest that invasion by these cells is differentially regulated--a possible factor in their distinct patterns of dissemination.

Suppression of basement membrane type IV collagen degradation and cell invasion in human melanoma cells expressing an antisense RNA for MMP-1.

During progression from benign nevus to vertical growth phase melanoma, melanocytes acquire the ability to invade into the dermis. This process requires rupture of the basal lamina and dissolution of dermal type I collagen. Metastases-derived human melanoma MIM cells have an invasive ability in vitro which is dependent on metalloproteinases. In the present study we analysed the role of type I collagenase (MMP-1) in melanoma invasion using MIM cells in which the constitutive expression of MMP-1 was suppressed by stable transfection with a plasmid vector expressing a 777 bp antisense fragment of MMP-1 genomic DNA. Two clones were isolated in which MMP-1 mRNA expression was blocked by 90-96% with a corresponding loss in protein synthesis. In their morphological appearance and growth rate in vitro these cells were indistinguishable from wild type cells or control cells transfected with the same vector expressing the MMP-1 fragment in the sense orientation. Their mRNA and protein levels for type IV collagenase (MMP-2) were unchanged as assessed by Northern and Western blot analyses and by gelatin zymography. However, when the invasive ability of the cells was measured, we found that in addition to type I collagen, invasion through type IV collagen and a reconstituted, type IV collagen-containing basement membrane (Matrigel) were also significantly inhibited as compared to normal or sense-transfected cells. The results indicate that despite the presence of functional MMP-2, degradation of type IV collagen matrices by the melanoma cells was dependent on expression of MMP-1.

Inhibition of carcinoma cell growth and metastasis by a vesicular stomatitis virus G-pseudotyped retrovector expressing type I insulin-like growth factor receptor antisense.

A replication-defective, vesicular stomatitis virus G-pseudotyped, Moloney murine leukemia virus retroviral vector (vLTR-IGF-IR(AS)) was generated in which a type I insulin-like growth factor receptor (IGF-IR) antisense fragment is expressed in a bicistronic mRNA with an enhanced green fluorescent protein (EGFP) reporter under the control of a potent long terminal repeat (LTR). The suitability of these retroparticles for gene therapy was tested with highly metastatic, carcinoma H-59 cells, which depend on IGF-IR expression for tumorigenicity and metastasis. Transduction with these, but not with control retroviral particles expressing EGFP only, resulted in a 70% reduction in IGF-IR levels and the loss of IGF-IR-regulated functions. Moreover, the ability of vLTR-IGF-IR(AS) retroparticle-transduced tumor cells to form experimental hepatic metastases was significantly reduced relative to controls. The results identify retrovector-mediated delivery of IGF-IR antisense as a potential strategy for cancer gene therapy.

Selection of a highly metastatic liver-colonizing subpopulation of Lewis lung carcinoma variant H-59 using murine hepatocyte monolayers.

We have previously described two highly metastatic variants of the Lewis lung carcinoma (3LL) with distinct patterns of organ-selective metastasis. Variant M-27 metastasizes exclusively to the lung regardless of the route of inoculation, whereas variant H-59 metastasizes primarily to the liver. The objective of this work was to investigate host-tumor cell interactions which determine the metastatic preference of these tumor cell lines. An in vitro adhesion assay using isotope-labelled tumor cells was utilized to compare the ability of the two cell lines to bind to murine hepatocytes. It was found that the proportion of H-59 cells which specifically bound to hepatocytes increased progressively for up to 30 min of incubation, at which time it peaked with as many as 30-50 per cent of the cells bound to the monolayers. The binding of M-27 cells was significantly lower and ranged from 4 to 8 per cent during incubation periods of up to 90 min. Hepatocyte monolayers were subsequently used to select a subpopulation of tumor H-59 enriched for highly adherent cells. This subpopulation was found to be highly metastatic to the liver, whereas the non-adherent fraction failed to give rise to hepatic metastases in most of the animals injected. DNA analysis using flow cytometry suggested that the selection was not based on cell-cycle related properties of the tumor cells. These results suggest that in the present tumor model, hepatic metastasis is closely related to and may be dependent on tumor cell binding to hepatocytes.

Tumor cell adhesion to frozen lymph node sections--an in vitro correlate of lymphatic metastasis.

Two variant sublines of the murine 3LL carcinoma with divergent potentials for lymphatic metastasis were used to assess the relationship between tumor cell potential for lymphatic metastasis and its ability to adhere specifically to lymphatic tissue. Using fresh cryostat sections of lymph nodes and spleens, it was found that tumor cell adhesion to the lymphatic tissue but not to control sections of the brain correlated well with their ability to metastasize lymphatically. On the other hand, there was no correlation between tumor cell attachment to isolated lymphocytes in vitro and their potential for lymphatic metastasis. When tumor cells were pretreated enzymatically or with the metabolic inhibitor tunicamycin with the aim of modulating cell surface carbohydrates, adhesion to the lymph node sections could be significantly reduced, implicating cell surface glycoproteins and in particular galactosyl groups in the binding. The results suggest that tumor cell attachment to lymph node cryostat sections could provide a useful tool in the study of host-tumor interactions in lymphatic metastasis.

Inhibition of carcinoma cell invasion and liver metastases formation by the cysteine proteinase inhibitor E-64.

Cysteine proteinases, in particular cathepsins B and L, have been implicated in tumor invasion and are thought to be important mediators of metastasis. Using two clonal sublines of the Lewis lung carcinoma with distinct patterns of metastasis, we previously reported that H-59 carcinoma cells, which are highly invasive and preferentially metastatic to the liver, express high levels of cathepsin L and lower levels of cathepsin B whereas M-27 cells which are less invasive and only moderately metastatic to the lung express cathepsin B only. In the present study, the role of these enzymes in invasion and metastasis, in particular the involvement of cysteine proteinases in liver metastasis of H-59 cells was further investigated. Using a reconstituted basement membrane (Matrigel) invasion assay we found that the cysteine proteinase inhibitor, E-64, blocked the invasion of H-59 cells under conditions which did not affect cell viability. A more minor but significant inhibitory effect (up to 32%) was also seen with the propeptide of cathepsin B, implicating this enzyme in the invasion process. Furthermore, treatment of H-59 cells with E-64 inhibited experimental liver metastases formation by up to 90%. On the other hand, invasion of M-27 cells could not be blocked by cysteine proteinase inhibitors even under conditions which resulted in complete abrogation of intracellular enzymatic activity, as assessed using synthetic substrates. Together, these results confirm our previous conclusion that the two carcinoma sublines utilize distinct proteolytic mechanisms for invasion and identify the cysteine proteinases as key mediators of H-59 carcinoma invasion and metastasis.